E : Immunofluorescence of hippocampal neurons transduced with GFP , GFP - TDP - 25 wild - type or mutant and treated with MG132 ( 10 µM , 14h , DIV5 + 8 ) . Representative images of GFP - TDP - 25 inclusions and diffuse cytoplasmic staining . Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei ( omitted here for clarity ) in MG132 treated cells compared to DMSO control . Counterstain to label the neuronal cytoskeleton ( MAP2 ) . Scale bar = 10 µm . Box plot as in Fig . 3C . GFP ( 1 . 23 ± 0 . 393 , n = 5 independent experiments ) vs GFP - TDP - 25 wt ( 2 . 11 ± 0 . 804 , n = 5 ) vs GFP - TDP - 25 mut ( 2 . 36 ± 1 . 16 , n = 5 ) : H ( 1 ) = 7 . 34 , df = 2 , p = 0 . 02548 , η² [ H ] = 0 . 445 , Kruskal - Wallis test . GFP vs GFP - TDP - 25 wt : * p = 0 . 048 , GFP vs GFP - TDP - 25 mut : * p = 0 . 048 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 690 , Pairwise Wilcoxon Rank Sum Tests with Benjamini - Hochberg correction .

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

E: Immunofluorescence of hippocampal neurons transduced with GFP, GFP-TDP-25 wild-type or mutant and treated with MG132 (10 µM, 14h, DIV5+8). Representative images of GFP-TDP-25 inclusions and diffuse cytoplasmic staining. Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei (omitted here for clarity) in MG132 treated cells compared to DMSO control. Counterstain to label the neuronal cytoskeleton (MAP2). Scale bar = 10 µm. Box plot as in Fig. 3C. GFP (1.23 ± 0.393, n=5 independent experiments) vs GFP-TDP-25 wt (2.11 ± 0.804, n=5) vs GFP-TDP-25 mut (2.36 ± 1.16, n=5): H(1)=7.34, df=2, p=0.02548, η²[H]=0.445, Kruskal-Wallis test. GFP vs GFP-TDP-25 wt: *p=0.048, GFP vs GFP-TDP-25 mut: *p=0.048, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.690, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.


A , Reconstructed MSOT images ( 800 nm ) without and with linear unmixing data in the neck and the upper and lower abdominal areas , showing the ROIs for the liver , kidney , iBAT , rpWAT , aorta , and SV . 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 neck / upper abdominal / lower abdominal : Hb : 2 . 5 / 3 . 5 / 0 . 9 ; HbO2 : 2 . 9 / 4 . 9 / 2 . 5 ; Lipid : 24000 / 19000 / 13000 ) 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

A, Reconstructed MSOT images (800 nm) without and with linear unmixing data in the neck and the upper and lower abdominal areas, showing the ROIs for the liver, kidney, iBAT, rpWAT, aorta, and SV. 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 neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 staining of control ( healthy ) and 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 staining 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 fluorescence microscopy and HE staining images of control and 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

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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel .

O 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) 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 ) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence . 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 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 the skin in vivo ( Douglas et al , 2018b ) and 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 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 and control ( Ctrl ) parasites in hydrogels with small ( D ) and 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and DAPI in P19 cells and E14 cortical cells 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 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 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 .

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

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 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 .

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

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 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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 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 % .

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-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

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 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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(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 cells transfeted for 24h with plasmids expressing GFP only or Trnp1 - IRES - GFP with antibodies indicated .

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

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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 , 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 .

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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 sequence including one mitosis of H2B - Venus in P19 cell expressing the indicated constructs .

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


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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 ) .

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

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(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 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 ) .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 ) .

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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 ) 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 ) 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 ) .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 .

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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 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 ) .

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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 the horizontal line ) .

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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 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 ) .

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 .

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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 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 .

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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 - 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 .

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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 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 .

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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 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 ) .

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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 ) and significance for transcripts in Kdm6apKO vs . control pancreas . Genes significant at q < 0 . 05 are shown as colored dots above the horizontal line .

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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 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 ) .

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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 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 .

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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 and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas .

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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 ) 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 .

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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 and H3K27ac ChIP - seq profiles in the Ppp4r4 - Serpina10 locus in Kdm6apKO pancreas .

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


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 .

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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.