E ) Suppression effect of GmLMM1 on flg22 - induced interaction of GmFLS2 with GmBAK1b ( E ) . Data information : experiments were performed two times E ) , as biological replicates , with similar results .

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E) Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1b (E). Data information: The experiments were performed two times E), as biological replicates, with similar results.


Single - marker association results for combined non - Jewish Jewish samples using Cochran - Mantel - Haenszel ( CMH ) . Each chromosome is depicted as a different color . red indicates threshold ( P 5 × 10 − 5 ) selected to define regions for evaluation in replication studies . P value thresholds for suggestive significant associations are 3 . 28 × 10 − 6 1 . 64 × 10 − 7 , respectively , on a Bonferroni correction for multiple testing . A modest correction factor ( λ = 1 . 056 ) is necessary to correct for stratification between cases controls , but it does not modify current results .

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Single-marker association results for the combined non-Jewish and Jewish samples using the Cochran-Mantel-Haenszel (CMH) test. Each chromosome is depicted as a different color. The red line indicates the threshold (P 5 × 10−5) selected to define regions for evaluation in the replication studies. The P value thresholds for suggestive and significant associations are 3.28 × 10−6 and 1.64 × 10−7, respectively, based on a Bonferroni correction for multiple testing. A modest correction factor (λ = 1.056) is necessary to correct for stratification between cases and controls, but it does not modify the current results.


( a - c ) We used quantitative real - time PCR to determine expression patterns of ATG5 ( a ) , ATG7 ( b ) ATG16L1 ( c ) in various human immune epithelial lines . ATG5 was broadly expressed in lines studied ( a ) , with between 1 . 5 - fourfold variations in expression level across lines . ATG7 expression ( b ) was broadly comparable to that of ATG5 , with exception of THP1 , which showed 35 - fold higher expression than SW480 . ATG16L1 expression ( c ) was also broadly similar to that of ATG5 ATG7 ( except for ATG7 expression in THP1 ) , with moderate variations across lines tested ( 1 . 5 - to 3 . 5 - fold ) .

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(a-c) We used quantitative real-time PCR to determine the expression patterns of ATG5 (a), ATG7 (b) and ATG16L1 (c) in various human immune and epithelial cell lines. ATG5 was broadly expressed in the cell lines studied (a), with between 1.5- and fourfold variations in expression level across the cell lines. ATG7 expression (b) was broadly comparable to that of ATG5, with the exception of THP1 cells, which showed 35-fold higher expression than SW480 cells. ATG16L1 expression (c) was also broadly similar to that of ATG5 and ATG7 (except for ATG7 expression in THP1 cells), with moderate variations across the cell lines tested (1.5- to 3.5-fold).


( d ) Expression of ATG16L1 in human immune . In a resting human immune panel ( Clontech ) , ATG16L1 showed a clear peak of expression in T compartment , with CD4 + CD8 + expressing high of transcript ( 15 - to 20 - fold more than control samples ) . We detected modest of ATG16L1 in CD19 + low in CD14 + monocytes unfractionated mononuclear . In cases , we performed real - time quantitative RT - PCR reactions in duplicate plotted means ; error bars represent 1 s . d . We normalized expression by comparison with GAPDH controls plotted arbitrary relative expression units , where SW480 or placental expression equals 1 ( for lines or , respectively ) . We isolated RNAs from resting in absence of stimulation or activation .

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(d) Expression of ATG16L1 in human primary immune cells. In a resting human immune cell RNA panel (Clontech), ATG16L1 showed a clear peak of expression in the T cell compartment, with CD4+ and CD8+ cells expressing high levels of transcript (15- to 20-fold more than control samples). We detected modest levels of ATG16L1 in CD19+ cells and low levels in CD14+ monocytes and unfractionated mononuclear cells. In all cases, we performed real-time quantitative RT-PCR reactions in duplicate and plotted the means; error bars represent 1 s.d. We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units, where the SW480 or placental RNA expression equals 1 (for cell lines or primary cells, respectively). We isolated all RNAs from resting cells in the absence of stimulation or activation.


( a ) We achieved specific siRNA knockdown of overexpressed , Flag - tagged ATG16L1 in HEK293 within 48 h of transfection with oligonucleotide duplexes . Flag - ATG16L1 was undetectable by blotting after treatment with specific siRNA constructs , but expression was maintained with control duplexes .

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(a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes. Flag-ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs, but expression was maintained with control duplexes.


( b ) Endogenous ATG16L1 mRNA knockdown ( mean relative abundance ± s . d . ) by siRNA 2 in HeLa within 48 h of transfection , as assessed by real - time quantitative RT - PCR normalized to GAPDH . Compared with control duplex , siRNA 2 yielded an 89 % reduction in transcripts . RT - PCR was performed in triplicate ; results represent two independent experiments .

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(b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance ± s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments.


c ) Knockdown of ATG16L1 prevented effective autophagy of S . typhimurium in HeLa . Forty - eight hours after cotransfection with control siRNA or duplex 2 LC3 - GFP plasmid , HeLa were infected for 1 h with Salmonella typhimurium SL1344 , fixed examined microscopically . Mean percentages ( ± s . e . m . ) of per encapsulated by LC3 + membranes ( autophagosomes ) are shown . were pooled from two separate experiments ; each counted a minimum of 100 infected . Significance was assessed by two - Students t - , assuming unequal variances .

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c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (± s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances.


( d ) Representative of intracellular S . typhimurium show normal autophagic encapsulation by LC3 + membranes in control siRNA - treated but loss of autophagic targeting after ATG16L1 knockdown . Channels are separated to show both LC3 - GFP fusion ( autophagosomal marker ) S . typhimurium SL1344 dsRed2 ( SL1344 ) , with a merged image in final column . Rows 2 4 show magnified views of boxed areas in rows 1 3 , respectively . were obtained using confocal microscopy are a flat projection of z stacks taken throughout plane of infected . Scale bars = 5 μm .

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(d) Representative images of intracellular S. typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells but loss of autophagic targeting after ATG16L1 knockdown. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. typhimurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3, respectively. Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell. Scale bars = 5 μm.


( B ) Volcano plots showing differentially expressed genes between untreated DLL4 & PDGFBB - treated mMuSCs , human myoblasts hiMPs . Red dots represent genes which display a positive fold - change in expression upon treatment with DLL4 & PDGF - BB whilst violet dots represent genes which are significantly downregulated . Differentially expressed genes required a P value of ≤ 0 . 05 to be considered significant .

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(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.


( C ) Heatmaps showing changes in expression of key myogenic ( MYOD , MYOGENIN ) , perivascular ( PDGFRB , NG2 , CD146 , ALPL ) NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate similarity of clusters as well as orders in which clusters were assembled .

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(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.


( B ) Phase contrast displaying of untreated treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric of treated untreated hiMPs . was quantified as circularity , where 1 = perfect circle 0 = ( experimental replicates = 3 ) . Statistical ( paired t ) was performed on first quartile to enhance detection of morphological changes ( error bars : SD ) .

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(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm. (C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).


( E ) Immunofluorescence of untreated treated hiMPs incubated with EdU for 2 hours . Scale bar : 75 μm . ( F ) Bar graphs quantifying EdU experiment shown in ( E ) ( experimental replicates = 3 ; error bars : ) . Statistical on an unpaired t - .

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(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.


( H ) Immunofluorescence of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in presence or absence of γ - secretase inhibitor L685458 immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying average of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : ) . Statistical significance on one - way ANOVA with Tukey ' s multiple comparison .

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(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.


( J ) Immunofluorescence panels showing human specific LAMIN A / C + ( nuclei ) SPECTRIN ( sarcolemma ) in tibialis anterior of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 ) untreated ( n = 3 ) N5 hiMPs . ( K ) of LAMIN A / C + grafted human across each muscle . Data information : full gene list for heatmaps in ( A ) ( D ) available in Appendix Table S4 .

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(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs. (K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.


( F ) Representative of CMFDA - stained untreated DLL4 & PDGF - BB - treated hiMPs within top perfusion channel , 15 minutes after delivery kept on OrganoFlow ® . ( G ) Bar graph quantifying adhesion in ( E ) . Statistical significance was calculated on a paired t - ( experimental replicates = 3 ) . Each point on plot represents number of adhered after 15 minutes within a single chip .

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(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.


( H ) Assessment of DLL4 & PDGF - BB - treated WT genetically corrected DMD hiMP migration through a layer of endothelial . Representative showing lower side of trans - well on which treated untreated hiMPs ( stained with transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying average number of CFDA - positive / mm2 , that have migrated through endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : ) . Statistical significance on one - way ANOVA with Bonferroni ' s multiple comparison .

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(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.


Sanger - sequencing chromatograms showing target region of sgAkt1E17K in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgAkt1E17K . EditR ( Kluesner et al . , 2018 ) was used to calculate frequency ( % ) of C - to - T conversion at C7 of protospacer targeted by sgAkt1E17K in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgAkt1E17K. EditR (Kluesner et al., 2018) was used to calculate the frequency (%) of C-to-T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WB1P - BE3 injected with Lenti - sgAkt1E17K - Myc ( n = 12 ) showed a reduced mammary - specific survival compared to WB1P - BE3 mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( 58 days after injection vs 72 days after injection , P < 0 . 01 by Mantel - Cox ) .

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Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc (n=12) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (58 days after injection vs 72 days after injection, P < 0.01 by Mantel-Cox test).


Sanger - sequencing chromatograms showing target region of sgAkt1E17K in 3 independent tumors from WB1P - BE3 injected with Lenti - sgAkt1E17K - Myc . Arrowheads highlight cytosines of protospacer that show base editing . EditR was used to calculate average frequency ( % ) of C - to - T conversion at C7 of protospacer in tumors from WB1P - BE3 injected with Lenti - sgNT - Myc or Lenti - sgAkt1E17K - Myc .

Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at C7 of the protospacer in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgAkt1E17K-Myc.


Sanger - sequencing chromatograms showing target regions of sgPik3caE542K , sgPik3caE545K sgPik3caE453K in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacers that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgPik3caE542K , Lenti - sgPik3caE545K Lenti - sgPik3caE453K .

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Sanger-sequencing chromatograms showing the target regions of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPik3caE542K, Lenti-sgPik3caE545K and Lenti-sgPik3caE453K.


EditR was used to calculate frequency ( % ) of C - to - T conversion at indicated target cytosines of protospacers in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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EditR was used to calculate the frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WB1P - BE3 injected with Lenti - sgPik3caE542K - Myc ( n = 10 ) , Lenti - sgPik3caE545K - Myc ( n = 10 ) Lenti - sgPik3caE453K - Myc ( n = 10 ) showed a reduced mammary - specific survival compared to WB1P - BE3 mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( respectively 47 , 44 49 days after injection vs 72 days after injection , P < 0 . 0001 by Mantel - Cox ) .

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Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPik3caE542K-Myc (n=10), Lenti-sgPik3caE545K-Myc (n=10) and Lenti-sgPik3caE453K-Myc (n=10) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (respectively 47, 44 and 49 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).


Sanger - sequencing chromatograms showing target region of sgPik3caE542K , sgPik3caE545K sgPik3caE453K in 3 independent tumors from WB1P - BE3 injected with corresponding Lenti - sgPik3ca - Myc vectors . Arrowheads highlight cytosines of protospacer that show base editing . EditR was used to calculate average frequency ( % ) of C - to - T conversion at indicated target cytosines of protospacers in tumors from WB1P - BE3 injected with Lenti - sgNT - Myc or Lenti - sgPik3caE542K - Myc , Lenti - sgPik3caE545K - Myc Lenti - sgPik3caE453K - Myc .

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Sanger-sequencing chromatograms showing the target region of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P-BE3 females injected with the corresponding Lenti-sgPik3ca-Myc vectors. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgPik3caE542K-Myc, Lenti-sgPik3caE545K-Myc and Lenti-sgPik3caE453K-Myc.


Sanger - sequencing chromatograms showing target region of sgPtenQ245 * in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgPtenQ245 * . EditR was used to calculate frequency ( % ) of C - to - T conversion at C4 of protospacer targeted by sgPtenQ245 * in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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Sanger-sequencing chromatograms showing the target region of sgPtenQ245* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPtenQ245*. EditR was used to calculate the frequency (%) of C-to-T conversion at C4 of the protospacer targeted by sgPtenQ245* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WB1P - BE3 injected with Lenti - sgPtenQ245 * - Myc ( n = 11 ) showed a reduced mammary - specific survival compared to WB1P - BE3 mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( 37 days after injection vs 72 days after injection , P < 0 . 0001 by Mantel - Cox ) .

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

Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPtenQ245*-Myc (n=11) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (37 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).


BE Analyzer ( Hwang et al . , 2018 ) was used to assess from next - generation sequencing data fraction of wild - type Pten alleles , base edited alleles or alleles with insertions / deletions ( indels ) in tumors from WB1P - BE3 injected with Lenti - sgNT - Myc or Lenti - sgPtenQ245 * - Myc . TIDE showing spectrum of indels of targeted Pten alleles in two independent representative tumors from WB1P - BE3 mice injected with Lenti - sgPtenQ245 * - Myc . For two tumors shown in ( F ) , Sanger - sequencing chromatograms showing target region of sgPtenQ245 * ( PCR products were subcloned for clarity ) . Arrowheads highlight cytosines of protospacer that show base editing . In lower example gene was inactivated by indels at both alleles , while in upper one by Q245 * base editing in one allele a deletion at second copy of gene .

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

BE Analyzer (Hwang et al., 2018) was used to assess from next-generation sequencing data the fraction of wild-type Pten alleles, base edited alleles or alleles with insertions/deletions (indels) in tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc or Lenti-sgPtenQ245*-Myc. TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P-BE3 mice injected with Lenti-sgPtenQ245*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgPtenQ245* (PCR products were subcloned for clarity). Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by indels at both alleles, while in the upper one by Q245* base editing in one allele and a deletion at the second copy of the gene.


Sanger - sequencing chromatograms showing target region of sgTrp53Q97 * in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgTrp53Q97 EditR was used to calculate frequency ( % ) of C - to - T conversion at C8 of protospacer targeted by sgTrp53Q97 * in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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

Sanger-sequencing chromatograms showing the target region of sgTrp53Q97* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgTrp53Q97 EditR was used to calculate the frequency (%) of C-to-T conversion at C8 of the protospacer targeted by sgTrp53Q97* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + injected with Lenti - sgPik3caE545K / sgTrp53Q97 * - Myc ( n = 6 ) showed a reduced mammary - specific survival compared to injected with Lenti - sgTrp53Q97 * - Myc ( n = 5 ) vectors ( 76 days after injection vs 101 days after injection , P < 0 . 05 by Mantel - Cox ) . injected with Lenti - sgNT - Myc ( n = 11 ) did not develop any palpable tumors during 150 days observation period .

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

Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ females injected with Lenti-sgPik3caE545K/sgTrp53Q97*-Myc (n=6) showed a reduced mammary tumor-specific survival compared to animals injected with Lenti-sgTrp53Q97*-Myc (n=5) vectors (76 days after injection vs 101 days after injection, P < 0.05 by Mantel-Cox test). Females injected with Lenti-sgNT-Myc (n=11) did not develop any palpable tumors during the 150 days observation period.


BE Analyzer was used to assess from next - generation sequencing data fraction of wild - type Trp53 alleles , base edited alleles or alleles with indels in tumors from WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + injected with Lenti - sgTrp53Q97 * - Myc . Tumors from WB1P - BE3 injected with Lenti - sgNT - Myc mice were used as control . TIDE showing spectrum of indels of targeted Trp53 alleles in two independent representative tumors from WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + mice injected with Lenti - sgTrp53Q97 * - Myc . For two tumors shown in ( F ) , Sanger - sequencing chromatograms showing target region of sgTrp53Q97 * . Arrowheads highlight cytosines of protospacer that show base editing . In lower example gene was inactivated by a deletion , while in upper one by Q97 * base editing . Of note , allele with indel also displays base editing at C6 of protospacer .

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

BE Analyzer was used to assess from next-generation sequencing data the fraction of wild-type Trp53 alleles, base edited alleles or alleles with indels in tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ animals injected with Lenti-sgTrp53Q97*-Myc. Tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc mice were used as control. TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ mice injected with Lenti-sgTrp53Q97*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgTrp53Q97*. Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by a deletion, while in the upper one by Q97* base editing. Of note, the allele with the indel also displays base editing at C6 of the protospacer.


U2OS modified to expressed endogenous GFP - tagged ATG2A ( green ) were grown in complete media ( CM ) or starved ( EBSS ) for 2 h before fixation immunostaining with antibodies against ATG2B ( red ) LC3B ( magenta ) analysed by confocal microscopy . Arrows mark ATG2A / LC3B / ATG2B positive structures . Scale bar 10µm . ( B - D ) were treated as in ( A ) stained with anti - WIPI2 ( red ) or ( C ) anti - ATG16L1 ( red ) or ( D ) GABARAP - L1 ( red ) . Arrows mark structures of interest . Scale bar 10µm .

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

U2OS cells modified to expressed endogenous GFP-tagged ATG2A (green) were grown in complete media (CM) or starved (EBSS) for 2 h before fixation and immunostaining with antibodies against ATG2B (red) and LC3B (magenta) and analysed by confocal microscopy. Arrows mark ATG2A/LC3B/ATG2B positive structures. Scale bar 10µm. (B-D) Cells were treated as in (A) and stained with anti-WIPI2 (red) or (C) anti-ATG16L1 (red) or (D) GABARAP-L1 (red). Arrows mark structures of interest. Scale bar 10µm.


U2OS WT or U2OS GFP - ATG2A knock - in ( KI ) were grown in CM or starvation media for 2 h , lysed incubated with anti - GFP nanobody beads coupled to agarose to immunoprecipitate ( IP ) GFP - ATG2A . IP samples 2 % input lysates were run on 4 - 12 % gradient gel processed for western blotting . Anti - ATG2A , anti - WIPI4 anti - LC3B , anti - GABARAP ( pan ) were used to probe for presence / absence of autophagy in immunoprecipitated samples . p - p70S6K ( T389 ) was used as a marker for starved total p70S6K as loading control .

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 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 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 O O

U2OS WT or U2OS GFP-ATG2A knock-in (KI) cells were grown in CM or starvation media for 2 h, lysed and incubated with anti-GFP nanobody beads coupled to agarose to immunoprecipitate (IP) GFP-ATG2A. IP samples and 2% input lysates were run on 4-12% gradient gel and processed for western blotting. Anti-ATG2A, anti-WIPI4 and anti-LC3B, anti-GABARAP (pan) were used to probe for the presence/absence of autophagy proteins in the immunoprecipitated samples. p-p70S6K (T389) was used as a marker for starved cells and total p70S6K as loading control.


Myc - tagged ATG2A - wild type ( WT ) ATG2A - mLIR ( orange ) were co - expressed with GFP - alone , GFP - LC3B or GFP - GABARAP in HEK293T , lysed anti - GFP nanobodies coupled to agarose were used to immunoprecipitate GFP - tagged . Samples were subjected to western blotting probed for presence of Myc - ATG2A in immunoprecipitated samples . Anti - p62 / SQSTM1 was used as an internal control for immunoprecipitated samples . As in ( B ) but using Myc - tagged ATG2B - WT or ATG2B - mLIR ( orange ) co - expressed with GFP - alone , GFP - LC3B or GFP - GABARAP . Samples were subjected to western blotting probed for presence of Myc - ATG2B in immunoprecipitated samples . Anti - p62 / SQSTM1 was used as an internal control for immunoprecipitated samples .

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

Myc-tagged ATG2A-wild type (WT) and ATG2A-mLIR (orange) were co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP in HEK293T cells, lysed and anti-GFP nanobodies coupled to agarose were used to immunoprecipitate GFP-tagged proteins. Samples were subjected to western blotting and probed for the presence of Myc-ATG2A in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   As in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR (orange) co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP. Samples were subjected to western blotting and probed for the presence of Myc-ATG2B in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.


HA - tagged ATG2A - WT , - mLIR ( orange ) - mYFS ( YFS / AAA ; purple ) were stably expressed in U2OS ATG2A / B double knock - out using retrovirus transduction . were transfected with GFP - GABARAP 24 h later grown in complete medium ( CM ) or starved for 2 h ( EBSS ) . were lysed anti - HA beads were used to immunoprecipitate HA - tagged ATG2A processed for western blot . Blots were then probed with antibodies against HA - tag ( ATG2A ) , anti - WIPI4 , anti - GFP for presence / absence in immunoprecipitated samples . blots are representative of at least n = 3 independent experiments . of WIPI4 GFP - GABARAP - ii co - precipitation with HA - ATG2A from ( D ) . bands were normalized against HA - ATG2A immunoprecipitate expressed as a fold change compared to HA - ATG2A - WT ( complete media ; CM ) . Each symbol represents an independent experiment ( n = 3 ) error bars mean ± SD .

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

HA-tagged ATG2A-WT, -mLIR (orange) and -mYFS (YFS/AAA; purple) were stably expressed in U2OS ATG2A/B double knock-out cells using retrovirus transduction. Cells were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and anti-HA beads were used to immunoprecipitate HA-tagged ATG2A and processed for western blot. Blots were then probed with antibodies against HA-tag (ATG2A), anti-WIPI4, and anti-GFP for the presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of WIPI4 and GFP-GABARAP-ii co-precipitation with HA-ATG2A from (D). bands were normalized against HA-ATG2A immunoprecipitate and expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) and error bars mean ± SD.


ATG2A / B do8uble knock - out ( DKO ) stably expressing HA - tagged ATG2A - WT , - mLIR ( orange ) or - mYFS ( YFS / AAA ; purple ) were transfected with GFP - GABARAP 24 h later grown in complete medium ( CM ) or starved for 2 h ( EBSS ) . were lysed GFP - GABARAP was immunoprecipitated using anti - GFP trap beads . Samples were processed for western blot probed with anti - HA - tag ( ATG2A ) , anti - WIPI4 , anti - GFP for their presence / absence in immunoprecipitated samples . blots are representative of at least n = 3 independent experiments . of HA - ATG2A WIPI4 co - precipitation with GFP - GABARAP from ( F ) . Results were expressed as a fold change compared to HA - ATG2A - WT ( complete media ; CM ) . Each symbol represents an independent experiment ( n = 3 ) with error bars mean ± SD .

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 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 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 I-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 O O O O O O O O O

ATG2A/B do8uble knock-out cells (DKO) stably expressing HA-tagged ATG2A-WT, -mLIR (orange) or -mYFS (YFS/AAA; purple) were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and GFP-GABARAP was immunoprecipitated using anti-GFP trap beads. Samples were processed for western blot and probed with anti-HA-tag (ATG2A), anti-WIPI4, and anti-GFP for their presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of HA-ATG2A and WIPI4 co-precipitation with GFP-GABARAP from (F). Results were expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) with error bars mean ± SD.


GFP alone , GFP - GABARAP or GFP - GABARAP with increasing concentrations of mCherry - WIPI4 were expressed in HEK293T for 24 h , lysed GFP - trap beads used to immunoprecipitate GFP alone or GFP - GABARAP . Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples . Blots are representative of n = 3 independent experiments . of ATG2A ( green , round symbols ) ATG2B ( grey squares ) co - precipitation with GFP - GABARAP in presence of increasing concentrations of mCherry - WIPI4 from ( H ) . Co - precipitation was normalised to GFP - GABARAP alone . error bars are mean ± SD of n = 3 independent experiments .

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

GFP alone, GFP-GABARAP or GFP-GABARAP with increasing concentrations of mCherry-WIPI4 were expressed in HEK293T cells for 24 h, lysed and GFP-trap beads used to immunoprecipitate GFP alone or GFP-GABARAP. Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples. Blots are representative of n=3 independent experiments.   Quantification of ATG2A (green line, round symbols) and ATG2B (grey line and squares) co-precipitation with GFP-GABARAP in the presence of increasing concentrations of mCherry-WIPI4 from (H). Co-precipitation was normalised to GFP-GABARAP alone. Line and error bars are mean ±SD of n=3 independent experiments.


U2OS ATG2A / B double knock - out ( DKO ) CRISPR / Cas9 stably expressing Tandem tagged LC3B ( mCherry - GFP - LC3B ) were retrovirally transduced to express vector , or HA - tagged ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) . were grown in complete medium ( CM ) or starved for 2 h ( EBSS ) or treated with CM plus BafilomycinA1 ( 200nM , 4 h ) , fixed analysed by confocal microscopy . Merged of GFP ( green ) mCherry ( red ) channels show presence of autophagosomes / phagophores ( GFP mCherry positive , yellow ) or autolysosomes ( mCherry only , red ) Scale bar 10µm . are representative of n = 3 independent experiments .

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

U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.


using flow cytometry of measuring GFP mCherry . were gated on GFP mCherry % mCherry positive gated used as an indication of autolysosome formation due to GFP quenching . Each symbol represents 1 independent experiment with 10 , 000 analysed per condition . A total of n = 3 independent experiments were performed , horizontal bar indicates mean ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification using flow cytometry of measuring GFP and mCherry fluorescence. Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching. Each symbol represents 1 independent experiment with 10,000 cells analysed per condition. A total of n=3 independent experiments were performed, and horizontal bar indicates mean ± SD.


U2OS ATG2A / B DKO reconstituted with vector only , HA - ATG2A - WT , - mLIR - mYFS were stimulated with complete medium , ( CM ) , 2 h starvation ( EBSS ) or 4 h BafilomycinA1 ( BafA1 , 200nM ) , lysed in total lysis buffer subjected to western blot . Blots were probed for presence of HA - tag ( ATG2A ) , p62 / SQSTM1 , LC3B , pan - GABARAP ( GABARAP , GABARAP - L1 GABARAP - L2 ) vinculin ( loading control ) .

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

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).


D , E p62 / SQSTM1 ( D ) LC3B - II ( E ) were normalized to loading control quantified as fold change of DKO . Each symbol represents an independent experiment . of at least n = 3 independent experiments is shown . Horizontal bar represents mean ± SD .

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

D, E p62/SQSTM1 (D) and LC3B-II (E) levels were normalized to loading control and quantified as fold change of DKO proteins levels. Each symbol represents an independent experiment. Quantification of at least n=3 independent experiments is shown. Horizontal bar represents mean ± SD.


U2OS ATG2A / B DKO reconstituted with vector only , HA - ATG2A - WT , - mLIR - mYFS were stimulated with complete medium , ( CM ) , 4 h starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) treatment . were centrifuged resuspended in PBS digitonin , spun , washed fractions incubated with proteinase K with without 0 . 1 % TritonX - 100 . Sampels were then subjected to western blotting using anti - p62 / SQSTM1 anti - HA ( ATG2A ) antibodies . p62 / SQSTM1 remaining was calculated using densitometry . Blots are representative of n = 3 independent experiments .

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

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.


U2OS ATG2A / B DKO reconstituted with vector only , HA - ATG2A - WT , - mLIR - mYFS stably expressing GFP - Syntaxin17 ( STX17 ) were stimulated starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) for 4 hours to stimulate autophagosome generation prevent their degradation in lysosome . were fixed immune - stained for LC3B ( magenta ) . DAPI was included ( blue ) to mark / nucleus . Confocal of LC3B GFP - STX17 ( green ) localization was performed . Closed arrows ( ATG2A / B DKO DKO + ATG2A mLIR ) highlight aggregate structures . Open arrows ( DKO + ATG2A - WT ATG2A - mYFS ) highlight STX17 / LC3B positive vesicles . Scale bar 10µm . of ( C ) expressed as a of with STX17 / LC3B positive vesicles . Each symbol represents a single field of with 5 - 10 per field . A total of 300 - 600 were analysed over n = 3 independent experiments . Data is shown as mean ± SD .

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

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.


( A - C ) U2OS ATG2A / B double knock out ( DKO ) or DKO reconstituted with ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) were starved for 2 h , fixed stained for LC3B ( green ) or WIPI2 ( magenta ) , ( B ) ATG9A ( magenta ) or ( C ) GABARAP ( Magenta ) imaged using a Zeiss 880 Airyscan super - resolution confocal microscope . are representatives of n = 3 independent experiments . Scale bars 10µm .

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(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.


Transmission electron of ATG2A / B DKO ( upper left ) , DKO + ATG2A - WT ( upper right ) , DKO + ATG2A - mLIR ( lower left ) DKO + ATG2A - mYFS ( lower right ) starved ( EBSS ) for 2 h . Clustered small vesicles ( open arrowheads ; DKO DKO - mLIR ) autophagosomes ( closed arrow heads , DKO + ATG2A - WT - mYFS ) indicated . are representative of n = 3 independent experiments . Scale bars 500nm . ER = endoplasmic reticulum ; N = Nucleus ; Mt = Mitochondria ; G = Golgi ; AP = Autophagosome .

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Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.


D Zygotic grk2i283 mutant at 4dpf compared to wild type ( N = 80 ) . Arrows indicate position of swim bladder . Scale bar ,

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D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm


A Western blot of different forms of Gli2a in MZgrk2 compared to wild type , shh mRNA dnPKA mRNA injected wild type cyclopamine ( Cyc ( A ) ) treated wild type 20hpf . Gli2a - FL are low relative to Gli2aR in wild type , but are elevated in response to pathway activation ( shh dnPKA mRNA injected ) . Gli2aR are increased while Gli2a - FL is undetectable in CycA treated MZgrk2 mutant . Probing same blot with rabbit anti - GRK3 ( which recognizes zebrafish Grk2 ) reveals a complete loss of full Grk2 in MZgrk2 . Probing with anti - γ - tubulin was performed as a loading control . Three biological replicates of this were performed .

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A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed.


B Phenotype of wild type , MZgrk2 , grk2 - GFP mRNA injected MZgrk2 at 24hpf ( n = 20 for each sample ) . lines indicate shape of somites ( middle panels ) separation of eyes ( right panels ) . Scale bar ,

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B Phenotype of wild type, MZgrk2, and grk2-GFP mRNA injected MZgrk2 embryos at 24hpf (n=20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200μm


C In situ hybridisation of ptch2 , nkx2 . 2a olig2 transcripts in wild type , MZgrk2 grk2 - GFP injected MZgrk2 24hpf . Each panel shows a full view of on left a cross sectional view of a somite on right . ( n = 30 for each sample ) . Scale bars , 200μm ( whole mounts ) , 50μm ( sections ) .

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C In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild type, MZgrk2 and grk2-GFP injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right. (n=30 for each sample). Scale bars, 200μm (whole mounts), 50μm (sections).


D Expression of Prox1a Engrailed ( Eng ) in somites of wild - type , MZgrk2 grk2 - GFP mRNA injected MZgrk2 at 30hpf . Each panel shows Prox1a in green , Eng in red merge with DAPI in blue . ( n = 10 for each sample ) . Scale bar , 50μm .

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D Expression of Prox1a and Engrailed proteins (Eng) in somites of wild-type, MZgrk2 and grk2-GFP mRNA injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merge images with DAPI staining in blue. (n=10 for each sample). Scale bar, 50μm.


A Prox1a Eng expression in myotome of 30hpf wild type MZgrk2 injected with mRNAs encoding Shh , dnPKA or mSmoA1 - GFP . Each panel shows Prox1a in green , Eng in red , merged with DAPI in blue . co - labelling for Prox1a Eng ( orange ) is indicative of MP differentiation ( n = 30 for each sample ) Scale bar , 50μm .

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A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh, dnPKA or mSmoA1-GFP. Each panel shows Prox1a in green, Eng in red, and the merged images with DAPI staining in blue. The co-labelling for Prox1a and Eng (orange) is indicative of MP differentiation (n=30 for each sample) Scale bar, 50μm.


B Western blot showing of Gli2a - FL Gli2a - R forms in 20hpf wild type MZgrk2 injected with mRNA encoding Shh , dnPKA or mSmoA1 - GFP , respectively . γ - tubulin was used as loading control . Three biological replicates of this were performed .

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B Western blot showing levels of Gli2a-FL and Gli2a-R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh, dnPKA or mSmoA1-GFP, respectively. γ-tubulin was used as loading control. Three biological replicates of this analysis were performed.


A Western blot of GRK2 , GLI1 , PTCH1 , GLI3 in Flp - In - 3T3 with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG . α - tubulin was used as loading control . Guide 2 was found to remove Grk2 efficiently used for subsequent experiments . GLI3 panel shows both full - ( GLI3FL ) repressor form of GLI3 ( GLI3R ) . ( n = 3 )

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A Western blot analysis of GRK2, GLI1, PTCH1, and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG. α-tubulin was used as loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R). (n=3)


B , C Hh reporter activity for wild type Grk2 - / - . were treated with PKA peptide inhibitor ( PKI ) mutant form of PKA peptide inhibitor ( PKI - M ) respectively , in presence or absence of SHH ( B ) were transfected with mSmoA1 - GFP expressing constructs in presence of SHH or SAG ( C ) . Data represent mean ± SD ( n = 3 ) . Unpaired Students t - was used for . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 n . s . ( not significant ) .

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B, C Hh reporter activity assay for wild type and Grk2-/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M) respectively, in presence or absence of SHH (B) Cells were transfected with mSmoA1-GFP expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


A Expression of Prox1a ( green ) eng2a : GFP reporter ( red ) in otherwise wild - type 30hpf injected with mRNA encoding wild type mutant forms of mouse Smo . Ectopic MPs are indicated by fibres co - labelled with Prox1a GFP ; ectopic MFFs are labeled only with GFP . are representative of in following proportions of each sample : 28 / 28 ( mSmo ; mSmoA1 ; mSmoSD ; mSmoKRA ) ; 9 / 28 ( mSmoA1SA ) ; 12 / 28 ( mSmoA1SD ) . Scale bar , 50μm .

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A Expression of Prox1a (green) and the eng2a:GFP reporter (red) in otherwise wild-type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo. Ectopic MPs are indicated by fibres co-labelled with Prox1a and GFP; ectopic MFFs are labeled only with GFP. Images are representative of embryos in the following proportions of each sample: 28/28 (mSmo; mSmoA1; mSmoSD; mSmoKRA); 9/28 (mSmoA1SA); 12/28 (mSmoA1SD). Scale bar, 50μm.


B Average number of Prox1a + ve slow fibres in wild type injected with mRNA encoding different forms of mSmo . Prox1 positive were quantified in 4 somites in each of 4 for each sample . error bars indicate SD . Unpaired Student ' s t - was used to determine statistical significance between uninjected various Smo mutants ( black asterisk ) ; between mSmoA1 mSmoA1SA or mSmoA1SD ( red asterisk ) . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 n . s . ( not significant ) .

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B Average number of Prox1a+ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo. Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample. The error bars indicate SD. Unpaired Student's t-test was used to determine the statistical significance between uninjected embryos and the various Smo mutants (black asterisk); and between mSmoA1 and mSmoA1SA or mSmoA1SD (red asterisk). ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


C Prox1a Eng expression in smohi1640 mutant injected with mRNA encoding mSmo ( n = 10 ) , mSmoSA ( n = 9 ) mSmo14SA ( n = 6 ) . Note full recovery of SSFs MPs compared to un - injected controls . Scale bar , 50μm .

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C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo (n=10), mSmoSA (n=9) and mSmo14SA (n=6). Note the full recovery of SSFs and MPs compared to the un-injected controls. Scale bar, 50μm.


D In situ hybridisation for transcripts of ptch2 , olig2 nkx2 . 2 in 24hpf smohi1640 mutant injected with mRNA encoding mSmo mSmoSA ( n = 6 for each sample ) . Scale bars , are 100μm ( lateral view ) , 50μm ( sections ) .

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D In situ hybridisation for transcripts of ptch2, olig2 and nkx2.2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA (n=6 for each sample). Scale bars, are 100μm (lateral view), 50μm (sections).


E Hh reporter of activity of wild type mutant forms of mSmo in Smo - / - MEFs in response to Shh or SAG stimulation . Note that mSmoA1 shows constitutive activity in absence of either Shh or SAG whereas mSmoKRA dose not ; mutants affecting phosphorylation failed to restore response to Shh or SAG . Data represent mean ± SD ( n = 3 ) . Unpaired Students t - was used for . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; n . s . ( not significant ) .

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E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo-/- MEFs in response to Shh or SAG stimulation. Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not; all mutants affecting phosphorylation failed to restore the response to Shh or SAG. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; and n.s. (not significant).


A Wild type 18hpf injected with mRNA encoding GFP - tagged mSmo ( green ) showing localisation to PC of myotomal labeled with anti - Acetylated tubulin ( AcTub ; red ) , stimulated in response to Shh injection ( n = 4 ) . Differences in PC distribution are due to morphological changes in myotome induced by ectopic expression of Shh as revealed by distribution of nuclei ( DAPI stained ; blue ) . Scale bar , 10μm .

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A Wild type 18hpf embryos injected with mRNA encoding GFP-tagged mSmo (green) showing localisation to the PC of myotomal cells labeled with anti-Acetylated tubulin (AcTub; red), stimulated in response to Shh injection (n = 4). Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei (DAPI stained; blue). Scale bar, 10μm.


B Notochord of wild type expressing GFP tagged wild - type mutant forms of mSmo . Note localisation to PC ( labeled with anti - AcTub ; red ) in each case ( n = 4 for each sample ) . Scale bar , 10μm .

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B Notochord cells of wild type embryos expressing GFP tagged wild-type and mutant forms of mSmo. Note the localisation to the PC (labeled with anti-AcTub; red) in each case (n=4 for each sample). Scale bar, 10μm.


C MZgrk2 18hpf injected with mRNA encoding GFP - tagged wild type mSmo or mSmoSA ( green ) showing localisation to PC ( labeled with anti - AcTub ; red ) in myotomal . More Smo is localised to PC in MZgrk2 mutants compared to wild type ( panel A ) . Scale bar , 10μm .

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C MZgrk2 18hpf embryos injected with mRNA encoding GFP-tagged wild type mSmo or mSmoSA (green) showing localisation to the PC (labeled with anti- AcTub; red) in myotomal cells. More Smo is localised to the PC in MZgrk2 mutants compared to wild type (panel A). Scale bar, 10μm.


B Prox1a ( green ) Eng ( red ) expression in myotome of 30hpf MZgrk2 that were injected with mRNA encoding : Grk2K220R - GFP ; Grk2K220M - GFP ; Grk2D335N - GFP ; Grk2ΔN7aa - GFP mGrk2L4AV7AL8A - GFP . Merged panels show with DAPI in blue . ( n = 20 for each sample ) . Scale bar , 50μm .

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B Prox1a (green) and Eng (red) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding: Grk2K220R-GFP; Grk2K220M-GFP; Grk2D335N-GFP; Grk2ΔN7aa-GFP and mGrk2L4AV7AL8A-GFP. Merged panels show nuclear staining with DAPI in blue. (n=20 for each sample). Scale bar, 50μm.


C Hh reporter activity in wild type Grk2 - / - 3T3 transfected with equivalent wild type mutant kinase domain forms of mouse Grk2 , Grk2K220R , Grk2K220M Grk2D335N after 24h Shh - N SAG treatment . Data represent mean ± SD ( n = 3 ) . Unpaired Students t - was used for . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; * P < 0 . 05 ; n . s . ( not significant ) .

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

C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).


Transgenic ESZscan4 _ Emerald were sorted total RNAs were extracted subjected to RT - qPCR . graph shows mRNA expression level of Arg2 in ES subpopulations .

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Transgenic ESZscan4_Emerald cells were sorted and total RNAs were extracted and subjected to RT-qPCR. The graph shows the mRNA expression level of Arg2 in ES subpopulations.


( B ) Transgenic ESZscan4 _ Emerald were sorted total were extracted subjected to immunoblotting . image is a representative of three independent biological western blotting on arg2 expression in sorted ESZscan4 _ Emerald control .

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(B)Transgenic ESZscan4_Emerald cells were sorted and total proteins were extracted and subjected to immunoblotting. The image is a representative of three independent biological western blotting analysis on arg2 expression in sorted ESZscan4_Emerald and control cells.


Transcript ( left panel ) expression ( right panel ) of Arg2 in ESZscan4 _ LNGFR after transfection with a non - silencing siRNA ( siCtrl ) or siRNA against Arg2 ( siArg2 ) .

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Transcript (left panel) and protein expression (right panel) levels of Arg2 in ESZscan4_LNGFR cells after transfection with a non-silencing siRNA (siCtrl) or siRNA against Arg2 (siArg2).


Imaging flow cytometry on Zscan4 + siArg2 Zscan4 + siCtrl . Samples were stained by labelling histone H3 . of H3 signal was measured in region by creating a mask defined by DAPI . texture feature of signal was analysed by using modulation method ( for details see Materials Methods ) . Two populations ( R1 R2 ) correspond to two different patterns of signal : homogenous clustered distribution , respectively .

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Imaging flow cytometry analysis on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Samples were stained by labelling histone H3 protein. The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining. The texture feature of fluorescent signal was analysed by using the modulation method (for details see Materials and Methods). Two cell populations (R1 and R2) correspond to two different patterns of fluorescent signal: homogenous and clustered distribution, respectively.


representative SeaHorse time courses of OCR ECAR respectively ; where are indicated following compounds added : oligomycin ( olig . ) , carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) , rotenone plus antimycin A ( R / A ) , glucose ( Glu ) , 2 - deoxyglucose ( 2DG ) . Each point in OCR ECAR time - courses is average ± S . D . of four technical replicates .

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

representative SeaHorse time courses of OCR and ECAR respectively; where are indicated the following compounds added: oligomycin (olig.), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone plus antimycin A (R/A), glucose (Glu), 2-deoxyglucose (2DG). Each point in the OCR and ECAR time-courses is the average ± S.D. of four technical replicates.


OCR / ECAR ; ( 4 technical replicates each ) ; Basal , ( resting OCR ) / ( ECAR + Glu ) ; Max , ( OCR + FCCP ) / ( ECAR + Olig . )

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

OCR/ECAR ratio; (4 technical replicates each); Basal, (resting OCR)/(ECAR +Glu); Max, (OCR+FCCP)/(ECAR+Olig.)


Cellular NAD+ and NADH content and NAD+/NADH.


Confocal microscopy of mitochondrial potential DYm . Pictures on right , representative imaging of loaded with tetramethylrhodamine , ethyl ester ( TMRE ) ; histogram on left , averaged / ± SD of n = 3 independent biological replicates ; * , P < 0 . 05 .

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Confocal microscopy analysis of the mitochondrial membrane potential DYm. Pictures on the right, representative imaging of cells loaded with tetramethylrhodamine, ethyl ester (TMRE); histogram on the left, averaged fluorescence intensity/cell ± SD of n=3 independent biological replicates; *, P < 0.05.


Representative confocal microscopy of loaded with MitoTracker Green ( pictures on left ) ; digital enlargements false colors rendering of details shown as contoured squares ( pictures on right ) .

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Representative confocal microscopy analysis of cells loaded with MitoTracker Green (pictures on the left); digital enlargements and false colors rendering of details shown as white contoured squares (pictures on the right).


Electron microscopy of sorted ESZscan4 _ Emerald control . Scale bar

Electron microscopy images of sorted ESZscan4_Emerald and control cells. Scale bar 1μm


Representative confocal microscopy of loaded with dichlorofluorescein - diacetate converted in dichlorofluorescein ( DCF ) ( pictures on left ) ; digital enlargements false - color rendering of details shown as contoured squares ( pictures on right ) ; histogram on left , averaged / ± of n = 3 independent biological replicates .

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

Representative confocal microscopy analysis of cells loaded with dichlorofluorescein-diacetate converted in dichlorofluorescein (DCF) (pictures on the left); digital enlargements and false-color rendering of details shown as white contoured squares (pictures on the right); histogram on the left, averaged fluorescence intensity/cell ± SEM of n=3 independent biological replicates.


Basal R / A - sensitive OCR measured as in Fig . 2A in absence in presence of a cocktail of inhibitors ( BPTES + etomoxir + UK5099 ) ; End . Sub . , OCR measured with endogenous substrates ; Exo . Sub . , OCR measured with glutamine ( Gln ) , fatty ( FA ) glucose ( Glc ) supplemented in medium . superimposed bars are means ± of n = 3 independent biological replicates ( 3 technical replicates each ) ;

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

Basal R/A-sensitive OCR measured as in Fig. 2A in the absence and in the presence of a cocktail of inhibitors (BPTES + etomoxir + UK5099); End. Sub., OCR measured with endogenous substrates; Exo. Sub., OCR measured with glutamine (Gln), fatty acids (FA) and glucose (Glc) supplemented in the assay medium. The superimposed bars are means ± SEM of n=3 independent biological replicates (3 technical replicates each);


Measurement of dependency flexibility of OCR to either of Gln , FA , Glc ; was carried out according to MitoFuel protocol ( see Material Methods ) ; superimposed bars ( whose sum corresponds to capacity ) are means ± of n = 3 independent biological replicates ( 3 technical replicates each ) ;

Measurement of dependency and flexibility of the OCR to either of Gln, FA, Glc; the assay was carried out according to the MitoFuel protocol (see Material and Methods); the superimposed bars (whose sum corresponds to the capacity) are means ± SEM of n=3 independent biological replicates (3 technical replicates each);


LC - MSMS of content of free aminoacids ; symbols of aminoacids are indicated in x - axis ; amount expressed as μmol / μg is indicated in y - axis . inset shows content of ornithine , citrulline arginine .

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LC-MSMS analysis of the cellular content of free aminoacids; the symbols of aminoacids are indicated in x-axis; the amount expressed as μmol/μg protein is indicated in y-axis. The inset shows the content of ornithine, citrulline and arginine.


( D ) LC - MSMS of content of acylcarnitines ; of acyl moiety is indicated by number of carbon atoms in abscissa ; bars values are means ± of n = 3 independent biological replicates ; inset shows content of carnitine ( C0 ) acetylcarnitine ( C2 ) .

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(D)LC-MSMS analysis of the cellular content of acylcarnitines; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa; bars values are means ± SEM of n=3 independent biological replicates; The inset shows the content of carnitine (C0) and acetylcarnitine (C2).


B , Multiplexed activation of Ascl1 , Lmx1a , NeuroD1 of Ascl1 , Lmx1a , Nr4a2 in astrocytic cultures . n = 3 , representative experiment from 2 - 3 independent experiments , additional data in supplement . Activation are depicted as fold change between transfected with without sgRNAs . were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

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

B, Multiplexed activation of Ascl1, Lmx1a, NeuroD1 and of Ascl1, Lmx1a, Nr4a2 in primary astrocytic cultures. n=3, representative experiment from 2-3 independent experiments, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


A , Representative photomicrographs taken from dorsal striatum 13 after AAV - injection . In mice injected with GFP control , virtually GFP positive depict an astrocytic , many GFP positive in ALNe - 218 ALN - treated show a neuron - like . Data information : Scale bars indicate 20 µm .

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A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In mice injected with GFP control virus, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.


B , Immunohistochemical showing GFP + / GFAP + double positive 13 wpi . Arrows indicate double positive GFP + / GFAP + , arrowheads indicate GFP + / GFAP - . of GFAP + / GFP + shows a significant decrease upon ALNe - 218 ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0064 , GFP vs . ALN P = 0 . 0002 ALN vs . ALNe - 218 P = 0 . 0127 ) . Multiple comparison ANOVA F ( 2 , 7 ) = 32 . 06 . C , Double immunostaining for GFP neuronal marker NeuN . Arrowheads indicate double positive GFP + / NeuN + 13 wpi . demonstrate a significant increase in NeuN + / GFP + upon ALNe - 218 ALN - induction ( GFP vs . ALNe - 218 P < 0 . 0001 , GFP vs . ALN P < 0 . 0001 ALN vs . ALNe - 218 P = 0 . 0012 ) . Multiple comparison ANOVA F ( 2 , 6 ) = 170 . 3 . Data information : Scale bars indicate 20 µm . Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . n = 3 - 4 mice per condition . Error bars represent mean ± SD .

O O 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 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 O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0064, GFP vs. ALN P=0.0002 and ALN vs. ALNe-218 P=0.0127). Multiple comparison ANOVA F(2,7)=32.06. C, Double immunostaining for GFP and the neuronal marker NeuN. Arrowheads indicate double positive GFP+/NeuN+ cell 13 wpi. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALNe-218 P<0.0001, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0012). Multiple comparison ANOVA F(2,6)=170.3. Data information: Scale bars indicate 20 µm. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 3-4 mice per condition. Error bars represent mean ± SD.


D , Double immunostaining for GFP TH as a marker for dopaminergic neurons in dorsal striatum of GFP - control , ALNe - 218 ALN - treated . Arrowheads indicate GFP + / TH - with neuronal . No GFP + / TH + could be detected in any of experimental groups . Scale bars indicate 20 µm . Data information : Scale bars indicate 20 µm .

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D, Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP-control, ALNe-218 and ALN-treated animals. Arrowheads indicate GFP+/TH- cells with neuronal morphology. No GFP+/TH+ cell could be detected in any of the experimental groups. Scale bars indicate 20 µm. Data information: Scale bars indicate 20 µm.


B , RT - qPCR of Ascl1 induction comparing activation capacity of full - versus split - dCas9 in Neuro2A ( data in fold change normalized to non - activated control ; dCas9 6116 ± 847 . 3 , split - dCas9 4415 ± 748 . 8 , n = 3 biological replicates , box - - whiskers plots indicate median , 25th - 75th percentile min / max whiskers ) .

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B, RT-qPCR analysis of Ascl1 induction comparing the activation capacity of full-length versus split-dCas9 in Neuro2A cells (data in fold change normalized to non-activated control; dCas9 6116 ± 847.3, split-dCas9 4415 ± 748.8, n=3 biological replicates, box-and-whiskers plots indicate median, 25th-75th percentile and min/max whiskers).


C , Immunocytochemistry of reprogrammed astrocytes 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa . Astrocytes are infected with two lentiviruses expressing dCas9 - VPR in a intein - split version similar to AAV - dCAS system but driven by a Tet - O promoter . Tet - O driven dsRed Ascl1 cDNA expressing construct were used as negative positive controls respectively . Arrows indicate single MAP2 positive background neurons , arrowheads indicate double positive induced neurons . Scale bar indicates 20 µm . Data information : Abbreviations : OE -

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

C, Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa. Astrocytes are infected with two lentiviruses expressing dCas9-VPR in a intein-split version similar to the AAV-dCAS system but driven by a Tet-O promoter. Tet-O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively. Arrows indicate single MAP2 positive background neurons, arrowheads indicate double positive induced neurons. Scale bar indicates 20 µm. Data information: Abbreviations: OE - overexpression


E , Multiplexed activation of Ascl1 / Lmx1a / NeuroD1 , Ascl1 / Lmx1a / Nr4a2 Ascl1 / Lmx1a / Nr4a2 / Pitx3 / FoxA2 in astrocytic . n = 2 - 3 biological replicates , one representative run is shown , additional data in supplement . Activation are depicted as fold change between transfected with without sgRNAs . were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

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

E, Multiplexed activation of Ascl1/Lmx1a/NeuroD1, Ascl1/Lmx1a/Nr4a2 and Ascl1/ Lmx1a/ Nr4a2/Pitx3/FoxA2 in primary astrocytic cells. n=2-3 biological replicates, one representative run is shown, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


A , Representative photomicrographs taken from dorsal striatum 13 after AAV - injection . In GFP control condition , virtually GFP positive depict an astrocytic , many GFP positive in ALNe - 218 ALN - treated show a neuron - like . Data information : Scale bars indicate 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

A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In the GFP control condition, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.


B , Immunohistochemical showing GFP + / GFAP + double positive 13 wpi . Arrows indicate double positive GFP + / GFAP + , arrowheads indicate GFP + / GFAP - . of GFAP + / GFP + shows a significant decrease upon ALNe - 218 ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0083 , GFP vs . ALN P < 0 . 0001 ALN vs . ALNe - 218 P = 0 . 0006 , multiple comparison ANOVA F ( 2 , 6 ) = 79 . 76 ) . C , Photomicrographs showing GFP + / NeuN + neurons 13 wpi . Arrowheads indicating GFP + / NeuN + induced neurons . demonstrate a significant increase in NeuN + / GFP + upon ALNe - 218 ALN - induction ( GFP vs . ALN P = 0 . 0008 , ALN vs . ALNe - 218 P = 0 . 0092 , multiple comparison ANOVA F ( 2 , 7 ) = 21 . 74 ) . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 3 - 4 mice ( b , c ) per condition . Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O 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 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 O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0083, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0006, multiple comparison ANOVA F(2,6)=79.76). C, Photomicrographs showing GFP+/NeuN+ neurons 13 wpi. Arrowheads indicating GFP+/NeuN+ induced neurons. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALN P=0.0008, ALN vs. ALNe-218 P=0.0092, multiple comparison ANOVA F(2,7)=21.74). Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n = 3-4 mice (b,c) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


D , In ALN - induced neurons , confocal is demonstrating a co - localization of GFP glutamic acid decarboxylase ( Gad65 / 67 ) , a marker specific for GABAergic neurons . E , of GAD6567 + / GFP + with neuronal shows that majority of ALN - induced neurons are colocalizing with this GABAergic marker both in AAV - dCAS as well as in dCAM setting . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 6 - 8 mice ( e ) per condition . Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, In ALN-induced neurons, confocal analysis is demonstrating a co-localization of GFP and glutamic acid decarboxylase (Gad65/67), a marker specific for GABAergic neurons. E, Quantification of GAD6567+/GFP+ cells with neuronal morphology shows that the majority of ALN-induced neurons are colocalizing with this GABAergic marker both in the AAV-dCAS as well as in the dCAM setting. Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n= 6-8 mice (e) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


A , Gait using CatWalk XT system reveals motor defects in 6 - OHDA lesion model . Both dCAM AAV - dCAS transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like average speed of tread . Naive vs . 6 - OHDA P = 0 . 0063 . AAV - dCAS : GFP vs . ALN P = 0 . 015 multiple comparison ANOVA F ( 2 , 17 ) = 12 . 81 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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A, Gait analysis using the CatWalk XT system reveals motor defects in 6-OHDA lesion model. Both dCAM and AAV-dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread. Naive vs. 6-OHDA P=0.0063. AAV-dCAS: GFP vs. ALN P=0.015 multiple comparison ANOVA F(2,17)=12.81) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


stride of front paws : B , Naive vs . 6 - OHDA P = 0 . 0164 . AAV - dCAS : GFP vs . ALN P = 0 . 005 , ALN vs . ALNe - 218 P = 0 . 0042 , multiple comparison ANOVA F ( 4 , 22 ) = 9 . 9 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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the stride length of front paws: B, Naive vs. 6-OHDA P=0.0164. AAV-dCAS: GFP vs. ALN P=0.005, ALN vs. ALNe-218 P=0.0042, multiple comparison ANOVA F(4,22)=9.9) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


duty cycle of left front paws . C , Naive vs . 6 - OHDA P = 0 . 0096 . dCAM : GFP vs . ALN P = 0 . 036 ALN vs . ALNe - 218 P = 0 . 0252 , multiple comparison ANOVA F ( 2 , 20 ) = 5 . 199 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

the duty cycle of left front paws. C, Naive vs. 6-OHDA P=0.0096. dCAM: GFP vs. ALN P=0.036 and ALN vs. ALNe-218 P=0.0252, multiple comparison ANOVA F(2,20)=5.199). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


D , In contrast to this , dopamine dependent drug - induced behavior does not show rescue effects : amphetamine - induced rotation : change in rotational behavior in lesioned upon treatment with dopamine releaser substance . Net rotation = rotation - rotation ( naïve vs . 6 - OHDA P = 0 . 09 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . CatWalk error bars represent mean ± SD . Rotation error bars represent mean ± .

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D, In contrast to this, dopamine dependent drug-induced behavior does not show rescue effects: amphetamine-induced rotation analysis: change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance. Net rotation = ipsilateral rotation-contralateral rotation (naïve vs. 6-OHDA P=0.09). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. CatWalk error bars represent mean ± SD. Rotation analysis error bars represent mean ± SEM.


( A ) Fractionation of compartments using a standard NP - 40 lysis protocol immunoblotting with chaperone specific antibodies

(A) Fractionation of nuclear and cytosolic compartments using a standard NP-40 lysis protocol and immunoblotting with chaperone specific antibodies


( B ) Immunofluorescence of histone chaperones in fixed using same antibodies used in ( A ) . are segregated into those undergoing replication those that are not . Scale bar represents 5 μm

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(B) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in (A). Cells are segregated into those undergoing replication and those that are not. Scale bar represents 5 μm