A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , lipid from chow HFD - fed mice . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value chow / HFD : Hb : 1 . 3 / 0 . 6 ; HbO2 : 1 . 8 / 0 . 9 ; Lipid : 27000 / 27000 ) . Scale bar : 4 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value chow/HFD: Hb: 1.3/0.6; HbO2: 1.8/0.9; Lipid: 27000/27000). Scale bar: 4 mm.


D , Tissue oxygenation ( sO2 ) total blood volume ( TBV ) results from liver , kidney , iBAT , rpWAT , Each dot represents data from one , in total 8 ( n = 8 ) . Data represent mean ( + / - 95 % confidence ) . unpaired t was used to verify statistical significance . Data information : In figure , A . U . = arbitrary units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Tissue oxygenation (sO2) and total blood volume (TBV) results from liver, kidney, iBAT, and rpWAT, Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units.


A , Oil - red - O of control ( healthy ) steatotic liver . Scale bar : 100 µm .

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

A, Oil-red-O staining of control (healthy) and steatotic liver. Scale bar: 100 µm.


B , Reconstructed MSOT image ( 800 nm ) with linear unmixing difference value of lipid in lower abdominal section . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid , jet for 700 nm - 930 nm difference . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : Hb : 1 . 1 / 0 . 5 ; HbO2 : 1 . 9 / 0 . 8 ; Lipid : 21000 / 24000 ; 700 - 900nm : 3000 / 3000 ) Scale bar : 4 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of lipid in lower abdominal section. Unmixing result: blue for Hb, red for HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5; HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.


A , HE of livers with grade 0 - 3 steatosis . Scale bar : 100 µm .

A, HE staining of livers with grade 0 - 3 steatosis. Scale bar: 100 µm.


E , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , lipid from chow HFD - fed mice . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value chow 1 month / chow 2 month / chow 3 month / HFD 1 month / HFD 2 month / HFD 3 month : Hb : 2 . 5 / 1 . 9 / 2 . 9 / 2 . 7 / 1 . 8 / 1 . 3 ; HbO2 : 2 . 8 / 2 . 6 / 2 . 8 / 3 . 1 / 2 . 1 / 1 . 9 ; Lipid : 27000 / 19000 / 29000 / 32000 / 26000 / 27000 ) . Scale bar : 4 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm.


F , Longitudinal track of lipid in mouse livers . Each represents data from one . Each data point is from a single measurement of each at each time point . have grade 3 steatosis at endpoint confirmed by histology .

O O O O O O O O O O O O O O O O O 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, Longitudinal track of lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each time point. All animals have grade 3 steatosis at the endpoint confirmed by histology.


A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of ICG from mice with without steatosis . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : 0 . 07 / 0 . 02 ) Scale bar : 4 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Reconstructed MSOT image (800 nm) with linear unmixing data of ICG from mice with and without steatosis. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: 0.07/0.02) Scale bar: 4 mm.


E , Representative microscopy HE of control steatotic liver . Scale bar : 100 µm .

E, Representative fluorescence microscopy and HE staining images of control and steatotic liver. Scale bar: 100 µm.


( A ) Ookinetes sandwiched between two hydrogel surfaces .

(A) Ookinetes sandwiched between two hydrogel surfaces.


( B ) of ookinetes moving for more than one within time of observation ( 5 - 10 min ) if sandwiched between soft , medium ( med ) or stiff hydrogel or glass . Bars represent mean error bars standard deviation of at least three independent experiments . ( C ) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of ookinetes moving for more than one parasite length within the time of observation (5-10 min) if sandwiched between soft, medium (med) or stiff hydrogel or glass. Bars represent the mean and error bars the standard deviation of at least three independent experiments. (C) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B.


( D ) of motile ookinetes after 20 - 26 hours of incubation between hydrogels compared to directly imaged after setting up experiment as shown in ( B ) . Shown is average ± S . D . from at least two independent experiments . ( E ) Speed of motile ookinetes after 20 - 26 hours of incubation between hydrogels . Speed on glass was not analyzed ( n . a . ) due to low fraction of motile ookinetes at this timepoint .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of motile ookinetes after 20-26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in (B). Shown is the average ± S.D. from at least two independent experiments. (E) Speed of motile ookinetes after 20-26 hours of incubation between hydrogels. Speed on glass was not analyzed (n.a.) due to the low fraction of motile ookinetes at this timepoint.


( F ) Migration patterns of ookinetes . Image : Overlay of tracked migration paths ( black ) DIC image showing ookinetes at end of recorded image . Graph : Quantitative of indicated different migration patterns on soft , medium ( med ) stiff hydrogels .

O O O O O O O O O O O O O O O O O O O O O O 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) Migration patterns of ookinetes. Image: Overlay of tracked migration paths (black) and DIC image showing ookinetes at the end of the recorded image sequence. Graph: Quantitative analysis of the indicated different migration patterns on soft, medium (med) and stiff hydrogels.


( G ) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints .

(G) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints.


( A ) Infected salivary were sandwiched between a soft PA hydrogel a glass coverslip inducing sporozoites to move into hydrogel .

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

(A) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel.


( B ) Overlay of sporozoite tracks ( black ) DIC image showing sporozoites inside a soft hydrogel at end of a recorded image . Scale bar , 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

(B) Overlay of sporozoite tracks (black) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence. Scale bar, 20 µm.


( C ) Speed of WT sporozoites moving in skin in vivo ( Douglas et al , 2018b ) in soft hydrogels with small or large pores .

O O O O 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) Speed of WT sporozoites moving in the skin in vivo (Douglas et al, 2018b) and in soft hydrogels with small or large pores.


( D , E ) Speed of indicated mutant control ( Ctrl ) in hydrogels with small ( D ) large ( E ) pores .

O O 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) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores.


( F , G ) Speed of drug - treated control in hydrogels with small ( F ) large ( G ) pores . Data information : Note faster speed in gels with larger pores . Data from at least two independent experiments . Numbers above bars indicate number of sporozoites analyzed . Significance determined by Mann - Whitney . Box - - whisker plots ( C - G ) depict 25 % quantile , median , 75 % quantile nearest observations within 1 . 5 times interquartile range ( whiskers ) . Outliers beyond this range are shown as black dots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Speed of drug-treated and control parasites in hydrogels with small (F) and large (G) pores. Data information: Note the faster speed in gels with larger pores. Data from at least two independent experiments. Numbers above bars indicate the number of sporozoites analyzed. Significance determined by Mann-Whitney test. Box-and-whisker plots (C-G) depict the 25% quantile, median, 75% quantile and nearest observations within 1.5 times the interquartile range (whiskers). Outliers beyond this range are shown as black dots.


( A ) of motile sporozoites on endothelial ( LSECs ) versus glass . Shown is average ± S . D . from three independent experiments . Significance determined using an unpaired 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

(A) Percentage of motile sporozoites on endothelial cells (LSECs) versus glass. Shown is average ± S.D. from three independent experiments. Significance determined using an unpaired t-test.


( B ) Left : Merged showing sporozoites on cultured HFF . Actin filaments of HFF were stained with phalloidin ( shown in red ) , nuclei were stained with Hoechst ( blue ) sporozoites expressed GFP ( green ) . Scale bar , 50 µm . Right : Maximum projection of sporozoites moving for 3 min on cultured HFF . Scale bar , 50 µm .

(B) Left: Merged fluorescence images showing sporozoites on cultured HFF cells. Actin filaments of HFF cells were stained with phalloidin (shown in red), nuclei were stained with Hoechst (blue) and sporozoites expressed GFP (green). Scale bar, 50 µm. Right: Maximum projection of sporozoites moving for 3 min on cultured HFF cells. Scale bar, 50 µm.


( C ) of motile sporozoites on confluent layers of endothelial ( HUVEC ) , fibroblasts ( HFF ) on glass . Shown is average ± S . D . from two independent experiments . No significant differences as determined by One - way of variance with Bonferroni ' s Multiple Comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of motile sporozoites on confluent layers of endothelial cells (HUVEC), fibroblasts (HFF) and on glass. Shown is average ± S.D. from two independent experiments. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


( D ) Sporozoite on planar uncoated hydrogels .

(D) Sporozoite on planar uncoated hydrogels.


( E ) Overlay of sporozoite tracks ( black ) DIC image showing sporozoites on a hydrogel . Scale bar , 50 µm . ( F ) of motile sporozoites on soft , medium ( med ) or stiff hydrogel or glass 30 min after activation with BSA . Shown is average ± S . D . from at least three independent experiments . Significance determined by One - way of variance with Bonferroni ' s Multiple Comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overlay of sporozoite tracks (black) and DIC image showing sporozoites on a hydrogel. Scale bar, 50 µm.(F) Percentage of motile sporozoites on soft, medium (med) or stiff hydrogel or glass 30 min after activation with BSA. Shown is the average ± S.D. from at least three independent experiments. Significance determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


( G ) Migration persistance as determined by number of circles performed within 100 s on hydrogels of different stiffnesses glass 30 min after activation with BSA . sporozoites moving for more than one within time of observation were analyzed , even if they stopped moving or detached during imaging . Significance determined by Kruskal - Wallis with Dunn ' s Multiple Comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA. All sporozoites moving for more than one parasite length within the time of observation were analyzed, even if they stopped moving or detached during imaging. Significance determined by Kruskal-Wallis test with Dunn's Multiple Comparison test.


( H ) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses . Note that not sporozoites analyzed in ( G ) complied with that requirement . To be able to analyze a high number of sporozoites , movies were taken 10 to 45 min after activation with BSA . Speed of sporozoites on soft hydrogels was not analyzed ( n . a . ) due to low fraction of motile sporozoites on these hydrogels . No significant differences as determined by One - way of variance with Bonferroni ' s Multiple Comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses. Note that not all sporozoites analyzed in (G) complied with that requirement. To be able to analyze a high number of sporozoites, movies were taken 10 to 45 min after activation with BSA. Speed of sporozoites on soft hydrogels was not analyzed (n.a.) due to the low fraction of motile sporozoites on these hydrogels. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


C , Immunostaining of endogenous Trnp1 DAPI in P19 E14 cortical at 1 day in vitro ( div ) .

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

C, Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro (div).


D , Co - IP with anti - GFP WB with anti - Trnp1 antibodies in lysates from P19 transfected with plasmids expressing Trnp1 - GFP fusion / or Trnp1 - IRES - RFP for 24h . Mw ( molecular weights ) : 55 kDa ( black ) ; 35 kDa ( blue ) ; 25 kDa ( red ) . Input : 0 , 1 % ; co - IP : immunoprecipitated .

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 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

D, Co-IP with anti-GFP and WB with anti-Trnp1 antibodies in lysates from P19 cells transfected with plasmids expressing Trnp1-GFP fusion protein and/or Trnp1-IRES-RFP for 24h. Mw (molecular weights): 55 kDa (black line); 35 kDa (blue line); 25 kDa (red line). Input: 0,1%; co-IP: all immunoprecipitated proteins.


E , Representative phase contrast of in vitro phase separation of recombinant YFP ( upper row ) or Trnp1 ( lower row ) at indicated in 50 mM Sorenson ' s buffer ( pH 7 , 6 ) containing 150 mM salt 2mM DTT with crowding agent dextran or when depicted .

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

E, Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP (upper row) or Trnp1 (lower row) at the indicated concentration in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted.


B , GFP Trnp1 immunostaining with DAPI labelling in E14 cortical transfected for 24 h with plasmids ( indicated by arrows ) expressing different truncated / mutant forms of Trnp1 GFP showing Trnp1 localization .

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-CONTROLLED_VAR O O B-MEASURED_VAR O O

B, GFP and Trnp1 immunostaining with DAPI labelling in E14 cortical cells transfected for 24 h with plasmids (indicated by arrows) expressing the different truncated/mutant forms of Trnp1 and GFP showing Trnp1 localization.


C , WB of enriched fractions of P19 transfected with different truncated / mutant forms of Trnp1 for 24h . Hist1 actin serve as loading controls for fractions , respectively . values under anti - Trnp1 blots indicate of signal relative to control set to 100 % after normalization by α - actin ( Image J ) . values under anti - actin anti - Hist1 blots indicate of signal considering that fractions together is 100 % .

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 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

C, WB of cytoplasmic and nuclear enriched fractions of P19 cells transfected with the different truncated/mutant forms of Trnp1 for 24h. Hist1 and actin serve as loading controls for nuclear and cytoplasmic fractions, respectively. The values under the anti-Trnp1 blots indicate the percentage of the signal relative to the control set to 100% after normalization by α-actin (Image J quantification). The values under the anti-actin and anti-Hist1 blots indicate the percentage of the signal considering that nuclear and cytoplasmic fractions together is 100%.


D , WB showing mutCC Trnp1 ∆ 1 - 16 Trnp1 turnover in P19 24 h after transfection in presence of cycloheximide ( CHX ) for time indicated below in hours ( h ) . GFP ( half live : 24h ) was used as a loading control .

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 O O O O O O O O O O

D, WB showing mutCC Trnp1 and ∆1-16 Trnp1 protein turnover in P19 cells 24 h after transfection in the presence of cycloheximide (CHX) for the time indicated below in hours (h). GFP (half live:24h) was used as a loading control.


E , Co - IP with anti - FLAG magnetic beads WB with anti - Trnp1 antibodies showing that FLAGTrnp1 interacts with GFP - fusion ∆ 1 - 16Trnp1 but not with other truncated Trnp1 indicating IDRs are crucial for Trnp1 homo - oligomerization . Samples were collected 24h after transfection of P19 with plasmids expressing indicated depicted in panel .

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-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

E, Co-IP with anti-FLAG magnetic beads and WB with anti-Trnp1 antibodies showing that FLAGTrnp1 interacts with the GFP-fusion and the ∆1-16Trnp1 proteins but not with other truncated Trnp1 proteins indicating IDRs are crucial for Trnp1 homo-oligomerization. Samples were collected 24h after transfection of P19 cells with plasmids expressing the indicated proteins depicted in the panel.


F , Representative phase contrast of in vitro phase separation of recombinant ∆ 1 - 16Trnp1 , ∆ 1 − 140Trnp1 ∆ 95 - 223Trnp1 at indicated concentrations in 50 mM Sorenson ' s buffer ( pH 7 , 6 ) containing 150 mM salt 2mM DTT plus Dextran or when indicated . G , Violin Dot Plots illustrating of of single droplets ( N = 700 ) formed by recombinant Trnp1 , ∆ 1 - 16Trnp1 , ∆ 1 − 140Trnp1 , ∆ 95 - 223Trnp1 at 25 μM in a representative experiment of three repetitions under conditions shown in F .

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

F, Representative phase contrast images of in vitro phase separation of the recombinant ∆1-16Trnp1, ∆1−140Trnp1 and ∆95-223Trnp1 proteins at the indicated concentrations in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT plus Dextran or RNA when indicated. G, Violin Dot Plots illustrating quantification of the area of single droplets (N=700) formed by the recombinant Trnp1, ∆1-16Trnp1, ∆1−140Trnp1, ∆95-223Trnp1 proteins at 25 μM concentration in a representative experiment of three repetitions under the conditions shown in F.


H , Histogram depicting of proliferating ( Ki67 + ) transfected for 24 h with plasmids expressing control ( GFP ) or Trnp1 - IRES - GFP its different mutant / truncated forms - IRES - GFP in dissociated E14 cortical . Biological replicates : GFP = 21 ; Trnp1 = 15 ; FLAGTrnp1 = 10 ; mutCC = 5 ; ∆ 1 - 16 = 7 ; ∆ 1 - 1 - 56 , ∆ 1 - 97 , ∆ 1 - 140 = 3 , ∆ 95 - 223 = 5 ∆ 140 - 223 = 4 .

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 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

H, Histogram depicting the percent of proliferating (Ki67+) cells transfected for 24 h with plasmids expressing control (GFP) or Trnp1-IRES-GFP and its different mutant/truncated forms-IRES-GFP in dissociated E14 cortical cells. Biological replicates: GFP=21; Trnp1=15; FLAGTrnp1=10; mutCC=5; ∆1-16=7; ∆1-1-56, ∆1-97, ∆1-140=3, ∆95-223=5 and ∆140-223=4.


A - C , showing examples of sections of E15 cerebral cortex 2 days after IUE of plasmids indicated in ( A - C ) with magnifications showing indicated immunostainings . Scale bars : 100 µm 20 µm for magnifications . Filled arrows show electroporated expressing Pax6 Ki67 . Open arrows show electroporated negative for Pax6 Ki67 . VZ : ventricular zone ; SVZ : subventricular zone ; IZ : intermediate zone ; CP : cortical plate ; V : ventricle . D , Bar dot plot showing distribution as of GFP + / bin after IUE as in A - C . A cortical column reaching from ventricular surface ( bottom in A ) to pial surface ( top in A ) was divided into equal sized bins with bin1 at ventricular surface bin 5 at pial surface .

O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Micrographs showing examples of sections of E15 cerebral cortex 2 days after IUE of the plasmids indicated in (A-C) with magnifications showing the indicated immunostainings. Scale bars: 100 µm and 20 µm for magnifications. Filled arrows show electroporated cells expressing Pax6 and Ki67 proteins. Open arrows show electroporated cells negative for Pax6 and Ki67 staining. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate; V: ventricle. D, Bar and dot plot showing the distribution as percent of GFP+ cells/bin after IUE and staining as in A-C. A cortical column reaching from the ventricular surface (bottom in A) to the pial surface (top in A) was divided into equal sized bins with bin1 at the ventricular surface and bin 5 at the pial surface.


E - G , Bar dot plots of proportion of proliferating ( Ki67 + ) ( E ) , number of NSCs ( Pax6 + ) ( F ) proportion of proliferating ( BrdU + ) TAPs ( Tbr2 + ) ( G ) as in A - C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆ 1 - 16Trnp1 ( E - F ) a significant decrease in proliferating TAPs upon Trnp1 IUE increase upon ∆ 1 - 16Trnp1 IUE compared to control ( G ) at E15 , 2 days after IUE .

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 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-CONTROLLED_VAR 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

E-G, Bar and dot plots of the proportion of proliferating (Ki67+) cells (E), the number of NSCs (Pax6+) (F) and the proportion of proliferating (BrdU+) TAPs (Tbr2+)(G) as in A-C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆1-16Trnp1 proteins (E- F) and a significant decrease in proliferating TAPs upon Trnp1 IUE and increase upon ∆1-16Trnp1 IUE compared to control (G) at E15, 2 days after IUE.


A , WB showing co - IP performed with antibodies indicated on top of lanes in lysates treated with nucleases indicated to right of panel from P19 transfected with plasmids expressing FLAGTrnp1 for 24h immunoblotted with anti - Trnp1 antibodies . As negative control no antibody was added to co - IP .

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

A, WB showing co-IP performed with the antibodies indicated on top of the lanes in nuclear lysates treated with the nucleases indicated to the right of the panel from P19 cells transfected with plasmids expressing FLAGTrnp1 for 24h immunoblotted with anti-Trnp1 antibodies. As negative control no antibody was added to the co-IP.


B - C , Volcano plot showing mean difference of iBAQ between FLAGTrnp1 ( B ) FLAG ∆ 1 - 16Trnp1 ( C ) interacting versus control plotted against p - value . used to experimentally confirm MS results are indicated with squares name in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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, Volcano plot showing the mean difference of the protein iBAQ between FLAGTrnp1 (B) and FLAG∆1-16Trnp1 (C) interacting proteins versus control plotted against the p-value. Proteins used to experimentally confirm the MS results are indicated with squares and the protein name in (B).


D , STRING of Trnp1 interactors disconnected nodules are not shown . cluster have been color coded ( green for association , red for splicing , blue for involved in nucleolus function yellow for ) .

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, STRING analysis of Trnp1 interactors The disconnected nodules are not shown. Proteins cluster have been color coded (green for chromatin association, red for RNA splicing, blue for proteins involved in nucleolus function and light yellow for the rest).


A , Superresolution confocal image of endogenous Nucleophosmin ( B23 ) , Nucleolin ( Ncl ) Trnp1 in E14 cortical at 1div . yellow indicates position of scan plot ( below confocal panels ) measuring grey value of each ( Trnp1 = grey ; Ncl = purple B23 = green ) .

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

A, Superresolution confocal image of endogenous Nucleophosmin (B23), Nucleolin (Ncl) and Trnp1 proteins in E14 cortical cells at 1div. The yellow line indicates the position of the intensity line scan plot (below confocal panels) measuring the grey value of each staining (Trnp1=grey; Ncl=purple and B23=green).


B , Co - IP experiments with lysates from P19 expressing FLAGTrnp1 for 24h . IPs WB with antibodies indicated show interaction between Trnp1 Ncl but not with Fbl1 consistent with MS data .

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 B-MEASURED_VAR O O O O O

B, Co-IP experiments with lysates from P19 cells expressing FLAGTrnp1 for 24h. IPs and WB with the antibodies indicated show interaction between Trnp1 and Ncl but not with Fbl1 consistent with the MS data.


Ultrastructural depicting in nucleolus ( arrows for nucleolar interstice ) ( C ) , condensed blocks ( arrows ) ( D ) speckles or interchromatin granule clusters ( arrows for dense cords ) Nu : nucleolus ; G : granular component ; D : dense fibrillar component ; FC : fibrillar center ; C : condensed .

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

Ultrastructural analysis depicting in the nucleolus (arrows for nucleolar interstice) (C), condensed chromatin blocks (arrows) (D) and nuclear speckles or interchromatin granule clusters (arrows for dense cords) Nu: nucleolus; G: granular component; D: dense fibrillar component; FC: fibrillar center; C: condensed chromatin.


( E ) in nucleus of P19 transfected with plasmids expressing Trnp1 - IRES - GFP or GFP in control FACS sorted fixed 24 h after transfection . Scale bars , 2 µm ( D ) 1 µm ( C E ) .

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

(E) in the nucleus of P19 cells transfected with plasmids expressing Trnp1-IRES-GFP or GFP in control cells FACS sorted and fixed 24 h after transfection. Scale bars, 2 µm (D) and 1 µm (C and E).


F , WB of lysates from P19 transfeted for 24h with plasmids expressing GFP only or Trnp1 - IRES - GFP with antibodies indicated .

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

F, WB of lysates from P19 cells transfeted for 24h with plasmids expressing GFP only or Trnp1-IRES-GFP with antibodies indicated.


A , Representative time - lapse of GFP - Fbl1 in P19 expressing plasmids indicated defining t = 0 when chromatids separate ( anaphase ) .

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, Representative time-lapse sequence of GFP-Fbl1 in P19 cells expressing the plasmids indicated defining t=0 when the chromatids separate (anaphase).


B - C , of number ( B ) total ( C ) of GFP - Fbl1 foci in P19 expressing constructs indicated for a period of 360 minutes . Band width represents grey areas represent time points with significant differences between control Trnp1 expressing conditions . ∆ 1 - 16Trnp1 expression does not show any statistical difference compared to control condition .

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

B-C, Analysis of the number (B) and the total area (C) of the GFP-Fbl1 foci in P19 cells expressing the constructs indicated for a period of 360 minutes. Band width represents SEM and grey areas represent time points with significant differences between the control and the Trnp1 expressing conditions. The ∆1-16Trnp1 expression does not show any statistical difference compared to the control condition.


D , Representative time - lapse including one mitosis of H2B - Venus in P19 expressing indicated constructs .

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

D, Representative time-lapse sequence including one mitosis of H2B-Venus in P19 cell expressing the indicated constructs.


E , Volcano Plot illustrating of M - phase from condensation to de - condensation of chromosomes in minutes . Each dot represents an from 3 different biological replicates . GFP = 61 , Trnp1 = 62 ∆ 1 - 16Trnp1 : 40 .

O O O O O O O O O O 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

E, Volcano Plot illustrating the length of M-phase from condensation to de-condensation of chromosomes in minutes. Each dot represents an individual cell from 3 different biological replicates. GFP=61 cells, Trnp1=62 cells and ∆1-16Trnp1: 40 cells.


F , Bar graph showing of expressing Trnp1 - IRES - GFP or GFP as control in each phase of mitosis indicated in different color as indicated at different times after release from synchronization in G2 . 100 per condition time were analyzed .

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

F, Bar graph showing the percentage of cells expressing Trnp1-IRES-GFP or GFP as control in each phase of the mitosis indicated in different color as indicated at different times after release from synchronization in G2. 100 cells per condition and time were analyzed.


G , Bar dot plots depicting proportion of GFP + NSCs ( Pax6 + ) in mitosis ( pHH3 + ) showing a significant decrease 24h after Trnp1 compared to control IUE . N = 4 brains from 2 - 3 different litters .

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

G, Bar and dot plots depicting the proportion of GFP+ NSCs (Pax6+) in mitosis (pHH3+) showing a significant decrease 24h after Trnp1 compared to control IUE. N= 4 brains from 2-3 different litters.


A - C , showing GFP ( green : electroporated ( A - C ) ) Ncl ( grey : nucleoli ( A ' , B ' C ' ) ) immunostaining of E15 cerebral cortex 2 days after IUE with plasmids indicated . Position of magnifications shown on right side are depicted by a square . V = ventricle ; VZ : ventricular zone . Yellow dotted shapes show examples of nucleoli in electroporated from VZ non - VZ .

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

A-C, Micrographs showing GFP (green: electroporated cells (A-C)) and Ncl (grey: nucleoli (A', B' and C')) immunostaining of E15 cerebral cortex 2 days after IUE with plasmids indicated. Position of magnifications shown on the right side are depicted by a white square. V= ventricle; VZ: ventricular zone. Yellow dotted shapes show examples of nucleoli in electroporated cells from the VZ and the non-VZ.


D - F Violin plots depicting ( D ) nucleoli number ( E ) nucleoli ( F ) from electroporated as in A - C with constructs areas indicated on x - axis . Note that in Trnp1 , but not ∆ 1 - 16Trnp1 , overexpression is sufficient to significantly increase nucleolar in non - VZ ( where most lack endogenous Trnp1 ) . N = 10 per region from 6 different control Trnp1 - IRES - GFP electroporated N = 15 per region from 3 - 4 different ∆ 1 - 16Trnp1 - IRES - GFP electroporated from at least 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 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 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

D-F Violin plots depicting the nuclear size (D) nucleoli number (E) and nucleoli size (F) from cells electroporated as in A-C with the constructs and the areas indicated on the x-axis. Note that in Trnp1, but not ∆1-16Trnp1, overexpression is sufficient to significantly increase nucleolar size in the non-VZ (where most cells lack endogenous Trnp1). N=10 cells per region from 6 different control and Trnp1-IRES-GFP electroporated embryos and N=15 per region from 3-4 different ∆1-16Trnp1-IRES-GFP electroporated embryos from at least 4 litters.


G - H , showing GFP ( green : electroporated ) , Pax6 ( magenta for NSCs ) Ncl ( grey for nucleoli ) immunostainings in sections of E15 cerebral cortex 2 days after IUE with plasmids indicated . Yellow dotted shapes show examples of nucleoli in electroporated yellow non dotted shapes show examples of nucleoli in non - electroporated from VZ non - VZ .

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 O O O O O O

G-H, Micrographs showing GFP (green: electroporated cells), Pax6 (magenta for NSCs) and Ncl (grey for nucleoli) immunostainings in sections of E15 cerebral cortex 2 days after IUE with plasmids indicated. Yellow dotted shapes show examples of nucleoli in electroporated and yellow non dotted shapes show examples of nucleoli in non-electroporated cells from the VZ and the non-VZ.


I - K Violin plots depicting ( D ) nucleoli number ( E ) nucleoli ( F ) from electroporated as in G - H with constructs areas indicated on x - axis . Note that Trnp1 knockdown significantly decreases nucleolar in VZ ( where most express endogenous Trnp1 ) . N = 10 per region from 7 per condition from at least 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 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-MEASURED_VAR O O O O O O O O O O O O O O O O O

I-K Violin plots depicting the nuclear size (D) nucleoli number (E) and nucleoli size (F) from cells electroporated as in G-H with the constructs and the areas indicated on the x-axis. Note that Trnp1 knockdown significantly decreases nucleolar size in the VZ (where most cells express endogenous Trnp1). N=10 cells per region from 7 embryos per condition from at least 4 litters.


A - C of sections of E15 cerebral cortex 2 days after IUE with plasmids magnifications indicated . V = ventricle ; VZ : ventricular zone . Scale bars : 20 µm 10 µm for magnifications . Yellow dotted shapes show examples of heterochromatin spots in nuclei of electroporated from VZ non - VZ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Micrographs of sections of E15 cerebral cortex 2 days after IUE with plasmids and magnifications indicated. V= ventricle; VZ: ventricular zone. Scale bars: 20 µm and 10 µm for magnifications. Yellow dotted shapes show examples of heterochromatin spots in nuclei of electroporated cells from the VZ and the non-VZ.


D - E , Violin plots depicting number ( D ) ( E ) of condensed spots from electroporated as in A - C . Note that Trnp1 overexpression is sufficient to significantly increase of condensed spots , while ∆ 1 - 16Trnp1 acts as a dominant negative reducing number of heterochromatin spots . N = 10 per region from 5 for GFP Trnp1 8 - 15 from 4 for ∆ 1 - 16Trnp1 of at least 4 litters . Each dot represents a .

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

D-E, Violin plots depicting the number (D) and size (E) of condensed chromatin spots from cells electroporated as in A-C. Note that Trnp1 overexpression is sufficient to significantly increase the size of condensed chromatin spots, while the ∆1-16Trnp1 acts as a dominant negative reducing the number and size of heterochromatin spots. N=10 cells per region from 5 embryos for GFP and Trnp1 and 8-15 cells from 4 embryos for ∆1-16Trnp1 of at least 4 litters. Each dot represents a cell.


G - H Translation using OPP - click reaction in P19 transfected for 24 hours with GFP , Trnp1 - IRES - GFP or ∆ 1 - 16Trnp1 - IRES - GFP plasmids analyzed by immunofluorescence ( G ) or FACs sorting ( H ) . Graph in ( G ) is representative from three different biological replicates . Each dot represents a . Control = 102 ; Trnp1 = 126 ; ∆ 1 - 16Trnp1 = 97 . Graph in ( H ) shows of 2 , 000 transfected from 1 representative experiment with 2 biological replicates for control 3 biological replicates for Trnp1 ∆ 1 - 16Trnp1 conditons . same FACs sorting experiment was repeated two more times . dotted vertical shows mean of OPP click signal for each condition .

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 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-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

G-H Translation assay using OPP-click reaction in P19 cells transfected for 24 hours with GFP, Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP plasmids and analyzed by immunofluorescence (G) or FACs sorting (H) . Graph in (G) is representative from three different biological replicates. Each dot represents a cell. Control=102; Trnp1=126; ∆1-16Trnp1=97. Graph in (H) shows analysis of 2,000 transfected cells from 1 representative experiment with 2 biological replicates for control and 3 biological replicates for Trnp1 and ∆1-16Trnp1 conditons. The same FACs sorting experiment was repeated two more times. The dotted vertical line shows the mean of the OPP click signal for each condition.


I , Bar dot plots depicting rRNA transcription for 18S , 28S , 45S 47S measured by qPCR in P19 transfected for 24h with plasmids expressing Trnp1 - IRES - GFP or ∆ 1 - 16Trnp1 - IRES - GFP compared to control expressing GFP . Each dot represents a biological replicate . N = 4 for each condition .

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

I, Bar and dot plots depicting rRNA transcription for the 18S, 28S, 45S and 47S measured by qPCR in P19 cells transfected for 24h with plasmids expressing Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP compared to control cells expressing GFP . Each dot represents a biological replicate. N=4 for each condition.


PI ( 5 ) P Regulates Autophagosome Biogenesis ( A B ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in HeLa treated with carrier alone , or in combination with PI ( 5 ) P di - C16 at indicated concentrations for 1 hr , in absence presence of 400 nM BAF ( treated in combination with lipids ) . Note that LC3 - I is often very faint compared with LC3 - II in HeLa under extraction conditions we used ( see Figure S1C ) . However , this was not a problem because one should relate LC3 - II to tubulin ( mean ± ) .

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

PI(5)P Regulates Autophagosome Biogenesis(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with carrier alone, or in combination with PI(5)P di-C16 at indicated concentrations for 1 hr, in the absence and presence of 400 nM BAF (treated in combination with lipids). Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we used (see Figure S1C). However, this was not a problem because one should relate LC3-II to tubulin (mean ± SEM).


PI ( 5 ) P Regulates Autophagosome Biogenesis ( C D ) HeLa stably expressing GFP - LC3 were treated as in ( A ) then left in complete media ( basal ) or starvation media ( HBSS ) for 2 hr , then fixed analyzed on a Cellomics ArrayScan system . of numbers of GFP - LC3 vesicles per in different conditions is shown in ( D ) ( mean ± ) .

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

PI(5)P Regulates Autophagosome Biogenesis(C and D) HeLa cells stably expressing GFP-LC3 were treated as in (A) and then left in complete media (basal) or starvation media (HBSS) for 2 hr, then fixed and analyzed on a Cellomics ArrayScan system. Quantification of numbers of GFP-LC3 vesicles per cell in the different conditions is shown in (D) (mean ± SEM).


( E ) HeLa stably expressing GFP - mRFP - LC3 were treated as in ( A ) analyzed on a Cellomics ArrayScan system . of numbers of autophagic vesicles ( AV ) or autolysosomes ( AL ) per in different conditions is shown in graph ( mean ± ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells stably expressing GFP-mRFP-LC3 were treated as in (A) and analyzed on a Cellomics ArrayScan system. Quantification of numbers of autophagic vesicles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph (mean ± SEM).


( F G ) Western blot of free ATG12 ATG5ATG12 with anti - HA antibody in HeLa transfected with HA - ATG12 ATG5 loaded with exogenous lipids . We quantified of ATG5ATG12 versus free ATG12 by direct infrared detection on an Odyssey Infrared Imaging System to assess ATG5 - ATG12 conjugation efficiency ( mean ± ) .

O 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 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

(F and G) Western blot analysis of free ATG12 and ATG5ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and loaded with exogenous lipids. We quantified the ratio of ATG5ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5-ATG12 conjugation efficiency (mean ± SEM).


( H ) HeLa transfected with GFP - PHD3X or GFP - PHD3X Znmut RFP - LC3 for 16 hr were left in complete media ( basal ) or starvation media ( HBSS ) for 1 hr , then fixed imaged on a confocal microscope . Empty arrowheads indicate PHD3X single - labeled structures , while filled arrowheads indicate PHD3X - LC3 - PHD3X - ATG16 - positive structures . Bar , 10 μm .

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

(H) HeLa cells transfected with GFP-PHD3X or GFP-PHD3X Znmut and RFP-LC3 for 16 hr were left in complete media (basal) or starvation media (HBSS) for 1 hr, then fixed and imaged on a confocal microscope. Empty arrowheads indicate PHD3X single-labeled structures, while filled arrowheads indicate PHD3X-LC3- and PHD3X-ATG16-positive structures. Bar, 10 μm.


( I ) HeLa transfected with GFP - PHD3X RFP - LC3 for 16 hr were left in HBSS for 1 hr , then fixed imaged on Elyra superresolution microscope . Final visualization was performed in Volocity 6 . 3 software using isosurface rendering of selected cropped regions of data sets . Note that this rendering means that vesicles positive for green red do not look yellow but have green red on surface . Representative cropped regions from different are shown . Bar , 1 . 13 μm . See also Figure S1 .

O O O O O O O O B-CONTROLLED_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 O 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) HeLa cells transfected with GFP-PHD3X and RFP-LC3 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed in Volocity 6.3 software using isosurface rendering of selected cropped regions of the data sets. Note that this rendering means that vesicles positive for green and red do not look yellow but have green and red on the surface. Representative cropped regions from different cells are shown. Bar, 1.13 μm. See also Figure S1.


( B C ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in HeLa treated with DMSO or increasing concentrations of YM - 201636 for 2 hr in presence or absence of BAF ( mean ± ) .

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

(B and C) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM-201636 for 2 hr in the presence or absence of BAF (mean ± SEM).


( D E ) HeLa transiently transfected with Strawberry - ATG16L1 were treated with YM - 201636 ( 100 nM , 2 hr ) in HBSS , then fixed stained for endogenous WIPI - 2 . Bar , 10 μm . ( E ) of ATG16L1 WIPI2 structures per , n = 20 ( mean ± ; n = 3 independent experiments ; ∗ ∗ ∗ p < 0 . 001 , 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O

(D and E) HeLa cells transiently transfected with Strawberry-ATG16L1 were treated with YM-201636 (100 nM, 2 hr) in HBSS, then fixed and stained for endogenous WIPI-2. Bar, 10 μm. (E) Quantification of ATG16L1 and WIPI2 structures per cell, n = 20 cells (mean ± SEM; n = 3 independent experiments; ∗∗∗p < 0.001, t test).


( F ) Western blot of free ATG12 ATG5 - ATG12 with anti - HA antibody in HeLa transfected with HA - ATG12 ATG5 treated with YM - 201636 ( 100 nM , 2 hr ) ( mean ± ) .

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

(F) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and treated with YM-201636 (100 nM, 2 hr) (mean ± SEM).


( G ) HeLa transfected with GFP - PHD3X Strawberry - ATG16L1 for 16 hr were left in HBSS for 1 hr , then fixed imaged on Elyra superresolution microscope . Final visualization was performed as previously described . Bar , 1 . 13 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

(G) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed as previously described. Bar, 1.13 μm.


( H I ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in incubated with YM - 201636 ( 100 nM , 2 hr ) loaded with exogenous PI ( 5 ) P for last 1 hr in presence or absence of BAF ( mean ± ) . See also Figure S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and I) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in cells incubated with YM-201636 (100 nM, 2 hr) and loaded with exogenous PI(5)P for the last 1 hr in the presence or absence of BAF (mean ± SEM). See also Figure S2.


( A B ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in HeLa transfected for 5 days with two rounds of control , PI5P4K2A , 2B , or 2C siRNA either left untreated or treated with BAF ( 200 nM , 16 hr ) ( mean ± ) .

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

(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM).


( C ) HeLa stably expressing GFP - mRFP - LC3 treated as in ( A ) were analyzed on a Cellomics ArrayScan system as previously described ( mean ± ) .

O O O O O O O O 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) HeLa cells stably expressing GFP-mRFP-LC3 treated as in (A) were analyzed on a Cellomics ArrayScan system as previously described (mean ± SEM).


( D ) Western blot of free ATG12 ATG5 - ATG12 with anti - HA antibody in HeLa treated with control , PI5P4K2A , 2B , 2C siRNA transfected with HA - ATG12 ATG5 for last 16 hr ( mean ± ) .

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

(D) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA and transfected with HA-ATG12 and ATG5 for the last 16 hr (mean ± SEM).


( E F ) HeLa transfected with GFP - PI5P4K2A , 2B , 2C , or catalytic - dead PI5P4K2C along with RFP - LC3 for 16 hr ( E ) or 30 hr ( F ) were fixed imaged on a confocal microscope . Bar , 10 μm . ( G ) of ( of total ) showing more than ten autophagic vesicles ( RFP - LC3 vesicles ) in different conditions from ( F ) is shown in graph ; n = 200 ( mean ± ) . See also Figure S3 .

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 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

(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or catalytic-dead PI5P4K2C along with RFP-LC3 for 16 hr (E) or 30 hr (F) were fixed and imaged on a confocal microscope. Bar, 10 μm.(G) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). See also Figure S3.


( A B ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in SKNSH MEFs treated for 2 hr with carrier alone or in combination with PI ( 5 ) P di - C16 ( 10 μM ) in absence presence of BAF ( mean ± ) .

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 O O O O O 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 and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5)P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM).


( C ) HeLa treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV - PI5P4K2C overexpression construct for 48 hr were transfected with EGFP - httQ74 for last 48 hr . of with EGFP - positive is shown in graph ; n = 500 ( mean ± ) . expressing control vector typically have 25 % of we set control at 100 % to enable comparison statistics from independent experiments .

O O O O O O B-CONTROLLED_VAR 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 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

(C) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV-PI5P4K2C overexpression construct for 48 hr were transfected with EGFP-httQ74 for the last 48 hr. Percentage of cells with EGFP-positive aggregates is shown in the graph; n = 500 cells (mean ± SEM). Cells expressing control vector typically have 25% of aggregates and we set the control at 100% to enable comparison and statistics from independent experiments.


( D E ) of with EGFP - positive was scored in HEK293 treated with PI5P4K2C siRNA for 5 days cotransfected with EGFP - httQ74 for last 48 hr ( D ) , or in COS7 SKNSH ( E ) , transfected with PI5P4K2C plasmid together with EGFP - httQ74 for last 48 hr .

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

(D and E) Percentage of cells with EGFP-positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP-httQ74 for the last 48 hr (D), or in COS7 and SKNSH cells (E), transfected with PI5P4K2C plasmid together with EGFP-httQ74 for the last 48 hr.


( F G ) WT ( Atg5 + / + ) or autophagy - deficient lacking key autophagy gene Atg5 ( Atg5 − / − ) MEFs were treated with PI5P4K2C siRNA ( 5 days ) or PI5P4K2C plasmid ( 48 hr ) together with EGFP - httQ74 for 48 hr . percentages of transfected with EGFP - httQ74 were assessed shown in graph ; n = 500 ( mean ± ) . Representative confocal are shown in ( G ) . See also Figures S3 S4 .

O O O O O O B-CONTROLLED_VAR 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 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 O O 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 and G) WT (Atg5+/+) or autophagy-deficient cells lacking the key autophagy gene Atg5 (Atg5−/−) MEFs were treated with PI5P4K2C siRNA (5 days) or PI5P4K2C plasmid (48 hr) together with EGFP-httQ74 for 48 hr. The percentages of transfected cells with EGFP-httQ74 aggregates were assessed and shown in the graph; n = 500 cells (mean ± SEM). Representative confocal images are shown in (G). See also Figures S3 and S4.


PI ( 5 ) P Triggers Autophagy in Absence of VPS34 Activation ( A B ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in HeLa pretreated with Wm ( 200 nM , 2 hr ) , then loaded with 10 μM PI ( 5 ) P for 1 hr ( in presence of Wm BAF ) ( mean ± ) .

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 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

PI(5)P Triggers Autophagy in the Absence of VPS34 Activation(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells pretreated with Wm (200 nM, 2 hr), then loaded with 10 μM PI(5)P for 1 hr (in the presence of Wm and BAF) (mean ± SEM).


( C ) of numbers of GFP - LC3 vesicles in HeLa stably expressing GFP - LC3 treated as in ( A ) then shifted to starvation media ( HBSS ) or complete media ( Basal ) for 2 hr ( in presence of Wm ) ( mean ± ) .

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

(C) Quantification of numbers of GFP-LC3 vesicles in HeLa cells stably expressing GFP-LC3 treated as in (A) and then shifted to starvation media (HBSS) or complete media (Basal) for 2 hr (in the presence of Wm) (mean ± SEM).


( D ) HeLa transfected with HA - ATG12 ATG5 treated with 200 nM Wm as in ( A ) subjected to western blot with anti - HA antibody to detect free ATG12 ATG5 - ATG12 ( mean ± ) . ATG5 - ATG12 conjugation was checked for endogenous in same conditions using an anti - ATG12 antibody ( bottom ) .

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

(D) HeLa cells transfected with HA-ATG12 and ATG5 treated with 200 nM Wm as in (A) and subjected to western blot analysis with anti-HA antibody to detect free ATG12 and the ATG5-ATG12 complex (mean ± SEM). ATG5-ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti-ATG12 antibody (bottom).


( E F ) Western blot of LC3 - II tubulin of LC3 - II / tubulin in HeLa transfected with control , PI5P4K2A , 2B , 2C siRNA treated with Wm ( 200 nM , 2 hr in presence of BAF ) ( mean ± ) .

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

(E and F) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected with control, PI5P4K2A, 2B, and 2C siRNA and treated with Wm (200 nM, 2 hr in the presence of BAF) (mean ± SEM).


( G H ) Western blot of free ATG12 ATG5 - ATG12 with anti - HA antibody in HeLa treated with control , PI5P4K2A , 2B , 2C siRNA , transfected with HA - ATG12 ATG5 for last 16 hr treated with Wm for 2 hr ( mean ± ) .

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

(G and H) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA, transfected with HA-ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr (mean ± SEM).


( I ) HeLa stably expressing GFP - LC3 treated with control , PI5P4K2A , 2B , 2C siRNA , were pretreated with Wm for 2 hr in complete medium then shifted in HBSS media for 2 hr ( in presence of Wm ) ( mean ± ) .

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

(I) HeLa cells stably expressing GFP-LC3 treated with control, PI5P4K2A, 2B, and 2C siRNA, were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr (in the presence of Wm) (mean ± SEM).


( J K ) HeLa stably expressing GFP - LC3 were transfected for 30 hr with myc - tagged empty vector , myc - MTMR3WT , myc - MTMR3C413S , incubated for 4 hr in HBSS in presence of BAF in presence or absence of Wm . were fixed , stained with anti - myc antibodies , imaged by confocal microscope . Asterisks indicate transfected . of numbers of GFP - LC3vesicles per is shown in ( K ) ( mean ± ) .

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

(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3vesicles per cell is shown in (K) (mean ± SEM).


( J K ) HeLa stably expressing GFP - LC3 were transfected for 30 hr with myc - tagged empty vector , myc - MTMR3WT , myc - MTMR3C413S , incubated for 4 hr in HBSS in presence of BAF in presence or absence of Wm . were fixed , stained with anti - myc antibodies , imaged by confocal microscope . Asterisks indicate transfected . of numbers of GFP - LC3 vesicles per is shown in ( K ) ( mean ± ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3 vesicles per cell is shown in (K) (mean ± SEM).


( L M ) HeLa transfected with GFP - PI5P4K2A , 2B , 2C , RFP - LC3 for 30 hr were loaded with 10 μM PI ( 3 ) P for 1 hr in complete medium , then fixed imaged on confocal microscope . Bar , 10 μm . of ( of total ) showing more than 10 RFP - LC3 vesicles in different conditions from ( J ) is shown in ( M ) ; n = 200 ( mean ± ) . See also Figure S5 .

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 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

(L and M) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, and RFP-LC3 for 30 hr were loaded with 10 μM PI(3)P for 1 hr in complete medium, and then fixed and imaged on confocal microscope. Bar, 10 μm. Quantification of cells (percentage of total) showing more than 10 RFP-LC3 vesicles in the different conditions from (J) is shown in (M); n = 200 cells (mean ± SEM). See also Figure S5.


( A ) HeLa transfected with GFP - WIPI2B or GFP - DFCP1 preloaded with indicated concentrations of PI ( 5 ) P for 1 hr , starved in HBSS for 1 hr , then incubated with Wm in HBSS were tracked by time - lapse microscopy for 10 min after addition of Wm . of WIPI2B or DFCP1vesicles ( of those at starting time [ T0 ] ) during treatments are shown in graphs .

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

(A) HeLa cells transfected with GFP-WIPI2B or GFP-DFCP1 preloaded with indicated concentrations of PI(5)P for 1 hr, starved in HBSS for 1 hr, and then incubated with Wm in HBSS were tracked by time-lapse microscopy for 10 min after the addition of Wm. Quantification of WIPI2B or DFCP1vesicles (percentage of those at the starting time [T0]) during the treatments are shown in the graphs.


( B - D ) HeLa treated with control , PI5P4K2A , 2B , 2C siRNA were transfected with GFP - WIPI2B ( B ) , GFP - WIPI2D ( C ) , or GFP - DFCP1 ( D ) ; starved in HBSS ( 1 hr ) ; then incubated with Wm in HBSS . WIPI2 or DFCP1 structures were tracked quantified as in ( A ) .

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 B-CONTROLLED_VAR O O O 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

(B-D) HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA were transfected with GFP-WIPI2B (B), GFP-WIPI2D (C), or GFP-DFCP1 (D); starved in HBSS (1 hr); and then incubated with Wm in HBSS. WIPI2 or DFCP1 structures were tracked and quantified as in (A).


( E ) Lysates from HeLa stably expressing GFP - WIPI2B were incubated with agarose beads coated with PI , PI ( 5 ) P , PI ( 3 ) P , eluted with SDS - PAGE sample buffer , recovered were assessed by western blotting using antibodies against GFP . Uncoated beads lysates from HeLa stably expressing GFP alone were used as internal controls .

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

(E) Lysates from HeLa cells stably expressing GFP-WIPI2B were incubated with agarose beads coated with PI, PI(5)P, and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls.


( F - H ) extracts from HeLa stably expressing GFP - WIPI2B were incubated for 3 hr with PI ( 5 ) P - containing liposomes ( F H ) or PI ( 3 ) P - containing liposomes ( G H ) before a pull - down experiment using indicated beads . PS - containing liposomes were used as internal controls for both competition assays in ( F G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Cell extracts from HeLa cells stably expressing GFP-WIPI2B were incubated for 3 hr with PI(5)P-containing liposomes (F and H) or PI(3)P-containing liposomes (G and H) before a pull-down experiment using the indicated beads. PS-containing liposomes were used as internal controls for both competition assays in (F and G).


( I ) Lysates from HeLa stably expressing GFP - WIPI2B WT GFP - WIPI2B FTTG mutant were incubated with agarose beads coated with PI ( 5 ) P PI ( 3 ) P , eluted with SDS - PAGE sample buffer , recovered were assessed by western blotting using antibodies against GFP . See also Figure S6 .

O 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 O O 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) Lysates from HeLa cells stably expressing GFP-WIPI2B WT and GFP-WIPI2B FTTG mutant were incubated with agarose beads coated with PI(5)P and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. See also Figure S6.


( A ) HeLa stably expressing GFP - LC3 were shifted to starvation media ( HBSS or glucose - free DMEM ) for 4 hr ( in presence of 200 nM Wm or 100 nM YM - 201636 ) . ( B ) of numbers of GFP - LC3 vesicles per in HeLa stably expressing GFP - LC3 treated as in ( A ) is shown in graph ( mean ± ) .

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

(A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).(B) Quantification of numbers of GFP-LC3 vesicles per cell in HeLa cells stably expressing GFP-LC3 treated as in (A) is shown in the graph (mean ± SEM).


( C ) Western blot of LC3 - II tubulin in MEF SKNSH were treated as in ( A ) in complete media or glucose - free media in presence of 100 nM YM - 201636 , 200 nM Wm , 400 nM BAF for 4 hr .

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

(C) Western blot analysis of LC3-II and tubulin levels in MEF and SKNSH cells were treated as in (A) in complete media or glucose-free media in the presence of 100 nM YM-201636, 200 nM Wm, and 400 nM BAF for 4 hr.


( D ) HeLa transfected with GFP - PHD3X Strawberry - ATG16L1 for 16 hr were left in complete media ( basal ) or glucose - free media ( glucose free ) for 4 hr in presence or absence of 100 nM YM - 201636 , then fixed imaged on a confocal microscope .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in complete media (basal) or glucose-free media (glucose free) for 4 hr in the presence or absence of 100 nM YM-201636, then fixed and imaged on a confocal microscope.


( E F ) HeLa transfected with GFP - PI5P4K2A , 2B , 2C , or PI5P4K2C catalytic dead RFP - LC3 for 30 hr ( E ) were starved for glucose for 4 hr , then fixed imaged on a confocal microscope . Bar , 10 μm . ( F ) of ( of total ) showing more than ten autophagic vesicles ( RFP - LC3 vesicles ) in different conditions from ( F ) is shown in graph ; n = 200 ( mean ± ) .

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 O O O O O O O O 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

(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or PI5P4K2C catalytic dead and RFP-LC3 for 30 hr (E) were starved for glucose for 4 hr, then fixed and imaged on a confocal microscope. Bar, 10 μm. (F) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM).


( G ) HeLa treated with CTR or MTMR3 siRNA for 5 days were transfected for last 16 hr with GFP - PHD3X , Strb - ATG16L1 , myc - MTMR3 WT . were starved for glucose for 4 hr , then fixed , stained with anti - myc antibodies , imaged on a confocal microscope . Bar , 10 μm .

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

(G) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP-PHD3X, Strb-ATG16L1, and myc-MTMR3 WT. Cells were starved for glucose for 4 hr, then fixed, stained with anti-myc antibodies, and imaged on a confocal microscope. Bar, 10 μm.


( H ) HeLa transfected for 16 hr with GFP - PHD3X Strb - ATG16L1 were starved for 4 hr in HBSS or glucose - free media , then fixed , stained for PI ( 3 ) P antibodies , imaged on a confocal microscope . Bar , 10 μm . See also Figure S7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells transfected for 16 hr with GFP-PHD3X and Strb-ATG16L1 were starved for 4 hr in HBSS or glucose-free media, then fixed, stained for PI(3)P antibodies, and imaged on a confocal microscope. Bar, 10 μm. See also Figure S7.


A . Representative immunofluorescence quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67 + ( red ) acinar nuclei co - with DAPI ( blue ) Amylase ( green ) . Arrows point to KI67 + acinar in Hnf1apKO mouse . Scale bar is 200 μm . Acinar proliferation is represented as average of KI67 + / Amylase + . Quantifications were performed on 3 random fields from 3 Pdx1Cre 3 Hnf1apKO mice . P - values are from two - Student ' s t .

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

A. Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67+ (red) acinar cell nuclei co-staining with DAPI (blue) and Amylase (green). Arrows point to KI67+ acinar cells in Hnf1apKO mouse. Scale bar is 200 μm. Acinar proliferation is represented as the average of the KI67+/Amylase+ cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice. P-values are from two-tailed Student's t test.


Representative H & E stainings of pancreata from KrasG12D Hnf1apKO ; KrasG12D mice . B - D . KrasG12D Hnf1apKO ; KrasG12D mice have normal at 7 days . E - J . At 21 days Hnf1apKO ; KrasG12D mice show acinar - to - metaplasia ( dashed encircled areas ) regions with desmoplastic reaction ( asterisk ) , which are not observed in KrasG12D mice ( E , H ) . K - P . At 8 KrasG12D pancreas show occasional abnormal structures ( dashed encircled areas in N which is a magnification of squared dotted box in K ) Hnf1apKO ; KrasG12D mice ( L , M , O , P ) present mucinous tubular complexes ( black arrows ) , more advanced PanINs with luminal budding ( open arrows ) including foci of spindle proliferation ( asterisks ) incipient infiltrative growth ( black dashed box in O ) . Data information : Black dashed boxes in ( E , F , K , L O ) indicate magnified areas in ( H , G , N , M P ) respectively . Scale bars indicate 100 µm ( C , E , F , K , L ) , 50 µm ( O ) 20 µm ( B , D , G , H - J , M , N , P ) .

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 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 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 O O 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 H&E stainings of pancreata from KrasG12D and Hnf1apKO;KrasG12D mice. B-D. KrasG12D and Hnf1apKO;KrasG12D mice have normal morphology at 7 days. E-J. At 21 days Hnf1apKO;KrasG12D mice show acinar-to-ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in KrasG12D mice (E, H). K-P. At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N which is a magnification of squared dotted box in K) and Hnf1apKO;KrasG12D mice (L,M,O,P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O). Data information: Black dashed boxes in (E,F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 100 µm (C,E,F,K,L), 50 µm (O) and 20 µm (B,D,G,H-J,M,N,P).


A . Fold change ( FC ) in transcripts in Hnf1aaKO vs . control pancreas , plotted against significance ( - Log10 q ; genes significant at q < 0 . 05 are shown as colored dots above horizontal ) .

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

A. Fold change (FC) in transcripts in Hnf1aaKO vs. control pancreas, plotted against significance (-Log10 q; genes significant at q<0.05 are shown as colored dots above the horizontal line).


B . GSEA showing that genes specific to differentiated acinar were downregulated in Hnf1aaKO pancreas , but not genes specific to islets or . Upregulated genes were enriched in genes specific to mesenchymal . Lineage - enriched genes were obtained from Muraro et al . ( Muraro et al . , 2016 ) .

B. GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from Muraro et al. (Muraro et al., 2016).


D . GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT , MAPK , KRAS , PI3K - AKT .

O O 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. GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K-AKT.


E . HNF1A promotes transcriptional activation of direct target genes . Left : HNF1A bound - genes were enriched among genes that showed downregulation in Hnf1a mutants , but not among upregulated genes . P - values odds ratios ( O . R . ) calculated by Fisher ' s exact . Right : Venn diagrams showing overlap of HNF1A - bound genes with genes that were downregulated upregulated in Hnf1a mutant pancreas .

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

E. HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A bound-genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P-values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A-bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.


A . Human orthologs of genes that were up - down - regulated in Hnf1aaKO pancreas were also up - down - regulated in human pancreas with low vs . high HNF1A expression ( lowest vs . highest expression deciles , respectively ) . A random list of 717 genes controlled for similar expression was used for comparison . Violin plots include median interquartile ranges . Dots are average values for each gene . Kruskall Wallis P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Human orthologs of genes that were up- and down-regulated in Hnf1aaKO pancreas were also up- and down-regulated in human pancreas with low vs. high HNF1A expression (lowest vs. highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskall Wallis P<0.0001.


D . HNF1A mRNA differed in HNF1A LoF control groups ( Kruskal - Wallis , P < 0 . 01 ) , despite considerable variability overlap between groups . E . KDM6A mRNA were downregulated in HNF1A LoF tumors ( Kruskal - Wallis , P < 0 . 001 ) . Data information : Box plots in ( D E ) show HNF1A KDM6A expression in HNF1A LoF tumors ( n = 26 ) Control 1 ( n = 39 ) Control 2 ( n = 57 ) tumors . horizontal central marks median . Box limits indicate first third quartiles whiskers extend to highest lowest data points within 1 . 5 x interquartile range outside box limits .

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 B-MEASURED_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 B-MEASURED_VAR I-MEASURED_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

D. HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal-Wallis, P<0.01), despite considerable variability and overlap between groups. E. KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal-Wallis, P<0.001).Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors (n=26) and Control 1 (n=39) and Control 2 (n=57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x interquartile range outside box limits.


H & E of pancreata from Kdm6apKO ; KrasG12D KrasG12D mice . A - C . 7 day - old KrasG12D Kdm6apKO ; KrasG12D mice showed normal . D - I . At 21 days , KrasG12D mice showed normal pancreas ( dotted at in D is shown at large magnification in G ) whereas Kdm6apKO ; KrasG12D mice already show acinar - to - metaplasia ( dashed encircled in F ) , spindle proliferation ( asterisks in E H ) , sarcomatoid architecture ( I ) desmoplastic reaction ( black arrowhead in H ) . J - O . KrasG12D mice present occasional acinar - to - metaplasia low grade PanINs at 8 ( M is a magnification of squared dotted box in J see Fig 1N ) whereas at same pancreas from Kdm6apKO ; KrasG12D mice show massive remodeling ( K ) , extensive acinar - to - metaplasia ( L dashed encircled in N ) , cancer with prominent spindle / mesenchymal proliferation , infiltrative growth ( black arrows asterisk respectively in O ) abundant stroma ( black arrowheads in N ) . Data information : Scale bars : 100 µm ( B , D , E , J , K ) or 20 µm ( A , C , F - I , L - N ) .

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 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 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

H&E staining of pancreata from Kdm6apKO;KrasG12D and KrasG12D mice. A-C. 7 day-old KrasG12D and Kdm6apKO;KrasG12D mice showed normal morphology. D-I. At 21 days, KrasG12D mice showed normal pancreas morphology (dotted area at in D is shown at large magnification in G) whereas Kdm6apKO;KrasG12D mice already show acinar-to-ductal metaplasia (dashed encircled area in F), spindle cell proliferation (asterisks in E and H), sarcomatoid architecture (I) and desmoplastic reaction (black arrowhead in H). J-O. KrasG12D mice present occasional acinar-to-ductal metaplasia and low grade PanINs at 8 weeks (M is a magnification of squared dotted box in J and see Fig 1N) whereas at the same age pancreas from Kdm6apKO;KrasG12D mice show massive remodeling (K), extensive acinar-to-ductal metaplasia (L and dashed encircled area in N), cancer with prominent spindle/mesenchymal proliferation, infiltrative growth (black arrows and asterisk respectively in O) and abundant stroma (black arrowheads in N). Data information: Scale bars: 100 µm (B,D,E,J,K) or 20 µm (A,C,F-I, L-N).


A . Fold change ( FC ) significance for transcripts in Kdm6apKO vs . control pancreas . Genes significant at q < 0 . 05 are shown as colored dots above horizontal .

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

A. Fold change (FC) and significance for transcripts in Kdm6apKO vs. control pancreas. Genes significant at q<0.05 are shown as colored dots above the horizontal line.


B . GSEA showing that genes specific to differentiated acinar were downregulated in KDM6A - deficient pancreas , but not genes specific to islets or . Upregulated genes were enriched in genes specific to mesenchymal . Lineage - enriched genes were obtained from ( Muraro et al . , 2016 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A-deficient pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from (Muraro et al., 2016).


D . GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas .

D. GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas.


E . KDM6A functions as a transcriptional activator of direct target genes in pancreatic . Left : Downregulated , but not upregulated , genes in Kdm6apKO pancreas were enriched for KDM6A binding . P - values odds ratios ( O . R . ) were calculated by Fisher ' s exact . Right : Venn diagrams showing overlap of down upregulated genes with KDM6A binding .

O O O O O O 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 B-MEASURED_VAR O O

E. KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells. Left: Downregulated, but not upregulated, genes in Kdm6apKO pancreas were enriched for KDM6A binding. P-values and odds ratios (O.R.) were calculated by Fisher's exact test. Right: Venn diagrams showing overlap of down and upregulated genes with KDM6A binding.


F . KDM6A - bound enhancers promoters showed increased H3K27me3 decreased H3K27ac in Kdm6apKO pancreas .

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

F. KDM6A-bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas.


G . Genes that were downregulated in Kdm6apKO pancreas ( KDM6A - dependent genes ) showed greatest changes in histone marks . Box plots show fold - changes of H3K27me3 ( left ) H3K27ac ( right ) signals in Kdm6apKO compared to control . Signals were analyzed in KDM6A bound regions in promoters enhancers of genes that were KDM6A dependent ( grey ; n = 420 regions ) independent ( ; n = 8035 regions ) . signals are average values from ChIP - seq experiments in two biological replicates . horizontal central marks median . Box limits indicate first third quartiles whiskers extend to highest lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - Mann - Whitney U .

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

G. Genes that were downregulated in Kdm6apKO pancreas (KDM6A-dependent genes) showed greatest changes in histone marks. Box plots show fold-changes of H3K27me3 (left) and H3K27ac (right) signals in Kdm6apKO compared to control. Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent (grey; n=420 regions) and independent (white; n=8035 regions). The signals are average values from ChIP-seq experiments in two biological replicates. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Mann-Whitney U test.


H . Changes in KDM6A , H3K27me3 H3K27ac ChIP - seq profiles in Ppp4r4 - Serpina10 locus in Kdm6apKO pancreas .

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

H. Changes in KDM6A, H3K27me3 and H3K27ac ChIP-seq profiles in the Ppp4r4-Serpina10 locus in Kdm6apKO pancreas.


I . qPCR of H3K27me3 , H3K27ac H3K4me1 changes in promoter enhancer regions highlighted in ( H ) ( R1 , R2 , R3 ) , Serpin10 mRNA . Error bars show + / - SD P - values are from two - Student ' s t , n = 3 .

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 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

I. qPCR of H3K27me3, H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in (H) (R1, R2, R3), and Serpin10 mRNA. Error bars show +/-SD and P-values are from two-tailed Student's t test, n=3.


J . KDM6A - bound genes that showed decreased expression in Kdm6apKO pancreas ( functional KDM6A binding sites ) showed simultaneous gain of H3K27me3 loss of H3K27ac .

O 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 B-MEASURED_VAR O O O O

J. KDM6A-bound genes that showed decreased expression in Kdm6apKO pancreas (functional KDM6A binding sites) showed simultaneous gain of H3K27me3 and loss and of H3K27ac.


B . Co - binding in functional KDM6A - bound enhancer promoter regions revealed that HNF1A was most enriched co - bound TF amongst three other acinar TFs . Binding regions of TAL1 in a non - pancreatic type random binding sites were used as negative controls . P - values was determined by Fisher ' s exact for peak comparisons using enhancer promoter regions as background .

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 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. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background.


C . most downregulated genes in Kdm6apKO pancreas are shown ranked by q - value , are almost invariably bound by HNF1A down regulated in Hnf1aaKO pancreas , or known to be direct HNF1A - dependent target genes from other studies ( red purple , respectively ) .

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

C. The most downregulated genes in Kdm6apKO pancreas are shown ranked by q-value, and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas, or known to be direct HNF1A-dependent target genes from other studies (red and purple, respectively).


D , E . GSEA on Hnf1aaKO Kdm6apKO ranked - ordered gene lists vs . their reciprocal up - or downregulated gene sets , demonstrated that KDM6A HNF1A regulate similar genes .

O O O O O O O O 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. GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked-ordered gene lists vs. their reciprocal up- or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes.


F . Expression changes in Hnf1aaKO Kdm6apKO pancreas , showing that genes bound by KDM6A downregulated in Kdm6apKO pancreas ( red dots ) were generally downregulated in Hnf1aaKO pancreas .

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

F. Expression changes in Hnf1aaKO and Kdm6apKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas (red dots) were generally downregulated in Hnf1aaKO pancreas.


G . HNF1A KDM6A co - occupy same regions in Pah , which is downregulated in Hnf1a Kdm6a knock - out pancreas .

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

G. HNF1A and KDM6A co-occupy the same regions in Pah, which is downregulated in Hnf1a and Kdm6a knock-out pancreas.


H . Genes that were co - bound by KDM6A HNF1A showed greatest downregulation in Kdm6apKO pancreas , compared with KDM6A - bound genes that were not bound by HNF1A . Box plots show median IQR of Log2 TPM fold - changes whiskers extend to highest lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - Student ' s t n = 4 replicates per condition .

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

H. Genes that were co-bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas, compared with KDM6A-bound genes that were not bound by HNF1A. Box plots show median and IQR of Log2 TPM fold-changes and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Student's t tests and n=4 replicates per condition.


A . Co - immunoprecipitation of endogenous HNF1A KDM6A followed by western blot demonstrated that HNF1A is in same as KDM6A .

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 B-MEASURED_VAR O

A. Co-immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A.


B . Western blot showing loss of HNF1A unchanged KDM6A in Hnf1a - / - pancreas .

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

B. Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a-/- pancreas.


C . Differential binding of KDM6A in Hnf1a - / - vs . wild type pancreas . Pink dots below zero ( 1873 sites ) show regions with reduced KDM6A binding pink dots above zero ( 118 sites ) are regions with increased binding at FDR < 0 . 05 .

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

C. Differential binding analysis of KDM6A in Hnf1a-/- vs. wild type pancreas. Pink dots below zero (1873 sites) show regions with reduced KDM6A binding and pink dots above zero (118 sites) are regions with increased binding at FDR<0.05.


Regions that show reduced KDM6A binding in Hnf1a - / - are strongly bound by HNF1A are highly enriched in HNF1 motifs . P - values in ( D ) were calculated with two - Mann - Whitney U

O O O O B-MEASURED_VAR 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

Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (D) were calculated with two-tailed Mann-Whitney U test


Regions that show reduced KDM6A binding in Hnf1a - / - are strongly bound by HNF1A are highly enriched in HNF1 motifs . P - values in ( E ) with Fisher ' s exact .

O O O O B-MEASURED_VAR 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

Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (E) with Fisher's exact test.


F . KDM6A binding is markedly reduced in HNF1A KDM6A co - bound regions in Hnf1a - / - pancreas , but not in other KDM6A bound regions .

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

F. KDM6A binding is markedly reduced in HNF1A and KDM6A co-bound regions in Hnf1a-/- pancreas, but not in other KDM6A bound regions.


G . Genes that loose KDM6A binding in Hnf1a - mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas are direct HNF1A target genes ( red dots ) .

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

G. Genes that loose KDM6A binding in Hnf1a-mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes (red dots).


violin plot depicting smaller genome of HAC isolates ( n = 820 ) than that of DPC ( n = 407 ) HPC ( n = 262 ) . Inside violins are box whisker plots , with dot showing median , box showing quartiles , whisker showing 95 % percentiles . P - values were calculated using unpaired t - ( * * , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The violin plot depicting the smaller genome size of HAC isolates (n=820) than that of DPC (n=407) and HPC (n=262). Inside the violins are the box and whisker plots, with the dot showing the median, the box showing the quartiles, and the whisker showing the 95% percentiles. P-values were calculated using unpaired t-test (**, P<0.05).


survival curve for zebrafish inoculated with representative S . suis isolates from HAC HPC . In total twenty - five representative isolates were tested , including fourteen isolates from HAC ten healthy - pig isolates from HPC , as well as SC19 in HAC responsible for 2005 human outbreak as a positive control . cumulative mortalities were calculated using combined data from isolates in each group . As expected , 14 isolates from HAC displayed a significantly higher mortality than HPC isolates ( P < 0 . 05 ) . Survival curve statistical were created using Kaplan - Meier SPSS 23 . 0 . P - values were calculated using Log rank . P < 0 . 05 was considered significant ( * * , P < 0 . 05 ; NS , P > 0 . 05 ) . No significant difference ( NS , P = 0 . 072 ) was found between HAC isolates SC19 .

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

The survival curve for zebrafish inoculated with representative S. suis isolates from HAC and HPC. In total twenty-five representative isolates were tested, including fourteen isolates from HAC and ten healthy-pig isolates from HPC, as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control. The cumulative mortalities were calculated using the combined data from the isolates in each group. As expected, all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates (P<0.05). Survival curve and statistical analysis were created using Kaplan-Meier and SPSS 23.0. P-values were calculated using Log rank test. P<0.05 was considered significant (**, P<0.05; NS, P>0.05). No significant difference (NS, P=0.072) was found between HAC isolates and SC19.