F . Plots of densitometric of amount of LCS - GR ( Golgi mature band ) over time , starting from 2 hr chase ( data are means of at least 3 experiments ± SD ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD).


G . GOLPH3 OE or KD HeLa expressing LCS - HA were treated with bafilomycin A1 ( BafA1 , 10 nM ) for 16 hr , fixed , processed for IF labelling : LCS ( green ) , LAMP1 ( red ) , DAPI ( blue ) . Arrowheads indicate LCS LAMP1 co - localisation . Scale bar , 20 µm .

O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.


H . WB of treated as in ( G ) . Densitometric of blot is plotted in top panel ( data are means of at least 3 experiments ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD).


I . GOLPH3 OE or KD HeLa expressing indicated HA - tagged glycoenzymes were fixed processed for IF . Asterisks indicate GOLPH3 OE . Scale bar , 20 µm .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


J . Number of glycoenzymes - HA positive spots per in GOLPH3 - KD ( left panel ) of with glycoenzymes - HA in ER in GOLPH3 - OE ( right panel ) are plotted ( n is indicated in graph ; data are means ± ; * * * p < 0 . 001 [ Student ' s t - ] ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).


A . Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [ 3H ] - sphingosine ( 2 hr ) chase ( indicated times ) , lipid extraction HPTLC separation radioactive counting ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - ] ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).


B . Effects of GOLPH3 KD or OE on sphingolipid as assessed by LC / MS of HeLa ( top ) PHFs ( middle ) . Effects of LCS KD or OE on sphingolipid as assessed by LC / MS of HeLa ( bottom ) ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with following IDs : ST001673 for HeLa , ST001672 for PHFs .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


C . Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI - MS after lipid extraction . Profiles are representative of 3 independent experiments . Untargeted lipidomics data from this study are available at Metabolomics Workbench database with following ID : ST001674 .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.


D . Histograms reporting of ShtxB or ChTxB positive in each condition as in Fig . 1F ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 [ Student ' s t - ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).


E . Effects of GOLPH3 KD or OE on ceramide as assessed by LC / MS of HeLa ( top ) PHFs ( middle ) . Effects of LCS KD or OE on ceramide as assessed by LC / MS of HeLa ( bottom ) . dotted black box highlights effects on C16 : 0 Cer ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with following IDs : ST001673 for HeLa , ST001672 for PHFs .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


A . lysates from HeLa KD or OE for either GOLPH3 or LCS were processed for SDS - PAGE Western blotting with antibodies aimed at evaluating activation status of mTOR pathway . Data are representative of at least 3 independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.


B . Clonogenic of HeLa in adherence . 250 HeLa KD for either GOLPH3 or LCS were allowed to form colonies for 10 days . Afterward , colonies were fixed stained as detailed in Materials Methods . graph on right reports number of colonies per well in each indicated condition ( data are means ± of 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 [ Student ' s t - ] ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).


C . Soft agar colony formation of HeLa transfected with different of either GOLPH3 ( left upper graph ) or LCS ( left bottom graph ) encoding plasmids . Soft agar colony formation of HeLa KD for either GOLPH3 or LCS ( right upper graph ) . Soft agar colony formation of HeLa KD for LCS GOLPH3 OE alone or in combination with LCS KD ( right bottom graph ) . Colonies were stained quantified as described in Materials Methods ; ( data points are shown in graphs ; data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 , n . s . = not significant [ Student ' s t - ] ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]).


D . lysates from parental ( left ) , GCS - KO ( middle ) , LCS - KO ( right ) HeLa , CTRL or GOLPH3 - OE were processed for SDS - PAGE WB with indicated antibodies . Data are representative of at least 3 independent experiments .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments.


E . Soft agar colony formation of parental LCS - KO HeLa expressing LCS - HA or GOLPH3 . Colony number was quantified ; ( data points are shown in graphs ; data are means ± SD of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - ] ) .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


F . of soft agar colony formation of LNCaP DU145 prostatic cancer lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS ( data are means ± of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - ] ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


D . Representative of NSCLC sections stained for GOLPH3 LCS . Scale bar , 100 µm .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O

D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.


( B ) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP ( magenta ) effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , ) . Dotted lines indicate electroporated of VZ . Single optical sections ; scale bars , 200 μm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( C ) Higher magnification of VZ SVZ of electroporated shown in ( B ) , with DAPI ( blue ) depicted in addition to PaprikaRFP ( Cas9 ) GFP . Boxes indicate areas shown at higher magnification in insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note presence of GFP in control ( top ) its absence upon Cas9 / gGFP electroporation ( bottom ) . are single optical sections . Scale bars , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(C) Higher magnification of the VZ and SVZ of the electroporated area shown in (B), with DAPI staining (blue) depicted in addition to PaprikaRFP (Cas9) and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( D ) of proportion of Cas9 - positive in VZ plus SVZ that are GFP - positive 48 h after control ( Con , ) or gGFP ( black ) Cas9 plasmid electroporation . Data are mean of 4 independent experiments ( 7 per condition in total , from 4 litters ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of the proportion of Cas9-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 plasmid electroporation. Data are the mean of 4 independent experiments (7 embryos per condition in total, from 4 litters).


( F ) Overview of electroporated areas of neocortices as revealed by mCherry ( magenta ) showing effects of Cas9 together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green , ) . Dotted lines indicate electroporated of VZ . Single optical sections ; scale bars , 200 μm .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence (magenta) showing the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( G ) Higher magnification of VZ SVZ of electroporated shown in ( F ) , with DAPI ( blue ) depicted in addition to mCherry GFP . Boxes indicate areas shown at higher magnification in insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note presence of GFP in control ( top ) its absence upon Cas9 / gGFP electroporation ( bottom ) . are single optical sections . Scale bars , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(G) Higher magnification of the VZ and SVZ of the electroporated area shown in (F), with DAPI staining (blue) depicted in addition to mCherry and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


H ) of proportion of mCherry - positive in VZ plus SVZ that are GFP - positive 48 h after control ( Con , ) or gGFP ( black ) Cas9 electroporation . Data are mean of 4 independent experiments ( 5 per condition in total , from 4 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

H) Quantification of the proportion of mCherry-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 protein electroporation. Data are the mean of 4 independent experiments (5 embryos per condition in total, from 4 litters).


( I ) VZ SVZ of electroporated areas showing Cas9 expression as revealed by PaprikaRFP ( magenta ) effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , ) ; blue , DAPI . Boxes indicate areas shown at higher magnification in insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note presence of GFP in control ( top ) its absence upon Cas9 / gGFP electroporation ( bottom ) . are single optical sections . Scale bars , 20 μm .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(I) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( J ) VZ SVZ of electroporated areas showing targeted as revealed by mCherry ( magenta ) effects of Cas9 together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green ) ; blue , DAPI . Boxes indicate areas shown at higher magnification in insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note presence of GFP in control ( top ) its absence upon Cas9 / gGFP electroporation ( bottom ) . are single optical sections . Scale bars , 20 μm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(J) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence (magenta) and the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


K ) of proportion of Cas9 - positive ( left ) mCherry - positive ( right ) in VZ plus SVZ that are GFP - positive 24 h after control ( Con , ) or gGFP ( black ) Cas9 plasmid ( left ) or ( right ) electroporation . Data are mean of 3 independent experiments each ( 3 per condition in total , from 3 litters ) . ( D , H , K ) Controls were set to 100 % gGFP conditions expressed relative to control ( D , 11 % ; H , 24 % ; K left , 40 % K right , 28 % ) . Error bars indicate SD ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K) Quantification of the proportion of Cas9-positive cells (left) and mCherry-positive cells (right) in the VZ plus SVZ that are GFP-positive 24 h after control (Con, white) or gGFP (black) Cas9 plasmid (left) or protein (right) electroporation. Data are the mean of 3 independent experiments each (3 embryos per condition in total, from 3 litters).(D, H, K) Controls were set to 100% and the gGFP conditions expressed relative to control (D, 11%; H, 24%; K left, 40% K right, 28%). Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.


( B ) Daughter of single aRGCs microinjected with either Cas9 / control gRNA ( top ) or Cas9 / gGFP ( bottom ) revealed by Dx - A555immunofluorescence ( magenta ) ; 1 , aRGC daughter ; 2 , BP daughter . Dashed lines indicate ventricular surface . are maximum projections of 20 ( Control ) 24 ( gGFP ) optical sections . Scale bars , 20 µm .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.


( C ) Single optical sections of 1 2 depicted in ( B ) , showing effects of Cas9 control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( as revealed by immunofluorescence , green ) . 1 2 are indicated by dotted lines ; note presence of GFP immunofluorescence in daughter in control its absence upon Cas9 / gGFP microinjection . Scale bars , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.


( D ) of proportion of daughter ( Dx - A555 + ) of microinjected that show GFP expression 24 h after control ( Con , ) or gGFP ( black ) microinjection . Control is set to 100 % gGFP condition expressed relative to control ( 39 % ) . Data are mean of two independent experiments ( 3 1 litter per experiment , total number of daughter scored : control 142 , gGFP 88 ) ; bars indicate variation of two values from mean ( P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).


( A - D ) Neocortex of mouseE13 . 5embryos was in utero electroporated with a plasmid encoding , under constitutive promoters , Cas9 _ T2A _ PaprikaRFP gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( Tbr2 ) , followed by at E15 . 5 . ( A ) Electroporated showing effects of Cas9 expression ( revealed by PaprikaRFPDAPI , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on aRGCs expression ( green , immunofluorescence ) ; blue , Cas9staining . Boxes indicate areas shown at higher magnification in insets ( 45 x 45 μm ) . Dotted lines indicate nuclei of progeny of electroporated Cas9 ; note presence of Tbr2 immunoreactivity in control ( top ) its absence upon Cas9 / gTbr2 plasmid electroporation ( bottom ) . Scale bars , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(A-D) Neocortex of mouseE13.5embryos was in utero electroporated with a plasmid encoding, under constitutive promoters, Cas9_T2A_PaprikaRFP and gRNA targeting either LacZ (Control, Con) or Tbr2 (Tbr2), followed by analysis at E15.5.(A) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFPDAPI, magenta) together with either control gRNA (top) or gTbr2 (bottom) on aRGCs expression (green, immunofluorescence); blue, Cas9staining. Boxes indicate areas shown at higher magnification in the insets (45 x 45 μm). Dotted lines indicate nuclei of progeny of electroporated Cas9; note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 plasmid electroporation (bottom). Scale bars, 20 μm.


( B ) of proportion of Cas9 + that are Tbr2 + 48 h after control ( Con , ) or gTbr2 ( black ) plasmid electroporation . Data are mean of 4 independent experiments ( 6 per condition , from 4 litters ) .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of the proportion of Cas9+ cells that are Tbr2+ 48 h after control (Con, white) or gTbr2 (black) plasmid electroporation. Data are the mean of 4 independent experiments (6 embryos per condition, from 4 litters).


( C ) Electroporated showing effects of Cas9 expression ( revealed by PaprikaRFP , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on abundance of mitotic progenitors as revealed by phosphohistone H3 ( PH3 ) immunofluorescence ( green ) . Apical basal mitoses are indicated by arrows arrowheads , respectively ; note reduction in mitotic Cas9 + BPs upon Cas9 / gTbr2 plasmid electroporation . Scale bars , 20 μm .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

(C) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFP fluorescence, magenta) together with either control gRNA (top) or gTbr2 (bottom) on the abundance of mitotic progenitors as revealed by phosphohistone H3 (PH3) immunofluorescence (green). Apical and basal mitoses are indicated by arrows and arrowheads, respectively; note the reduction in mitotic Cas9+ BPs upon Cas9/gTbr2 plasmid electroporation. Scale bars, 20 μm.


( D ) of apical basal Cas9 + mitoses ( as revealed by PH3 immunofluorescence ) in a unit 48 h after Cas9 / gTbr2 plasmid electroporation . Data are expressed as of data obtained upon control plasmid electroporation are mean of 3 independent experiments ( 3 per condition in total , from 3 litters ) ; note reduction in basal ( black , 68 % ) but not apical ( ) mitoses .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of apical and basal Cas9+ mitoses (as revealed by PH3 immunofluorescence) in a unit area 48 h after Cas9/gTbr2 plasmid electroporation. Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments (3 embryos per condition in total, from 3 litters); note the reduction in basal (black, 68%) but not apical (white) mitoses.


( E - H ) Single aRGCs in neocortex in organotypic slices of telencephalon of E14 . 5 mouse were microinjected with recombinant Cas9 together with gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( gTbr2 ) with Dextran 10 , 000 - Alexa 555 ( Dx - A555 ) , followed by after 48 h of culture . ( E ) Progeny of single microinjected aRGCs ( revealed by Dx - A555immunofluorescence , magenta , dotted lines ) showing effects of Cas9 together with either control gRNA ( top ) or gTbr2 ( bottom ) on Tbr2 expression ( green , immunofluorescence ) ; blue , DAPI . Note presence of Tbr2 immunoreactivity in control ( top ) its absence upon Cas9 / gTbr2 ( bottom ) microinjection . are single optical sections . Scale bars , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(E-H) Single aRGCs in neocortex in organotypic slices of telencephalon of E14.5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ (Control, Con) or Tbr2 (gTbr2) and with Dextran 10,000-Alexa 555 (Dx-A555), followed by analysis after 48 h of culture.(E) Progeny of single microinjected aRGCs (revealed by Dx-A555immunofluorescence, magenta, dotted lines) showing the effects of Cas9 together with either control gRNA (top) or gTbr2 (bottom) on Tbr2 expression (green, immunofluorescence); blue, DAPI staining. Note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 (bottom) protein microinjection. Images are single optical sections. Scale bars, 5 µm.


( F ) of proportion of progeny ( Dx - A555 + ) of microinjected aRGCs that show Tbr2 expression 48 h after control ( Con , ) or gTbr2 ( black ) microinjection . Data are mean of 3 independent experiments ( 3 per condition ; total number of scored : control 45 , gTbr2 41 ) .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

(F) Quantification of the proportion of the progeny (Dx-A555+) of microinjected aRGCs that show Tbr2 expression 48 h after control (Con, white) or gTbr2 (black) microinjection. Data are the mean of 3 independent experiments (3 embryos per condition; total number of cells scored: control 45, gTbr2 41).


( G ) Distribution across cortical wall of progeny ( revealed by Dx - A555immunofluorescence , magenta ; arrows , aRGC ; arrowheads , BPs ; open arrow , neuron ) of single control ( left ) or gTbr2 ( right ) microinjected aRGCs . Dashed lines indicate ventricular surface ( bottom ) basal lamina ( top ) . are maximum projections of 67 ( Control ) 94 ( gTbr2 ) single optical sections . Scale bars , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(G) Distribution across the cortical wall of the progeny (revealed by Dx-A555immunofluorescence, magenta; arrows, aRGC; arrowheads, BPs; open arrow, neuron) of single control (left) or gTbr2 (right) microinjected aRGCs. Dashed lines indicate ventricular surface (bottom) and basal lamina (top). Images are maximum intensity projections of 67 (Control) and 94 (gTbr2) single optical sections. Scale bars, 20 µm.


( H ) of distribution of progeny ( Dx - A555 + ) of microinjected aRGCs in VZ , SVZ intermediate zone / cortical plate ( CP + IZ ) 48 h after control ( Con , ) or gTbr2 ( black ) microinjection ( total number of scored : control 37 , gTbr2 51 , pooled from at least 3 independent experiments ) . Note that upon gTbr2 microinjection distribution of across various cortical zones is significantly different ( VZ : control 77 % , gTbr2 39 : SVZ : control 20 % , gTbr2 37 % ; IZ / CP : control 3 % , gTbr2 24 % , * , P < 0 . 05 ) . ( B , F ) Controls were set to 100 % gTbr2 conditions expressed relative to control ( B , 48 % ; F , 50 % ) . ( B , D , F ) Error bars indicate SD ( B , D , F ) ; * , P < 0 . 05 ; * * , P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantification of the distribution of the progeny (Dx-A555+) of microinjected aRGCs in the VZ, SVZ and intermediate zone/cortical plate (CP+IZ) 48 h after control (Con, white) or gTbr2 (black) microinjection (total number of cells scored: control 37, gTbr2 51, pooled from at least 3 independent experiments). Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different (VZ: control 77%, gTbr2 39: SVZ: control 20%, gTbr2 37%; IZ/CP: control 3%, gTbr2 24%, *, P<0.05).(B, F) Controls were set to 100% and the gTbr2 conditions expressed relative to control (B, 48%; F, 50%).(B, D, F) Error bars indicate SD (B, D, F); *, P<0.05; **, P<0.01.


Figure 4 . Sequencing of CRISPR / Cas9 - induced disruption of Tbr2 locus . ( A - C ) Next - generation sequencing of amplicons containing Tbr2 locus 4 off - target loci generated from PaprikaRFP - expressing isolated at E15 . 5 from neocortex of mouse in utero electroporated at E13 . 5 with plasmids encoding Cas9 _ T2A _ PaprikaRFP either gTbr2 ( black ) or control gRNA ( Con , ) as in Fig 3 A - D . ( A ) of frequency of mismatches to mouse genome spanning 30 bp upstream downstream of Tbr2 target site off - target sites ( OT1 - 4 ) . Data are expressed as of total number of reads are mean of two independent experiments ( 8 5 per condition , respectively ) . ( B , C ) of one of experiments of ( A ) for deletions ( B ) insertions ( C ) in region spanning 80 bp upstream downstream of Tbr2 target site upon Cas9 / gTbr2 plasmid electroporation .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O

Figure 4. Sequencing analysis of the CRISPR/Cas9-induced disruption of the Tbr2 locus.(A-C) Next-generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off-target loci generated from PaprikaRFP-expressing cells isolated at E15.5 from neocortex of mouse embryos in utero electroporated at E13.5 with plasmids encoding Cas9_T2A_PaprikaRFP and either gTbr2 (black) or control gRNA (Con, white) as in Fig 3 A-D.(A) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off-target sites (OT1-4). Data are expressed as percentage of the total number of reads and are the mean of two independent experiments (8 and 5 embryos per condition, respectively).(B, C) Analysis of one of the experiments of (A) for deletions (B) and insertions (C) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9/gTbr2 plasmid electroporation.


A of ULK1 in MEFs exposed to hypoxia ( 1 % O2 ) for indicated times . actin , Western

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blot


B Quantification of the ratio of ULK1 to actin in (A).


C of ULK1 in MEFs treated with FCCP ( 20 μM ) for indicated times .

O O B-MEASURED_VAR O O O O O O O O O O O O O

C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actin


D Quantification of the ratio of ULK1 to actin in (C).


E MEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 6 h then fixed by 4 % paraformaldehyde . were stained with anti ‐ TIM23 ( mouse ) anti ‐ ULK1 ( rabbit ) antibodies , then Alexa Fluor ‐ 488 ‐ labeled donkey anti ‐ rabbit IgG Alexa Fluor ‐ 555 ‐ labeled donkey anti ‐ mouse IgG secondary antibodies before by immunofluorescence microscopy . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm.


F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia ( 1 % O2 ) for 12 h . PNS , post ‐ supernatant ; Cyto , cytosol ; Mito , mitochondria .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP ( 20 μM ) for 6 h . PNS , post ‐ supernatant ; Cyto , cytosol ; Mito , mitochondria .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


A HeLa were co − transfected with FUNDC1 − Myc FLAG − ULK1 . were lysed immunoprecipitated with anti − Myc antibody 24 h after transfection .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection.


B FLAG − FUNDC1 was co − transfected with GFP − vector or GFP − ULK1 for 24 h in HeLa , then , lysates were immunoprecipitated with anti − GFP antibody .

O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

B FLAG−FUNDC1 was co−transfected with GFP−vector or GFP−ULK1 for 24 h in HeLa cells, and then, cell lysates were immunoprecipitated with anti−GFP antibody.


CMEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 3 h , followed by immunoprecipitation using anti ‐ ULK1 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody.


D expression of FUNDC1 is induced by 10 ng / ml tetracycline ( Tet ) in FUNDC1 ‐ inducible HeLa . were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) for 24 h . Then , lysates were prepared for immunoblotting using indicated antibodies .

O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies.


EULK1 + / + were transfected with FUNDC1 ‐ Myc , ULK1 - / - were transfected with FUNDC1 ‐ Myc alone , FUNDC1 ‐ Myc HA ‐ ULK1 , or FUNDC1 ‐ Myc HA ‐ ULK1 ( K46N ) . were harvested lysed for immunoblots24 h after transfection .

O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

EULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1-/- cells were transfected with FUNDC1‐Myc alone, FUNDC1‐Myc and HA‐ULK1, or FUNDC1‐Myc and HA‐ULK1 (K46N). Cells were harvested and lysed for immunoblots24 h after transfection.


F Purified GST ‐ FUNDC1 GST ‐ FUNDC1 ( S17A ) were subjected to an in vitro kinase with Myc ‐ ULK1 immunoprecipitated by anti ‐ Myc antibody from Myc ‐ ULK1 ‐ transfected about 24 h post ‐ transfection . Phosphorylated FUNDC1 was detected by anti ‐ p ‐ S17 ‐ FUNDC1 antibody . red asterisks mark target bands .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

F Purified GST‐FUNDC1 and GST‐FUNDC1 (S17A) were subjected to an in vitro kinase assay with Myc‐ULK1 immunoprecipitated by anti‐Myc antibody from Myc‐ULK1‐transfected cells about 24 h post‐transfection. Phosphorylated FUNDC1 was detected by anti‐p‐S17‐FUNDC1 antibody. The red asterisks mark the target bands.


G Western blotULK1 , FUNDC1 , phosphorylated FUNDC1 in MEFs exposed to hypoxia ( 1 % O2 ) for indicated times .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O

G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times.


HWestern blot of kinetics of ULK1 , FUNDC1 , phosphorylated in MEFs treated with FCCP ( 20 μM ) for indicated times .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actin


I , J single mutant which can abolish ULK1 FUNDC1 interaction was identified . HeLa were co ‐ transfected with Flag ‐ ULK1 indicated constructs . 24 h after transfection , were lysed for immunoprecipitation by anti ‐ Flag antibody .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

I, J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified. HeLa cells were co‐transfected with Flag‐ULK1 and the indicated constructs. 24 h after transfection, cells were lysed for immunoprecipitation by anti‐Flag antibody.


KMEFs were transfected with FUNDC1 ‐ Myc its mutants after endogenous FUNDC1 was knocked down by siRNA . 36 h after transfection , were harvested in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) then lysed for immunoblotting with indicated antibodies .

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies.


L MEFs were co ‐ transfected with FUNDC1 ‐ Myc or its mutants MitoDsred after endogenous FUNDC1 was knocked down by siRNA . 24 h post ‐ transfection , were treated with hypoxic ( 1 % O2 ) conditions for 12 h or FCCP ( 20 μM ) for 6 h before fixed by 4 % paraformaldehyde stained with indicated antibodies . Scale bar , 10 μm .

O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.


AULK1 + / + ULK1 − / − were cultured under hypoxic ( 1 % O2 ) condition for 12 h or 24 h in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . lysates were immunoblotted .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


B ULK1 + / + ULK1 − / − were treated with FCCP ( 20 μM ) for indicated times in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . lysates were immunoblotted .

O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


C ULK1 + / + ULK1 − / − were transfected with FUNDC1 − Myc for 24 h then harvested in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . lysates were immunoblotted .

O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


D ULK1 − / − were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . lysates were immunoblotted .

O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.


EULK1 − / − were transfected with HA − ULK1 or HA − ULK1 then treated with FCCP ( 20 μM ) for 24 h . were harvested in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . lysates were immunoblotted .

O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


F FUNDC1 ‐ knockdown MEFs were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17A ) for indicated times . lysates were used for immunoblotting .

O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting.


G ULK1 + / + were transfected with FUNDC1 ‐ Myc , ULK1 − / − were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) . 24 h after transfection , were fixed with 4 % paraformaldehyde stained with anti ‐ Myc ( mouse ) anti ‐ LC3 ( rabbit ) antibodies for immunofluorescence microscopy . Scale bar , 10 μm .

O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm.


H ULK1 − / − were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . lysates were immunoblotted .

O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.


I ULK1 − / − were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . lysates were detected with citrate synthase activity kit .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.


J HeLa were transfected with FUNDC1 ‐ Myc , FUNDC1 - Myc ( S17A ) , or FUNDC1 - Myc ( S17D ) . 24 h after transfection , lysates were immunoprecipitated with anti ‐ LC3 antibody .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.


A ULK1 + / + or ULK1 − / − were cultured under hypoxic ( 1 % O2 ) or normoxic conditions in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − were transfected with HA ‐ ULK1 , ULK1 − / − FUNDC1 ‐ KD were transfected with HA ‐ ULK1 FUNDC1 ‐ Myc , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . were harvested lysed for immunoblotting assays .

O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


B ULK1 + / + or ULK1 − / − were treated with or without FCCP ( 20 μM ) in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − were transfected with HA ‐ ULK1 ULK1 − / − FUNDC1 ‐ KD were transfected with HA ‐ ULK1 FUNDC1 ‐ Myc then treated with FCCP ( 20 μM ) for 24 h in absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . were harvested lysed for immunoblotting assays .

O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


C ULK1 + / + , ULK1 − / − FUNDC1 ‐ KD , or ULK1 − / − FUNDC1 ‐ KD rescued by introduction of HA ‐ ULK1 FUNDC1 ‐ Myc were cultured under hypoxic ( 1 % O2 ) conditions for 24 h or with FCCP ( 20 μM ) for 24 h . Samples were analyzed by electron microscopy . Scale bar , 2 μm . yellow asterisks mark mitochondria . red arrows indicate double ‐ membraned autophagic structures . yellow arrow denotes autolysosomes .

O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.


D - E mitochondrial coverage mitochondrial − containing AVs shown in ( C ) were quantified . Recon , reconstitution .

O O O O O O O O O O O O O O O O O O O O O

D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution.


A Representative immunofluorescence microscopy of GFP of small intestinal tissue of LGR5 ‐ GFP knock ‐ in ( LGR5 ‐ GFPki ) mice . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm . B of GFP of intestinal progenitor ( ISPCs ) at indicated positions in basal crypts of LGR5 ‐ GFPkimice ( n = 80 crypts from four mice ) . Each dot presents one . Red lines : mean values ± . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5‐GFP knock‐in (LGR5‐GFPki) mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.B Quantification of GFP staining intensity of intestinal stem and progenitor cells (ISPCs) at indicated positions in basal crypts of LGR5‐GFPkimice (n = 80 crypts from four mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


C Representative immunofluorescence microscopy of Msi1 of small intestinal tissue of young mTerc + / + mice . Dashed outlines a crypt . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm . D of Msi1 of ISPCs at indicated positions in basal crypts of young mTerc + / + mice ( n = 150 crypts from three mice ) . Each dot presents one . Red lines : mean values ± . Unpaired two ‐ Student ' s t ‐ .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc+/+mice. Dashed line outlines a crypt. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc+/+mice (n = 150 crypts from three mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


E Representative FACS plots showing gating of LGR5hi LGR5lo populations within LGR5 + ( GFP ‐ positive ) gating .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O

E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5+ (GFP‐positive) cell gating.


F Representative FACS plots showing gating of LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , LGR5lo ‐ low populations within LGR5hi gating .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O

F Representative FACS plots showing gating of LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations within the LGR5hi gating.


G - I mRNA expression of indicated genes relative to GAPDH in LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , LGR5lo ‐ low populations ( n = 3 mice ) . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I mRNA expression of indicated genes relative to GAPDH in LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations (n = 3 mice). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A mRNA expression of indicated components of canonical Wnt / β ‐ catenin pathway in freshly isolated small intestinal crypts of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice ‐ matched mTerc + / + mice ( n = 5 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 5 mice per group).


B Axin2 mRNA expression in LGR5 + of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice mTerc + / + mice ( n = 3 mice per group ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


C mRNA expression of indicated components of canonical Wnt / β ‐ catenin pathway of p53 target gene p21 in cultured crypts of 2 ‐ month ‐ old G3 mTerc − / − mice mTerc + / + mice ( n = 3 mice per group ) .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


A - J Flow cytometry of freshly isolated crypt from small intestine of young ( 2 - 3 month ) old ( 12 - 16 month ) LGR5 ‐ GFPki , mTerc + / + mice LGR5 ‐ GFPki , G3 mTerc − / − mice ( n = 4 mice per group ) . ( A , B , D , E , H , I ) Representative FACS plots depicting of LGR5 + . Note reduction in LGR5 + in G3 mTerc − / − mice with remaining showing almost exclusively weak expression of GFP ( LGR5lo ) that within fraction of LGR5hi , with high LGR5 ‐ reporter activity ( LGR5hi ‐ high ) are preferentially depleted in response to IR . ( C , F ) of ( C ) number of LGR5 + ( F ) number of LGR5hi LGR5lo . ( G ) of of LGR5hi LGR5lo within fraction of LGR5 + . ( J ) of survival rate of LGR5hi ‐ high LGR5hi ‐ low within fraction of LGR5hi comparing old G3 mTerc − / − mice to mTerc + / + mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

A-J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young (2-3 month) and old (12-16 month) LGR5‐GFPki, mTerc+/+mice and LGR5‐GFPki, G3 mTerc−/−mice (n = 4 mice per group). (A, B, D, E, H, I) Representative FACS plots depicting the analysis of LGR5+ cells. Note the reduction in LGR5+ cells in G3 mTerc−/−mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo) and that within the fraction of LGR5hi cells, the cells with high LGR5‐reporter activity (LGR5hi‐high) are preferentially depleted in response to IR. (C, F) Quantification of (C) the number of LGR5+ cells and (F) the number of LGR5hi and LGR5lo cells. (G) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5+ cells. (J) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing old G3 mTerc−/−mice to mTerc+/+mice.


K - O Immunofluorescence microscopy of GFP in basal crypts of 9 ‐ month ‐ old LGR5 ‐ GFPki , G3 mTerc − / − mice ‐ matched LGR5 ‐ GFPki , mTerc + / + mice ( n = 2 mice per group ) . ( K , L ) Representative pictures are given . Dashed lines outline crypts . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm . ( M ) of absolute number of GFP + ISPCs at indicated positions at crypt base per 50 crypts . ( N ) histogram depicts depletion rate ( % ) of LGR5 + in LGR5 ‐ GFPki , G3 mTerc − / − mice compared to ‐ matched LGR5 ‐ GFPki , mTerc + / + mice at indicated positions in basal crypts . ( O ) of GFP of ISPCs at indicated positions in positively stained basal crypts . Note that of position 4 is equal in mTerc + / + mice G3 mTerc − / − mice is significantly lower than position 1 2 of mTerc + / + mice .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-O Immunofluorescence microscopy staining of GFP in basal crypts of 9‐month‐old LGR5‐GFPki, G3 mTerc−/−mice and age‐matched LGR5‐GFPki, mTerc+/+mice (n = 2 mice per group). (K, L) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (M) Quantification of absolute number of GFP+ISPCs at indicated positions at the crypt base per 50 crypts. (N) The histogram depicts the depletion rate (%) of LGR5+ cells in LGR5‐GFPki, G3 mTerc−/−mice compared to age‐matched LGR5‐GFPki, mTerc+/+mice at indicated positions in the basal crypts. (O) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts. Note that the staining intensity of position 4 cells is equal in mTerc+/+mice and G3 mTerc−/−mice and is significantly lower than position 1 and 2 cells of mTerc+/+mice.


A - I of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( A , B ) H & amp ; amp ; E , ( C , F , I ) of absolute numbers of ISPCs at indicated positions in crypt base per 50 crypts as determined by counting of ( C ) spindle ‐ shaped in between adjacent to Paneth . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of (A, B) H&amp;E staining, (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( D , E ) PCNA . Arrowheads numbers indicate position of ISPCs in basal crypts . Scale bars : 20 μm . ( C , F , I ) of absolute numbers of ISPCs at indicated positions in crypt base per 50 crypts as determined by counting of positive for ( F ) PCNA . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 4-5 mice per group). Representative pictures of (D, E) PCNA staining. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for (F) PCNA . Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( ( G , H ) Msi1 are given . Arrowheads numbers indicate position of ISPCs in basal crypts . Scale bars : 20 μm . ( C , F , I ) of absolute numbers of ISPCs at indicated positions in crypt base per 50 crypts as determined by counting of ( I ) Msi1 . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of ((G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


J , K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9 ‐ month ‐ old G3 mTerc − / − mice ( K ) ‐ matched mTerc + / + ( J ) mice . Arrowheads point to positive . Dashed lines outline crypts . Note that remaining ISPCs in G3 mTerc − / − mice were mostly located in position 4 above Paneth . Scale bar : 20 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9‐month‐old G3 mTerc−/− mice (K) and age‐matched mTerc+/+ (J) mice. Arrowheads point to positive cells. Dashed lines outline the crypts. Note that the remaining ISPCs in G3 mTerc−/− mice were mostly located in position 4 above the Paneth cells. Scale bar: 20 μm.


A - I Three ‐ month ‐ old mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR ( n = 5 mice per group ) . ( A - F ) Representative pictures of PCNA . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm . Note depletion of PCNA + CBC in position 1 2 in between Paneth at 3 - 48 h after IR regeneration of these at 4 - 6 days after IR . ( G ) of PCNA + ISPCs at indicated positions in basal crypts at indicated time points after IR . Mean values ± are given .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (A-F) Representative pictures of PCNA staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. Note the depletion of PCNA+ CBC cells in position 1 and 2 in between the Paneth cells at 3-48 h after IR and the regeneration of these cells at 4-6 days after IR. (G) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts at the indicated time points after IR. Mean values ± SEM are given.


A - I Three ‐ month ‐ old mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR ( n = 5 mice per group ) . ( H , I ) Representative pictures of Olfm4 in situ hybridization . Arrowheads point to positive . Dashed lines outline crypts in irradiated samples . Scale bar : 20 μm . Note selective survival of ISPCs above Paneth at 24 h after IR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (H, I) Representative pictures of Olfm4 in situ hybridization. Arrowheads point to positive cells. Dashed lines outline the crypts in irradiated samples. Scale bar: 20 μm. Note the selective survival of ISPCs above the Paneth cells at 24 h after IR.


J - X Three ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR . ( J - N ) Representative pictures of GFP at indicated time points after irradiation . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-X Three‐month‐old LGR5‐GFPki, mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (J-N) Representative pictures of GFP staining at indicated time points after irradiation. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.


J - X Three ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR . ( O - X ) Flow cytometry of freshly isolated basal crypt at indicated time points after IR ( n = 4 - 10 mice per group ) . ( O ) histogram depicts number of LGR5 + in freshly isolated basal crypts at indicated time point after IR . ( P , Q ) Flow cytometry was used to determine of ( P ) LGR5hi ( Q ) LGR5lo within fraction of LGR5 + . ( R ‐ W ) Representative FACS plots of small intestinal crypt of non ‐ irradiated mice , 24 h after irradiation . Note reduction in LGR5 + in irradiated mice with remaining showing almost exclusively weak expression of GFP ( LGR5lo ) . ( X ) of survival rate of LGR5hi ‐ high LGR5hi ‐ low within fraction of LGR5hi comparing irradiated mice to non ‐ irradiated mice . Note preferential depletion of LGR5hi ‐ high within LGR5hi population upon IR . Mean values ± are given . NIR , non ‐ irradiated ; IR , irradiated .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

J-X Three‐month‐old LGR5‐GFPki, mTerc+/+mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (O-X) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR (n = 4-10 mice per group). (O) The histogram depicts the number of LGR5+ cells in freshly isolated basal crypts at the indicated time point after IR. (P, Q) Flow cytometry analysis was used to determine the percentage of (P) LGR5hi cells and (Q) LGR5lo cells within the fraction of LGR5+ cells. (R‐W) Representative FACS plots of small intestinal crypt cells of non‐irradiated mice, 24 h after irradiation. Note the reduction in LGR5+ cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo). (X) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing irradiated mice to non‐irradiated mice. Note the preferential depletion of LGR5hi‐high cells within LGR5hi cell population upon IR. Mean values ± SEM are given. NIR, non‐irradiated; IR, irradiated.


B , C LGR5hi LGR5lo were freshly isolated from 3 ‐ month ‐ old LGR5 ‐ GFPki mice 6 h after 12 Gy γ ‐ irradiation ( IR ) or from non ‐ irradiated ( NIR ) mice ( n = 3 mice per group ) . ( B ) qPCR of relative mRNA expression of apoptosis ‐ related genes compared to GAPDH . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (B) qPCR analysis of relative mRNA expression of apoptosis‐related genes compared to GAPDH. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


B , C LGR5hi LGR5lo were freshly isolated from 3 ‐ month ‐ old LGR5 ‐ GFPki mice 6 h after 12 Gy γ ‐ irradiation ( IR ) or from non ‐ irradiated ( NIR ) mice ( n = 3 mice per group ) . ( C ) Representative Western blot of lysates for expression of phospho ‐ p53 caspase ‐ 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (C) Representative Western blot analysis of cell lysates for the expression of phospho‐p53 and cleaved caspase‐3.


D , E Three ‐ month ‐ old mTerc + / + mice were γ ‐ irradiated with 12 Gy . Small intestinal tissue was collected at 3 h after IR ( n = 3 mice per group ) . ( D ) Representative picture of TUNEL . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm . ( E ) of TUNEL ‐ positive ISPCs at indicated positions in basal crypts . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E Three‐month‐old mTerc+/+ mice were γ‐irradiated with 12 Gy. Small intestinal tissue was collected at 3 h after IR (n = 3 mice per group). (D) Representative picture of TUNEL staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (E) Percentage of TUNEL‐positive ISPCs at indicated positions in basal crypts. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


F - P Two ‐ month ‐ old LGR5 ‐ GFPki , p53 + / + mice LGR5 ‐ GFPki , p53 − / − mice were exposed to 12 Gy γ ‐ irradiation ( n = 4 mice per group ) . Basal crypts were isolated at 24 h after IR . ( F - H ) Flow cytometry of survival rate of LGR5 + ( F ) , LGR5hi LGR5lo ( G ) , LGR5hi ‐ high LGR5hi ‐ low ( H ) of irradiated mice ( 24 h after IR ) compared to non ‐ irradiated mice ( NIR ) . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ . ( I - P ) Representative FACS plots of LGR5 + in freshly isolated basal crypts from irradiated non ‐ irradiated mice of indicated genotypes . Note stronger rescue of survival rate of LGR5hi ‐ high than LGR5hi ‐ low upon p53 deletion . NIR , non ‐ irradiated ; IR , irradiated .

O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O

F-P Two‐month‐old LGR5‐GFPki, p53+/+ mice and LGR5‐GFPki, p53−/− mice were exposed to 12 Gy γ‐irradiation (n = 4 mice per group). Basal crypts were isolated at 24 h after IR. (F-H) Flow cytometry analysis of the survival rate of LGR5+ cells (F), LGR5hi and LGR5lo cells (G), and LGR5hi‐high and LGR5hi‐low cells (H) of irradiated mice (24 h after IR) compared to non‐irradiated mice (NIR). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. (I-P) Representative FACS plots of LGR5+ cells in freshly isolated basal crypts from irradiated and non‐irradiated mice of indicated genotypes. Note the stronger rescue of survival rate of LGR5hi‐high than the LGR5hi‐low cells upon p53 deletion. NIR, non‐irradiated; IR, irradiated.


Crypts of 2 ‐ to 3 ‐ month ‐ old LGR5ki mice were cultured γ ‐ irradiated with 6 Gy on day 10 of culture . Sixteen hours before irradiation , were exposed to indicated treatment ( n = 4 independent experiments per group ) . A - J Representative FACS plots of indicated treatments are given . NIR , non ‐ irradiated ; IR , irradiated . K - P Flow cytometry of of LGR5 + , LGR5hi , LGR5lo of irradiated cultures ( 24 h after IR ) compared to in non ‐ irradiated controls ( NIR ) set to 100 % . ( K - M ) Note that inhibition of Wnt signaling by DKK1 treatment or R ‐ spondin reduction ( 50 % Rspo ) partially rescued decrease in LGR5 + in irradiated cultures . ( N - P ) Note that activation of Wnt signaling by 6 ‐ BIO treatment aggravated decrease in LGR5 + in irradiated cultures . Mean values ± are given . Unpaired two ‐ Student ' s t ‐ .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Crypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture. Sixteen hours before irradiation, cells were exposed to the indicated treatment (n = 4 independent experiments per group).A-J Representative FACS plots of indicated treatments are given. NIR, non‐irradiated; IR, irradiated.K-P Flow cytometry analysis of the percentage of LGR5+, LGR5hi, and LGR5lo cells of irradiated cultures (24 h after IR) compared to the percentage in non‐irradiated controls (NIR) set to 100%. (K-M) Note that inhibition of Wnt signaling by DKK1 treatment or R‐spondin reduction (50% Rspo) partially rescued the decrease in LGR5+ cells in irradiated cultures. (N-P) Note that activation of Wnt signaling by 6‐BIO treatment aggravated the decrease in LGR5+ cells in irradiated cultures. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - G Two ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were i . v . injected with LRP6 neutralization antibody , IgG serves as a negative control . Thirty ‐ six hours after injection , small intestinal tissue was collected for GFPimmunofluorescence microscopy . ( A - F ) Representative pictures are given . Dashed lines outline crypts . Arrowheads numbers indicate positions in crypts . Scale bar : 20 μm . ( G ) of GFP of at indicated positions in basal crypts ( n = 3 mice per group , fifty positively stained were measured per mouse ) . Note GFP was substantially reduced in anti ‐ LRP6 antibody ‐ injected group with intact maintenance of ISPCs at crypt bottom as shown by DAPI .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A-G Two‐month‐old LGR5‐GFPki, mTerc+/+mice were i.v. injected with LRP6 neutralization antibody, and IgG serves as a negative control. Thirty‐six hours after injection, small intestinal tissue was collected for GFPimmunofluorescence microscopy staining. (A-F) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm. (G) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, fifty positively stained cells were measured per mouse). Note the GFP staining intensity was substantially reduced in anti‐LRP6 antibody‐injected group with intact maintenance of ISPCs at the crypt bottom as shown by DAPI staining.


H - L Two ‐ month ‐ old mTerc + / + mice were i . v . injected with anti ‐ LRP6 antibody 12 h before γ ‐ irradiation . Small intestinal tissue was collected at 24 h after IR ( n = 3 - 4 mice per group ) . IgG serves as a negative control . ( H - K ) Representative pictures of PCNA of small intestine of non ‐ irradiated mice ( H , I ) or 6 Gy ‐ irradiated mice ( J , K ) . Note anti ‐ LRP6 antibody injection did not induce loss of CBC at position 1 2 in non ‐ irradiated mice . Arrowheads numbers indicate ISPC positions in crypts . Scale bar : 20 μm . ( L ) of PCNA + ISPCs at indicated positions in basal crypts 24 h after IR with indicated doses or non ‐ irradiated mice .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

H-L Two‐month‐old mTerc+/+ mice were i.v. injected with anti‐LRP6 antibody 12 h before γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3-4 mice per group). IgG serves as a negative control. (H-K) Representative pictures of PCNA staining of small intestine of non‐irradiated mice (H, I) or 6 Gy‐irradiated mice (J, K). Note the anti‐LRP6 antibody injection did not induce loss of CBC cells at position 1 and 2 in non‐irradiated mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (L) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR with indicated doses or non‐irradiated mice.


M - R Two ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were i . v . injected with LRP6 neutralization antibody 12 h before 12 Gy γ ‐ irradiation . IgG serves as a negative control . Small intestinal tissue was collected at 24 h after IR ( n = 4 mice per group ) . ( M - P ) Representative FACS plots of indicated treatments . ( Q ) Flow cytometry of number of LGR5 + ISPCs of irradiated mice compared to number in non ‐ irradiated mice ( NIR ) set to 100 % . ( R ) of number of LGR5 + , LGR5hi , LGR5lo of mice with indicated treatment by flow cytometry . Note that total LGR5 + frequency was not significantly different between anti ‐ LRP6 antibody ‐ injected control IgG ‐ injected mice , though LGR5hi fraction was reduced in LRP6 antibody ‐ injected group .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O

M-R Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.v. injected with LRP6 neutralization antibody 12 h before 12 Gy γ‐irradiation. IgG serves as a negative control. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (M-P) Representative FACS plots of indicated treatments. (Q) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (R) Quantification of the number of LGR5+, LGR5hi, and LGR5lo cells of mice with indicated treatment by flow cytometry. Note that the total LGR5+cell frequency was not significantly different between anti‐LRP6 antibody‐injected and control IgG‐injected mice, though the LGR5hi fraction was reduced in the LRP6 antibody‐injected group.


A - C GFPimmunofluorescence microscopy of small intestine from 2 ‐ month ‐ old LGR5 ‐ GFPki , APC + / + mice LGR5 ‐ GFPki , APCmin / + mice . ( A ) of GFP of at indicated positions in basal crypts ( n = 3 mice per group , 50 positively stained were measured per mouse ) . Note GFP was increased in APCmin / + mice , particularly in position 4 5 . ( B , C ) Representative pictures are given . Dashed lines outline crypts . Arrowheads numbers indicate positions in crypts . Scale bar : 20 μm .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C GFPimmunofluorescence microscopy staining of small intestine from 2‐month‐old LGR5‐GFPki, APC+/+ mice and LGR5‐GFPki, APCmin/+mice. (A) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, 50 positively stained cells were measured per mouse). Note the GFP staining intensity was increased in APCmin/+ mice, particularly in position 4 and 5 cells. (B, C) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm.


D - F Two ‐ month ‐ old APC + / + mice APCmin / + mice were exposed to 6 Gy γ ‐ irradiation . Small intestinal tissue was collected at 24 h after IR ( n = 3 mice per group ) . ( D ) of PCNA + ISPCs at indicated positions in basal crypts 24 h after IR . IR , irradiated . ( E , F ) Representative pictures of PCNA are given . Arrowheads numbers indicate positions in crypts . Scale bar : 20 μm .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

D-F Two‐month‐old APC+/+ mice and APCmin/+ mice were exposed to 6 Gy γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3 mice per group). (D) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR. IR, irradiated. (E, F) Representative pictures of PCNA staining are given. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm.


G - K Two ‐ month ‐ old LGR5 ‐ GFPki , APC + / + mice LGR5 ‐ GFPki , APCmin / + mice were exposed to 6 Gy γ ‐ irradiation . Small intestinal tissue was collected at 24 h after IR ( n = 4 mice per group ) . ( G ) Flow cytometry of number of LGR5 + ISPCs of irradiated mice compared to number in non ‐ irradiated mice ( NIR ) set to 100 % . ( H - K ) Representative FACS plots of indicated groups are given .

O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-K Two‐month‐old LGR5‐GFPki, APC+/+ mice and LGR5‐GFPki, APCmin/+ mice were exposed to 6 Gy γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (G) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (H-K) Representative FACS plots of indicated groups are given.


B Dose response curves for viability of parental ( in red ) or selected clones ( in black ) upon treatment with NFR for 48h . green dashed box highlight in physiologically relevant range . Curves are mean ± s . e . m . of 3 independent experiments performed in triplicate . P values were calculated using 2way ANOVA of variance between parental each clone . Bar graph represents EC50 ( half maximal effective ) of dose responses . Data are mean ± s . e . m . of 3 independent experiments performed in triplicate . P values were calculated using 1way ANOVA of variance ( P value in red ) followed by Dunnett ' s multiple comparison . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Dose response curves for viability of parental cells (in red) or selected clones (in black) upon treatment with NFR for 48h. The green dashed box highlight concentration in the physiologically relevant range. Curves are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone. Bar graph represents the EC50 (half maximal effective concentration) of the dose responses. Data are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 1way ANOVA analysis of variance (P value in red) followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01.


C parental population NFR resistant clones were analyzed for eEF2K mRNA expression by real - time PCR relative to β - actin ( mean s . e . m . from 3 independent batches of are shown ) . P values were calculated using 1way ANOVA of variance followed by Dunnett ' s multiple comparison . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real-time PCR relative to β-actin (mean and s.e.m. from 3 independent batches of cells are shown). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.


D parental population representative NFR resistant clones were analyzed by WB for eFF2K total level . Tubulin is used as loading control . Histogram represents mean s . e . m of relative eEF2K level quantified from WB of 4 different batches of collected at different dates ( see also source data ) . P values were calculated using 1way ANOVA of variance followed by Dunnett ' s multiple comparison . * * * P value ≤ 0 . 001 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level. Tubulin is used as loading control. Histogram represents mean and s.e.m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates (see also source data). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *** P value ≤ 0.001.


A Immunoblot of eEF2 Thr56 phosphorylation ( P - eEF2 ) in HeLa treated for 6h with increasing doses of NFR , compared with 10 μg / ml tunicamycin ( TM ) , 200 nM rapamycin ( Rapa . ) or 1h starvation ( Starv . ) . Tubulin is used as loading control .

O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Immunoblot analysis of eEF2 Thr56 phosphorylation (P-eEF2) in HeLa cells treated for 6h with increasing doses of NFR, and compared with 10 μg/ml tunicamycin (TM), 200 nM rapamycin (Rapa.) or 1h starvation (Starv.). Tubulin is used as loading control.


B eEF2K WT KO MEF treated for 6h with indicated of NFR , 200 nM rapamycin ( Rapa . ) , 1h starvation ( Starv . ) or 1 mM of AMPK activator AICAR , were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 ( Thr56 ) .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, 200 nM rapamycin (Rapa.), 1h starvation (Starv.) or 1 mM of the AMPK activator AICAR, were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 (Thr56).


C Three representative NFR - resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level eEF2 phosphorylation upon NFR compared to parental HeLa . For treatments , medium was replaced for 6h with medium containing DMSO ( Mock ) , increasing doses of NFR or 10 μg / ml tunicamycin ( TM ) , or for 1h with PBS for starvation ( Starv . ) . Tubulin is used as loading control .

O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Three representative NFR-resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells. For treatments, medium was replaced for 6h with medium containing DMSO (Mock), increasing doses of NFR or 10 μg/ml tunicamycin (TM), or for 1h with PBS for starvation (Starv.). Tubulin is used as loading control.


D Immunoblot of eEF2 Thr56 phosphorylation in HeLa treated for 6h with increasing doses of different HIV - PIs as indicated or NFR metabolite M8 ( hydroxy - tert - butylamide ) . Molecular structures of different HIV - PIs used are indicated . Each panel is representative of at least 3 independent experiments .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6h with increasing doses of different HIV-PIs as indicated or the NFR metabolite M8 (hydroxy-tert-butylamide). Molecular structures of the different HIV-PIs used are indicated. Each panel is representative of at least 3 independent experiments.


B eEF2K WT KO MEF treated for 6h with indicated of NFR , Rapamycin ( Rapa ) , AICAR or starved for indicated time in PBS ( Starv . ) , were analyzed by immunoblot using indicated antibodies . Tubulin is used as loading control .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for indicated time in PBS (Starv.), were analyzed by immunoblot using indicated antibodies. Tubulin is used as loading control.


C A potent mTOR inhibition does not impair NFR - mediated eEF2 phosphorylation . WT MEFs were treated with indicated concentrations of Rapamycin or with vehicle ( Mock ) . After 30 minutes , indicated doses of NFR were added were incubated for additional 6h analyzed for phosphorylated S6R eEF2 . Tubulin is used as loading control .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

C A potent mTOR inhibition does not impair NFR-mediated eEF2 phosphorylation. WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle (Mock). After 30 minutes, indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2. Tubulin is used as loading control.


D NFR - mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO . AMPKα1α2 WT dKO MEF treated for 6h with indicated of NFR , Rapamycin ( Rapa ) , AICAR or starved for 1h in PBS ( Starv . ) , were analyzed by immuoblot with indicated antibodies . * anti - total AMPKα antibody give an unspecific band with a slightly higher molecular in dKO . Tubulin is used as loading control . Each panel is representative of at least 3 independent experiments .

O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D NFR-mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO. AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for 1h in PBS (Starv.), were analyzed by immuoblot with indicated antibodies. * anti-total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells. Tubulin is used as loading control. Each panel is representative of at least 3 independent experiments.