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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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 ) 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 ) .

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

(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 ) .

O 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 B-CONTROLLED_VAR 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 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 ) .

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

(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 .

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

(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 .

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

(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 ) .

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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.


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 .

O O O O 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

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 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 .

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 . 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 ) .

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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 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 .

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

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

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 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 .

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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 and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A .

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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 and unchanged KDM6A in Hnf1a - / - pancreas .

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


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 .

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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 - / - 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

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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 - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( E ) with Fisher ' s exact test .

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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 and KDM6A co - bound regions in Hnf1a - / - pancreas , but not in other KDM6A bound regions .

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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 and are direct HNF1A target genes ( red dots ) .

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


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 ) .

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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).


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 .

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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.


A ) Shear - dilation relations of Tek - Cre : : Lama5 - / - and Lama4 - / - mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek - Cre : : Lama5 - / - arteries and an enhanced response of Lama4 - / - arteries . Data shown are mean changes in vessel diameter ( Δ µm ) ± s . e . m . from 8 experiments with 16 wild type and 16 KO arteries ; KO and wild type were analysed as pairs in each experiment . * * P < 0 . 01 , paired t - test .

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A) Shear-dilation relations of Tek-Cre::Lama5-/- and Lama4-/- mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek-Cre::Lama5-/- arteries and an enhanced response of Lama4-/- arteries. Data shown are mean changes in vessel diameter (Δ µm) ± s.e.m. from 8 experiments with 16 wild type and 16 KO arteries; KO and wild type were analysed as pairs in each experiment. **P<0.01, paired t-test.


The dose - response curves of arteries from wild type , Tek - Cre : : Lama5 - / - ( B ) and Lama4 - / - mice ( C ) and wild type littermates stimulated with methacholine do not show significant differences ( n . s . ) . Data are expressed as percent relaxation of the maximum force developed in presence of 0 . 3 µM U46619 , and are mean values ± s . e . m from 7 experiments with 1 wild type and 1 KO artery in each experiment .

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The dose-response curves of arteries from wild type, Tek-Cre::Lama5-/- (B) and Lama4-/- mice (C) and wild type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as percent relaxation of the maximum force developed in presence of 0.3 µM U46619, and are mean values ± s.e.m from 7 experiments with 1 wild type and 1 KO artery in each experiment.


A ) Three - dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice , and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers . DAPI staining permits identification of endothelial cell nuclei , which lie perpendicular to the nuclei of the smooth muscle cells . Laminin α5 is absent from endothelial basement membranes of Tek - Cre : : Lama5 - / - arteries ( arrow ) but is still present in smooth muscle basement membranes ( asterisk ) , while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes . Lama4 - / - arteries lack laminin α4 in both endothelial ( arrow ) and smooth muscle ( asterisk ) basement membranes , but laminin α5 is still detectable . Scale bars are 10 μm .

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A) Three-dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek-Cre::Lama5-/- arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4-/- arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm.


B ) Scanning electron microscopy images of endothelial cell - denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - arteries . The arrows indicate artificial ruptures due to preparation procedure . Scale bars are 100 nm .

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B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm.


C ) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters ( - 24 . 3 % ) in Tek - Cre : : Lama5 - / - arteries and larger diameters ( + 27 . 6 % ) in Lama4 - / - arteries compared to wild type controls . Auto - fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter . Scale bar is 100 µm . Data are means ± s . e . m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype . * P < 0 . 05 unpaired t - test .

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C) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.


A ) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511 , compared to the non - endothelial cell laminin 111 and fibronectin , showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411 .

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A) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411.


B ) Enhanced fold change ( Δ ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear . mRNA data are means ± s . e . m of 4 independent experiments with triplicates / experiment , paired t - test .

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B) Enhanced fold change (Δ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test.


B ) Enhanced fold change ( Δ ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear . Protein quantification data are means ± s . e . m of 7 independent experiments , Mann - Whitney test .

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B) Enhanced fold change (Δ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test.


C ) Angle histogram of HUAEC orientation after 120 min of 10dyn / cm2 shear stress showing the percent cells at 10 - 90 ° in relation to the direction of flow . Cells were plated on 25 nM laminin 511 and laminin 111 . Data are means ± s . e . m of 6 independent experiments , t - test .

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C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test.


D , E ) Inhibition assays performed at 25nM laminin 511 ( D ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D, E) Inhibition assays performed at 25nM laminin 511 (D) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


D , E ) Inhibition assays performed at 25nM laminin 111 , laminin 411 or fibronectin ( E ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

D, E) Inhibition assays performed at 25nM laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


( A ) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions ( arrow heads ) . Scale bar is 10 µm .

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(A) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions (arrow heads). Scale bar is 10 µm.


B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test .

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B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Correpsonding quantification of sizes ( C ) and frequency distribution ( D ) of adhesion complexes per endothelial cell . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test .

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B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Correpsonding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


E , F ) The endothelium of excised wild type , Tek - Cre : : Lama5 - / - aortae were analysed by AFM , revealing reduced cortical stiffness in Tek - Cre : : Lama5 - / - vessels ( - 9 . 5 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test .

E, F) The endothelium of excised wild type, Tek-Cre::Lama5-/- aortae were analysed by AFM, revealing reduced cortical stiffness in Tek-Cre::Lama5-/- vessels (-9.5%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


E , F ) The endothelium of excised wild type and Lama4 - / - aortae were analysed by AFM , revealing increased cortical stiffness in Lama4 - / - vessels ( + 2 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test .

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E, F) The endothelium of excised wild type and Lama4-/- aortae were analysed by AFM, revealing increased cortical stiffness in Lama4-/- vessels (+2%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


G ) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111 , reveal increased cortical stiffness in cells on laminin 511 . Data are mean ± s . e . m from 3 experiments with triplicates / experiments , * P < 0 . 05 , unpaired t - test .

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G) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111, reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m from 3 experiments with triplicates/experiments, *P<0.05, unpaired t-test.


A ) A dual pipette - pulling assay was employed to measure cell - cell adhesion strength in HUAECs bound to laminin - coated beads ( asterisk ) or in the absence of beads . Scale bar is 10 µm . B ) Quantification shows higher adhesion strength between cells bound to laminin 511 - coated beads compared to laminin 111 - coated beads or cells not incubated with beads . Measurements could not be made with cells incubated with laminin 411 - coated beads since it was impossible to find cell - bead complexes . Values are means ± s . e . m from 15 cells from 3 independent experiments , unpaired t - test with Welch ' s correction .

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A) A dual pipette-pulling assay was employed to measure cell-cell adhesion strength in HUAECs bound to laminin-coated beads (asterisk) or in the absence of beads. Scale bar is 10 µm. B) Quantification shows higher adhesion strength between cells bound to laminin 511-coated beads compared to laminin 111-coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411-coated beads since it was impossible to find cell-bead complexes. Values are means ± s.e.m from 15 cells from 3 independent experiments, unpaired t-test with Welch's correction.


C ) Dual pipette - pulling assay performed in the presence of VE - cadherin blocking antibody , isotope control and soluble laminin 511 , showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions . Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511 - coated beads .

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C) Dual pipette-pulling assay performed in the presence of VE-cadherin blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511-coated beads.


D ) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody ; cell - cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK , SRC or FAK inhibitors . Values are means ± s . e . m from 11 cells from 3 independent experiments , unpaired t - test with Welch ' s correction . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , n . s . = not significant , n . d . = not determined .

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D) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody; cell-cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors. Values are means ± s.e.m from 11 cells from 3 independent experiments, unpaired t-test with Welch's correction. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. = not significant, n.d. = not determined.


A ) Immunofluorescence - based antibody feeding assay performed using HUAECs seeded on laminin 411 , 511 or 111 - coated coverslips , reveals more VE - cadherin at junctions and less in vesicles in cells plated on laminin 511 ( arrow heads mark VE - cadherin positive vesicles ) . Scale bar is 10 µm . B ) Quantification of VE - cadherin positive vesicles / cell in HUAECs plated on laminin 411 , 511 or 111 . Values are means ± s . e . m from 300 cells from 3 independent experiments , unpaired t - test .

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A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test.


C ) Western blot of biotinylated ( surface ) VE - cadherin in HUAECs plated on different laminins versus total VE - cadherin ( input ) confirms more junctionally located VE - cadherin in cells bound to laminin 511 .

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C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511.


D ) Quantification of Western blots expressed as relative signal proportions ( surface VE - cadherin / total VE - cadherin ) . Values shown are means ± s . e . m of 6 experiments , unpaired t - test .

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D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test.


E ) Western blot of p120 catenincoimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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E) Western blot of p120 catenincoimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


E ) Western blot of p120 catenin coimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins , and ( F ) corresponding quantification of the p120 signal intensity relative to the total VE - cadherin signal . Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


A ) Double immunofluorescence staining for phosphorylated myosin light chain II ( pMLC II ) and VE - cadherin in mesenteric resistance artery endothelium from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice ( arrow heads ) . Scale bar is 10µm . B ) Quantification of the pMLC II signal along VE - cadherin signal revealed significantly less overlap in Tek - Cre : : Lama5 - / - and more overlap in Lama4 - / - endothelium , compared with wild type controls . The data are normalized to the skeletal length of the VE - cadherin signal . Values shown are means ± s . e . m resulting from quantification of 4 field of view per arteries , 4 mice / genotype . * P < 0 . 05 , * * P < 0 . 01 , paired t - test .

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A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.


A , B . Aggregate number of detected and undetected cysteines ( A ) and lysines ( B ) in 3 , 840 CpDAA - containing proteins .

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A, B. Aggregate number of detected and undetected cysteines (A) and lysines (B) in 3,840 CpDAA-containing proteins.


C . Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220 - 479 of CASP8 ( UniProt ID Q14790 ) . Dashed horizontal line marks deleterious threshold of 25 .

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C. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25.


E . Activity of recombinant caspase - 8 protein assayed using fluorogenic IETD - AFC substrate . Percentage activity shown relative to wild - type ( WT ) protein for three replicate experiments , bars and error bars as mean ± SD .

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E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.


( A ) Amylose pulldown from benzonase - treated HEK293T cell lysates expressing 2xMBP - BRCA2NT in untreated or irradiated cells ( 6Gy ; + IR ) . DDX5 and BRCA2NT ( MBP ) detected by immunoblot . StainFree images of the gels before transfer were used as loading

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(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control