Figure 5Volcano plot of DEGs in female ESCs ( # A4 , # A10 , # A11 ) versus males ( # A6 , # A13 , # A17 ) . The definition of up ( red dots ) or down ( blue dots ) genes is as follows : the ratio of up or down genes is > 1 . 5 and < two - thirds , respectively , with q - value < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Volcano plot of DEGs in female ESCs (#A4, #A10, #A11) versus males (#A6, #A13, #A17). The definition of up (red dots) or down (blue dots) genes is as follows: the ratio of up or down genes is > 1.5 and < two-thirds, respectively, with q-value< 0.05.


Figure 6Brcc3 expression confirmed by RNA - seq [ biological replicates ( n = 3 ) with three different cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] . Brcc3 expression confirmed by RT - qPCR [ biological replicates ( n = 3 ) with three different cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] . Brcc3 expression confirmed by western blotting [ biological replicates ( n = 3 ) with three different cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] . Confirmation of Brcc3 expression from knockdown samples in by RT - qPCR ( technical duplicates ) Confirmation of Brcc3 expression from knockdown samples in western blottingNanog HR assay with Brcc3 knockdown ( three biological replicates ) . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown , as shown in Figure 2C . The combined number of GFP - positive cells and total number of colonies are shown ( please note that total cell number of # A13 for drug screening was 3 . 67 × 107 because of contamination of 1 out of 12 dishes , compared with the other colonies for which 4 × 107 cells were used ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Brcc3 expression confirmed by RNA-seq [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].Brcc3 expression confirmed by RT-qPCR [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].Brcc3 expression confirmed by western blotting [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].Confirmation of Brcc3 expression from knockdown samples in by RT-qPCR (technical duplicates)Confirmation of Brcc3 expression from knockdown samples in western blottingNanog HR assay with Brcc3 knockdown (three biological replicates). The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown, as shown in Figure 2C. The combined number of GFP-positive cells and total number of colonies are shown (please note that total cell number of #A13 for drug screening was 3.67×107 because of contamination of 1 out of 12 dishes, compared with the other colonies for which 4×107 cells were used).


Figure 7Kaplan - Meier curves for the overall survival of breast cancer patients based on BRCC3 ( A ) , XIST ( B ) , and BRCC3 / XIST ( C ) expression levels . HR : hazard ratio .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Kaplan-Meier curves for the overall survival of breast cancer patients based on BRCC3 (A), XIST (B), and BRCC3/XIST(C) expression levels. HR: hazard ratio.


Figure 1Trajectory plots of siRNA - transfected HUVECs under VEGF and / or Sema3E gradients for 12 h . Red trajectories indicate cells with displacement to the negative y - axis at the end of analyses . Rose plots of cell trajectories in ( A ) . Leaflets are grouped into 10 ° segments . The radius of each leaflet represents the cumulative number of cells migrating in the corresponding angular bin . P values for the Rayleigh test represent non - random distributions of cell endpoints . Forward migration index ( FMI ) along the y - and x - axes during cell migration . n = 90 cells per group . Data represent mean ± SEM . * * p < 0 . 01 ; * * * p < 0 . 001 ; NS , not significant , by Tukey - Kramer test .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Trajectory plots of siRNA-transfected HUVECs under VEGF and/or Sema3E gradients for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses.Rose plots of cell trajectories in (A). Leaflets are grouped into 10° segments. The radius of each leaflet represents the cumulative number of cells migrating in the corresponding angular bin. P values for the Rayleigh test represent non-random distributions of cell endpoints.Forward migration index (FMI) along the y- and x-axes during cell migration. n = 90 cells per group. Data represent mean ± SEM. **p < 0.01; ***p < 0.001; NS, not significant, by Tukey-Kramer test.


Figure 2Confocal ( left three columns ) and super - resolution ( right - most columns ) images of subcellular distributions of ectopic RhoJ ( red ) , endogenous VEGFR2 ( green ) and PlexinD1 ( left , white ; right , blue ) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs . Note the colocalization of WT - RhoJ and CA - RhoJ ( Q79L ) with PlexinD1 in perinuclear endosomes and DN - RhoJ ( T35N ) with VEGFR2 in cytoplasmic and nuclear puncta . Light - blue asterisks in the PlexinD1 panels indicate nuclei . See also Appendix Fig S2 for the full images of super - resolution microscopy . Scale bar , 5 µm ( left ) ; 1 µm ( right ) . Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and / or PlexinD1 . n = 10 cells per group . Data represent mean . Co - immunoprecipitation ( co - IP ) from 293T cells 48 h after transfection . WT - and CA - RhoJ associated with PlexinD1 , while DN - RhoJ associated with VEGFR2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Confocal (left three columns) and super-resolution (right-most columns) images of subcellular distributions of ectopic RhoJ (red), endogenous VEGFR2 (green) and PlexinD1 (left, white; right, blue) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs. Note the colocalization of WT-RhoJ and CA-RhoJ (Q79L) with PlexinD1 in perinuclear endosomes and DN-RhoJ (T35N) with VEGFR2 in cytoplasmic and nuclear puncta. Light-blue asterisks in the PlexinD1 panels indicate nuclei. See also Appendix Fig S2 for the full images of super-resolution microscopy. Scale bar, 5 µm (left); 1 µm (right).Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and/or PlexinD1. n = 10 cells per group. Data represent mean.Co-immunoprecipitation (co-IP) from 293T cells 48 h after transfection. WT- and CA-RhoJ associated with PlexinD1, while DN-RhoJ associated with VEGFR2.


Figure 3Co - IP from 293T cells 48 h after transfection . RhoJ - PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30 - min stimulation with Sema3E . Arrows indicate PD1 Full ( upper , 250 kDa ) and PD1 △ RBD ( lower , 1 . 7 kDa smaller than PD1 Full ) . PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation . Scale bar , 10 µm . Super - resolution images of subcellular distributions of ectopic WT - RhoJ ( red ) and endogenous PlexinD1 ( green ) in HUVECs 24 h after transfection . RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation . Scale bar , 1 µm . Labeling of siRNA - transfected HUVECs with phalloidin ( white ) 30 min after Sema3E stimulation . Scale bar , 50 µm . Quantification of areas of individual HUVECs . n > 100 cells per group . Data represent mean ± SEM . * * * p < 0 . 001 ; NS , not significant , by Tukey - Kramer test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Co-IP from 293T cells 48 h after transfection. RhoJ-PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30-min stimulation with Sema3E. Arrows indicate PD1 Full (upper, 250 kDa) and PD1△RBD (lower, 1.7 kDa smaller than PD1 Full).PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation. Scale bar, 10 µm.Super-resolution images of subcellular distributions of ectopic WT-RhoJ (red) and endogenous PlexinD1 (green) in HUVECs 24 h after transfection. RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation. Scale bar, 1 µm.Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm.Quantification of areas of individual HUVECs. n > 100 cells per group. Data represent mean ± SEM. ***p < 0.001; NS, not significant, by Tukey-Kramer test.


Figure 4Co - IP from HUVECs . Blots for β - actin shown in ( A ) were used to confirm equal loading in ( B ) , as the same cell lysates were analyzed in both experiments . Immunoblots in HUVECs . Immunoblots in siRNA - transfected HUVECs . Co - IP from siRNA - transfected HUVECs . The graph shows relative levels of PlexinD1 - VEGFR2 association , in which the values at 0 min are set to 1 . n = 3 per group . Super - resolution images of receptor colocalization in siRNA - transfected HUVECs 30 min after Sema3E stimulation . Left panels show immunolabeling of VEGFR2 ( green ) , PlexinD1 ( red ) , and Nrp1 ( white ) , with light - blue asterisks indicating nuclei . Middle panels show magnified views of white boxes in the left panels . Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels . Scale bar , 10 µm . The graph shows the proportion of receptor complexes in PlexinD1 - positive vesicles . n = 10 cells per group . Trajectory plots ( left ) and rose plots ( right ) of HUVECs pre - treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h . Red trajectories indicate cells with displacement to the negative y - axis at the end of analyses . P values for the Rayleigh test represent non - random distributions of cell endpoints . The graphs show FMI along the y - and x - axes during cell migration . n = 90 cells per group .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O B-PANEL_START O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Co-IP from HUVECs. Blots for β-actin shown in (A) were used to confirm equal loading in (B), as the same cell lysates were analyzed in both experiments.Immunoblots in HUVECs.Immunoblots in siRNA-transfected HUVECs.Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light-blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in PlexinD1-positive vesicles. n = 10 cells per group.Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.


Figure 5Super - resolution images of receptor colocalization in siRNA - transfected HUVECs 30 min after VEGF stimulation . VEGFR2 ( green ) , PlexinD1 ( red ) , and Nrp1 ( white ) are shown as in Fig 4E . Scale bar , 10 µm . The graph shows the proportion of receptor complexes in the PlexinD1 - positive vesicles . Data of " no stimuli " are the same from Fig 4E . n = 10 cells per group . Co - IP from siRNA - transfected HUVECs . The graph shows relative levels of PlexinD1 - VEGFR2 association , in which the values at 0 min are set to 1 . n = 3 per group . Immunoblots in siRNA - transfected HUVECs . Blots for β - actin shown in ( C ) were used to confirm equal loading in ( D ) , as the same cell lysates were analyzed in these experiments . The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min . n = 3 per group . Immunoblots in siRNA - transfected HUVECs . Note the early and transient activation of PLCγ , Erk1 / 2 , and Akt , but not p38 MAPK , in RhoJ - knockdown ECs . Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ - expressing vectors . The graph shows protein levels of VEGFR2 relative to those of the control set as 100 % . n = 3 per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after VEGF stimulation. VEGFR2 (green), PlexinD1 (red), and Nrp1 (white) are shown as in Fig 4E. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in the PlexinD1-positive vesicles. Data of "no stimuli" are the same from Fig 4E. n = 10 cells per group.Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.Immunoblots in siRNA-transfected HUVECs. Blots for β-actin shown in (C) were used to confirm equal loading in (D), as the same cell lysates were analyzed in these experiments. The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min. n = 3 per group.Immunoblots in siRNA-transfected HUVECs. Note the early and transient activation of PLCγ, Erk1/2, and Akt, but not p38 MAPK, in RhoJ-knockdown ECs.Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ-expressing vectors. The graph shows protein levels of VEGFR2 relative to those of the control set as 100%. n = 3 per group.


Figure 6Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model . Upper panels are snapshots of time - lapse imaging in which EC nuclei are labeled by SYTO dye ( green ) . In analyzed regions framed by dotted lines , the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan . Initial discrete labeling becomes intermixed over time ( see also Movie EV1 ) . Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation . Scale bar , 100 µQuantification of the kinetics of EC migration . Values of forward and reverse migration for RhojWT / WT are normalized to 1 . n = 33 vessel branches per group . Quantification of vessel elongation ( left graph ) . n = 33 vessel branches per group . Scatter plot shows relationship between " vessel elongation " and " reverse migration " in RhojGFP / GFP aortic rings as evaluated by a Pearson correlation coefficient . Labeling for YFP / GFP ( green ) , CD31 ( red ) , and ETS transcription factor ERG ( blue ) in retinal vascular fronts of P5 CAG - MerCreMer : R26R - EYFPflox / WT and CAG - MerCreMer : Rhojfloxflox mice after intraperitoneal ( i . p . ) injections of 10 µg of 4 - hydroxytamoxifen ( 4OHT ) at P1 . Scale bar , 50 µm . Relative contribution of YFP / GFP - positive ECs to the tip positions . n = 16 per group . Values for CAG - MerCreMer : R26R - EYPflox / WT are normalized to 1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µQuantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group.Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient.Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.


Figure 7Labeling for GFP ( green ) and CD31 ( red ) in retina of P4 RhojGFP / GFP mouse . Scale bar , 200 µm ( left ) ; 20 µm ( right ) . Labeling for EdU ( green ) , ERG ( red ) , and CD31 ( gray ) in retinas of P4 RhojWT / WT and RhojGFP / GFP mice 2 h after i . p . EdU injections . Scale bar , 20 µm . The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts . n = 12 per group . Labeling for CD31 in retinas of P4 and P7 RhojWT / WT and RhojGFP / GFP mice . Scale bars , 500 µm . Morphometric analyses of retinal vessels in RhojWT / WT ( P4 , n = 18 ; P7 , n = 8 ) and RhojGFP / GFP ( P4 , n = 16 ; P7 , n = 11 ) mice . Labeling for CD31 in retinas of P4 Rhojflox / flox and Pdgfb - iCreERT2 : Rhojflox / flox ( RhojiΔEC ) mice after single i . p . injection of 100 µg of 4OHT at P1 . Scale bar , 500 µm . Morphometric analyses of retinal vessels in P4 Rhojflox / flox ( n = 7 ) and Rhoji ∆ EC ( n = 6 ) mice . Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas . n = 3 per group . Labeling for GFP ( green ) and CD31 ( red ) in retina of P18 OIR - RhojGFP / WT mouse . Scale bar , 200 µLabeling for CD31 in retinas of P18 OIR - Rhojflox / flox and OIR - Rhoji ∆ EC mice after daily i . p . injection of 200 µg of 4OHT from P12 . Scale bar , 500 µm . Quantification of areas of neovascular tufts in P18 OIR - Rhojflox / flox and OIR - Rhoji ∆ EC mice . n = 6 per group .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Labeling for GFP (green) and CD31 (red) in retina of P4 RhojGFP/GFP mouse. Scale bar, 200 µm (left); 20 µm (right).Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.Morphometric analyses of retinal vessels in RhojWT/WT (P4, n = 18; P7, n = 8) and RhojGFP/GFP (P4, n = 16; P7, n = 11) mice.Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.Morphometric analyses of retinal vessels in P4 Rhojflox/flox (n = 7) and Rhoji∆EC (n = 6) mice.Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas. n = 3 per group.Labeling for GFP (green) and CD31 (red) in retina of P18 OIR-RhojGFP/WT mouse. Scale bar, 200 µLabeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.


Figure 2A - D LSM images of proximity ligation assay ( PLA ) dots composed of VEGFR3 and phosphorylated tyrosine ( p - Tyr ) on cross - sections through the jugular lymph sac / primordial thoracic duct ( jls / pTD ) of E13 . 5 control and ILK K . O . embryos . Scale bars : 10 µm . E - H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine ( p - Tyr ) on cross - sections through the jls / pTD of E13 . 5 control and ILK K . O . embryos . Scale bars : 10 µm . I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . embryos ( n = 9 embryos per genotype ) , * P = 0 . 022 . J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . embryos ( n = 3 embryos per genotype ) . K Quantification of the total number of PLA dots indicating both VEGFR2 ( left ) and VEGFR3 ( right ) with phosphorylated tyrosine ( p - Tyr ) per jls / pTD section of E13 . 5 control or ILK K . O . embryos ( n = 3 embryos per genotype ) , * P = 0 . 005 ( VEGFR2 / p - Tyr in ILK K . O . versus VEGFR3 / p - Tyr in ILK K . O . ) , * P = 0 . 013 ( VEGFR3 / p - Tyr in control versus VEGFR3 / p - Tyr in ILK K . O . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D LSM images of proximity ligation assay (PLA) dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. E-H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 9 embryos per genotype), *P = 0.022. J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype). K Quantification of the total number of PLA dots indicating both VEGFR2 (left) and VEGFR3 (right) with phosphorylated tyrosine (p-Tyr) per jls/pTD section of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype), *P = 0.005 (VEGFR2/p-Tyr in ILK K.O. versus VEGFR3/p-Tyr in ILK K.O.), *P = 0.013 (VEGFR3/p-Tyr in control versus VEGFR3/p-Tyr in ILK K.O.)


Figure 3LSM images of cross - sections through the jugular lymph sac / primordial thoracic duct ( jls / pTD ) of an E13 . 5 control embryo stained for surface VEGFR3 and β1 integrin . Scale bar : 10 µmLSM images of proximity ligation assay ( PLA ) dots ( arrows ) composed of VEGFR3 and β1 integrin on cross - sections through the jls / pTD of E13 . 5 control and ILK K . O . embryos . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots normalised to Lyve1 - positive area of control or ILK K . O . embryos ( n = 6 embryos per genotype ) ,

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3LSM images of cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 µmLSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1-positive area of control or ILK K.O. embryos (n = 6 embryos per genotype),


Figure 4Bright - field image of an E13 . 5 Flk1 - Cre ; Ilk ∆ / + ; Itgb1 ∆ / + mouse embryo ( referred to as " Control " ) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells , and a LSM image of a stained cross - section through its jugular lymph sac / primordial thoracic duct ( jls / pTD ) . Scale bars : 500 and 100 µm , respectively . C , D Bright - field image of an E13 . 5 Flk1 - Cre ; Ilk ∆ / ∆ ; Itgb1 ∆ / + embryo ( referred to as " ILK & β1 integrin K . O . " ) with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells , and a LSM image of a stained cross - section through its jls / pTD . Scale bars : 500 and 100 µm , respectively . E - H LSM images of cross - sections through the jls / pTD of E13 . 5 control and ILK & β1 integrin K . O . embryos stained for the proliferation marker phospho - Histone H3 . Arrows point to phospho - Histone H3 - positive LECLSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine ( p - Tyr ) on stained cross - sections through the jls / pTD of control and ILK & β1 integrin K . O . embryos . Arrows point to PLA dots within the Lyve1 stained area . Scale bars : 10 µmNumber of LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O . embryosLEC proliferation as determined by the number of phospho - Histone H3 - positive LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O . embryosQuantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK & β1 integrin K . O .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Bright-field image of an E13.5 Flk1-Cre;Ilk∆/+;Itgb1∆/+ mouse embryo (referred to as "Control") with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jugular lymph sac / primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 µm, respectively. C, D Bright-field image of an E13.5 Flk1-Cre;Ilk∆/∆;Itgb1∆/+ embryo (referred to as "ILK & β1 integrin K.O.") with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jls/pTD. Scale bars: 500 and 100 µm, respectively. E-H LSM images of cross-sections through the jls/pTD of E13.5 control and ILK & β1 integrin K.O. embryos stained for the proliferation marker phospho-Histone H3. Arrows point to phospho-Histone H3-positive LECLSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µmNumber of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryosLEC proliferation as determined by the number of phospho-Histone H3-positive LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryosQuantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos


Figure 5LSM images of a whole mount stained adult Prox1 - CreERT2 ; Ilk + / + mouse ear ( referred to as " Adult Control " ) and Prox1 - CreERT2 ; Ilk ∆ / ∆ mouse ear ( referred to as " Adult ILK K . O . " ) , with higher magnification images indicated by dashed lines . Scale bars : 500 and LSM images of a whole - mount stained adult control and ILK K . O . ear , with higher magnification images indicated by dashed lines . Arrows point to proliferating LECs . Scale bars : 100 and 20 µm , respectivelyDermal lymph vessel density as determined by VEGFR3 - positive area normalised to the total analysed area of adult control or ILK K . O . mice ( n = 4 control and n = 5 ILK K . O . mice ) , LEC proliferation as determined by phospho - Histone H3 - positive LECs normalised to analysed VEGFR3 - positive area in the skin of adult control or ILK K . O . mice ( with n = 7 control and n = 5 ILK K . O . mice ) , VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K . O . mice ( n = 3 mice per genotype ) LSM images of a whole mount stained adult control and ILK K . O . cornea , with detailed images on their right ( L " , M " ) . Arrows point to lymph vessels protruding into the cornea ( encircled by a dotted line ) . Scale bars : 500 µm and 100 µm , re Number of lymph vessels in control or ILK K . O . corneas ( n = 5 and n = 7 corneas ) ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5LSM images of a whole mount stained adult Prox1-CreERT2;Ilk+/+ mouse ear (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse ear (referred to as "Adult ILK K.O."), with higher magnification images indicated by dashed lines. Scale bars: 500 and LSM images of a whole-mount stained adult control and ILK K.O. ear, with higher magnification images indicated by dashed lines. Arrows point to proliferating LECs. Scale bars: 100 and 20 µm, respectivelyDermal lymph vessel density as determined by VEGFR3-positive area normalised to the total analysed area of adult control or ILK K.O. mice (n = 4 control and n = 5 ILK K.O. mice),LEC proliferation as determined by phospho-Histone H3-positive LECs normalised to analysed VEGFR3-positive area in the skin of adult control or ILK K.O. mice (with n = 7 control and n = 5 ILK K.O. mice),VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype)LSM images of a whole mount stained adult control and ILK K.O. cornea, with detailed images on their right (L", M"). Arrows point to lymph vessels protruding into the cornea (encircled by a dotted line). Scale bars: 500 µm and 100 µm, re Number of lymph vessels in control or ILK K.O. corneas (n = 5 and n = 7 corneas),


Figure 6LSM images of cross - sections through the heart of an adult Prox1 - CreERT2 ; Ilk + / + mouse ( referred to as " Adult Control " ) and Prox1 - CreERT2 ; Ilk ∆ / ∆ mouse ( referred to as " Adult ILK K . O . " ) showing the outer lateral region of the left ventricle ( LV ) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1 - positive and CD31 - positive area ( normalised to total analysed myocardial area ) , respectively ( n = 4 control and n = 5 ILK K . O . mice ) LSM images of cross - sections through the heart of an adult control and ILK K . O . mouse four weeks after myocardial ischemia and reperfusion ( MI / R ) , showing the outer lateral region of the LV . Scale bars : 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1 - positive and CD31 - positive area ( normalised to total analysed myocardial area ) , respectively . ( n = 5 control and n = 7 ILK K . O . mice ) , P = 0 . 051 ( cardiac lymph vessel density in adult control versus adult ILK K . O . ) , * P = 0 . 025 ( cardiac blood vessel density in adult control versus adult ILK K . O . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6LSM images of cross-sections through the heart of an adult Prox1-CreERT2;Ilk+/+ mouse (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse (referred to as "Adult ILK K.O.") showing the outer lateral region of the left ventricle (LV) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively (n = 4 control and n = 5 ILK K.O. mice)LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.)


Figure 7Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA . Scale bars : 50 µmLEC proliferation as determined by the number of BrdU - positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF - C Cys156Ser ( n = 3 independent transfections per siRNA ) , * P = 0 . 032 ( control versus ILK - 1 ) , * P = 0 . 005 ( control versus ILK - 2 ) , * P = 0 . 0003 ( control versus ILK - 3 ) VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF - C Cys156Ser ( n = 4 ( control siRNA , ILK - 1 siRNA , and ILK - 3 siRNA ) , or n = 8 ( ILK - 2 siRNA ) independent transfections per siRNA ) , * P = 0 . 0001 ( control versus each siRNA ) LSM images of VEGFR3 / β1 integrin PLA dots in human LECs transfected with control or ILK siRNA . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA ) , P = 0 . 234 ( control versus ILK - 1 ) , * P = 0 . 024 ( control versus ILK - 2 ) , * P = 0 . 001 ( control versus ILK - 3 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µmLEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3)VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA)LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)


Figure 8LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes . Red dots are PLA dots composed of VEGFR3 and β1 integrin . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots per human LEC with ( + ) or without ( - ) mechanical stretch ( n = 6 independent stretch chambers ) , Western blot ( WB ) image of human LECs that were either kept unstretched or stretched for 30 minutes , and used for immunoprecipitation ( IP ) of HA - tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with ( + ) or without ( - ) mechanical stretch ; normalised to the respective amount of HA - tagged β1 integrin ( n = 3 ( unstretched ) or n = 5 ( stretched ) independent stretch

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (-) mechanical stretch (n = 6 independent stretch chambers),Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers


Figure 1C , D Two types of TNTs were detected in H1299 ( C ) and A549 ( D ) cells , Type 1 , containing F - actin with little or no detectable microtubules ( white box ) and Type2 , containing both microtubules and F - actin ( yellow box ) . Anti - β - tubulin antibody ( green ) and phalloidin ( red ) were used for fluorescent staining . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . Boxed areas are enlarged and shown in panels on the right .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.


Figure 2C , D TNT formation ( marked by black triangles and white arrowheads ) was imaged in control and shM2 H1299 ( C ) and shM2 A549 ( D ) cells by confocal microscopy . Scale bar : 60 μm . Boxed areas are enlarged and shown at the bottom of the corresponding images . E , F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 ( E ) and shM2 A549 ( F ) cells . 13 images per group ( E ) and 10 per group ( F ) were captured for quantification . Three independent experiments were conducted . G Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were performedH , I H1299 cells stably expressing the Ctr or GFP - M2 were cultured in the presence of 20 μM 2 - DG for 24 h to induce TNT formation . TNTs were imaged by confocal microscopy ( A ) and the percentage of cells forming TNTs was quantified ( B ) . n = 21 , 20 and 19 images were captured from Ctr , GFP - M2 # 1 and GFP - M2 # 2 , respectively . Three independent experiments were performed . Scale bar : 60 μm . Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C, D TNT formation (marked by black triangles and white arrowheads) was imaged in control and shM2 H1299 (C) and shM2 A549 (D) cells by confocal microscopy. Scale bar: 60 μm. Boxed areas are enlarged and shown at the bottom of the corresponding images. E, F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 (E) and shM2 A549 (F) cells. 13 images per group (E) and 10 per group (F) were captured for quantification. Three independent experiments were conducted. G Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performedH,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm. Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


Figure 3A Time - lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito - GFP were connected by TNTs containing mitochondria . Cells were seeded in collagen - coated dishes and cultured for 24 h before time - lapse life cell images were acquired by confocal microscopy ( also see Movies EV1 and EV2 ) . Fluorescent images ( with bright field ) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells . Scale bar : 20 μm . The moving mitochondria are marked by the white arrows . Boxed areas ( shCtr in yellow dotted boxes ; shM2 in red dotted boxes ) are enlarged and shown in the middle panel . B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . The white arrows marked TNTs , whereas the white triangles marked mitochondria inside TNTs . Scale bar : 25 μm . C Data information : nuclei were stained with Hoechst 33342 ( blue ) . C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells . The percentage of TNTs containing mitochondria was determined . n = 168 , 187 and 192 for shCtr , shM2 # 1 and shM2 # 2 groups , respectively ; three independent experiments . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or fE Representative flow cytometry plot of shCtr and shM2 cells following co - culture for 2 days . Experiments were conducted under three conditions : co - cultured in direct contact in normal glucose medium or in galactose medium or co - cultured through a filter . F The percentage of mCherry - expressing recipient cells containing Mito - GFP was quantified . Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or fewer recipient cells contained Mito - GFP , and this group is not included in the graph to avoid an overcrowded figure . Three independent experiments were conducted . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue).C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fE Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter.F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


Figure 4A Down - regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton . Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . Scale bar : 25 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . C Down - regulating MICAL2PV enhances the mitochondrial localization of Miro1 , Miro2 and KIF5B . Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting . The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β - tubulin protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 μm. Data information: nuclei were stained with Hoechst 33342 (blue).C Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1, Miro2 and KIF5B. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β-tubulin protein.


Figure 5E MICAL2PV interacts with the C - terminal domain of Miro2 containing the transmembrane region . H1299 cells were co - transfected with plasmids encoding Flag - MICAL2PV and Myc - tagged Miro2 mutants as indicated . Following transfection , cells were lysed for immunoprecipitation using the anti - Flag antibody and Western blotting . Please note the Myc - Miro2 ( 410 - 618 ) protein band in lane 4 migrated as a faint band , slightly faster than the IgG light chain . F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration . H1299 cells were co - transfected with Myc - tagged Miro2 and GFP - tagged MCAL2PV ( GFP - M2 ) or control vector . Following transfection , cells were fixed and stained with anti - Tom20 ( red ) and anti - Myc antibodies ( magenta ) . Confocal microscopic images were taken under the same objective and the same microscope settings . In cells co - expressing the GFP vector and Myc - Miro2 ( upper panels ) , most of Miro2 signals ( magenta ) are co - colocalized with mitochondria ( red Tom20 signals ) . However , a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co - expressing GFP - M2 and Myc - Miro2 ( marked by the yellow arrowheads ) . Mitochondria in cells expressing GFP - M2 show the perinuclear distribution pattern , whereas mitochondria in cells transfected by Myc - Miro2 without GFP - M2 ( marked by the yellow stars ) exhibit an infiltration pattern into the cortical cytoplasm . Scale bar : 20 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E MICAL2PV interacts with the C-terminal domain of Miro2 containing the transmembrane region. H1299 cells were co-transfected with plasmids encoding Flag-MICAL2PV and Myc-tagged Miro2 mutants as indicated. Following transfection, cells were lysed for immunoprecipitation using the anti-Flag antibody and Western blotting. Please note the Myc-Miro2 (410-618) protein band in lane 4 migrated as a faint band, slightly faster than the IgG light chain.F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration. H1299 cells were co-transfected with Myc-tagged Miro2 and GFP-tagged MCAL2PV (GFP-M2) or control vector. Following transfection, cells were fixed and stained with anti-Tom20 (red) and anti-Myc antibodies (magenta). Confocal microscopic images were taken under the same objective and the same microscope settings. In cells co-expressing the GFP vector and Myc-Miro2 (upper panels), most of Miro2 signals (magenta) are co-colocalized with mitochondria (red Tom20 signals). However, a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co-expressing GFP-M2 and Myc-Miro2 (marked by the yellow arrowheads). Mitochondria in cells expressing GFP-M2 show the perinuclear distribution pattern, whereas mitochondria in cells transfected by Myc-Miro2 without GFP-M2 (marked by the yellow stars) exhibit an infiltration pattern into the cortical cytoplasm. Scale bar: 20 μm.


Figure 6A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . Following transfection , cells were stained with anti - Tom20 antibody ( green ) and phalloidin ( red ) . Confocal microscopic images are shown . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . F Down - regulating Miro2 inhibits mitochondrial transport in shM2 cells . Mito - GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . These cells were then co - cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito - GFP was quantified . Twenty thousand events were analyzed for each condition Data information : Data were analyzed using two - way ANOVA with Bonferroni post - test and presented as mean ± SEM ; * P < 0 . 05 , * * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant . Three independent experiments were conducted .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).F Down-regulating Miro2 inhibits mitochondrial transport in shM2 cells. Mito-GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. These cells were then co-cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito-GFP was quantified. Twenty thousand events were analyzed for each condition Data information: Data were analyzed using two-way ANOVA with Bonferroni post-test and presented as mean ± SEM; * P < 0.05, *** P < 0.01, *** P < 0.001, ns, not significant. Three independent experiments were conducted.


Figure 7A Stable H1299 cells ( shCtr or shM2 ) were treated by 5 - FU ( 50 μM , 24 h ) and then cultured alone or co - cultured with non - drug treated vehicle - treated cells stably expressing mito - mCherry in the absence or presence of Cyto B ( 500 nM , 24 h ) . The percentage of dead cells in each group was quantified . Three independent experiments were conducted . Data information : data were analyzed using two - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant . D Confocal microscopic images of TNTs in each group , white arrowheads and yellow triangle in enlarged views ( zoom - in panels , zoom ) marked the TNTs formed between non - drug treated ( mCherry positive ) and drug - treated cells . Scale bar : 25 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.D Confocal microscopic images of TNTs in each group, white arrowheads and yellow triangle in enlarged views (zoom-in panels, zoom) marked the TNTs formed between non-drug treated (mCherry positive) and drug-treated cells. Scale bar: 25 μm.


Figure 8D MICAL2PV mutants containing the MO domain are active in depolymerizing F - actin . Following transfection of plasmids expressing different HA - MICAL2PV mutants ( # 1 , # 2 , or # 4 ) into shM2 cells , confocal microscopy was performed following staining with phalloidin ( red ) and anti - HA antibody ( green ) . F - actin signals were reduced in cells expressing different mutants ( HA - positive green cells marked by dashed lines ) . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . G , H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation . Stable shM2 H1299 ( G ) or A549 ( H ) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site ( MOmut : amino acid residues 93 - 98 GxGxxG mutated to WxWxxW ) or the control vector ( Ctr ) . Following transfection , Western blotting and quantification of TNT formation were carried out as in panel E . 10 images were captured for quantification in each group . Three independent experiments were performed . Data information : Data were analyzed using a one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green). F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).G, H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation. Stable shM2 H1299 (G) or A549 (H) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site (MOmut: amino acid residues 93-98 GxGxxG mutated to WxWxxW) or the control vector (Ctr). Following transfection, Western blotting and quantification of TNT formation were carried out as in panel E. 10 images were captured for quantification in each group. Three independent experiments were performed. Data information: Data were analyzed using a one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


Figure 1 ( A ) Quantification of the amount of LC3 ‐ II protein in mouse Rab ‐ knockdown cells . Representative blots are shown in supplementary Fig S1C online . Error bars represent the means and s . e . m . of three independent experiments . ( B ) Quantification of the amount of p62 protein in candidates Rab ‐ knockdown cells . Representative blots are shown in supplementary Fig S1D online . Error bars represent the means and s . e . m . of three independent experiments . ( C , D ) Control and Rab12 ‐ knockdown MEFs were cultured under N or S conditions , fixed and then immunostained with the antibodies indicated . Representative images are shown in supplementary Fig S2 online . The mean numbers of LC3 ‐ positive ( C ) or Atg16L1 ‐ positive ( D ) dots per cell are shown . Error bars represent the means and s . e . m . of representative data ( n ≥ 80 ) from three independent experiments . ( E ) MEFs that had been transfected with the control siRNA or Rab12 siRNA were cultured as in ( C , D ) , and their lysates were analysed by immunoblotting with the antibodies indicated . ( F ) Quantification of ( E ) . ( G ) Control and Rab12 ‐ knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h . Cell lysates were analysed by immunoblotting with the antibodies indicated , and the intensity of the LC3 ‐ II band was quantified . The normalized amount ( arbitrary units ) of LC3 ‐ II in lanes 1 , 2 , 3 , 4 , 5 and 6 is 1 . 0 , 2 . 3 , 4 . 3 , 0 . 9 , 1 . 6 and 3 . 8 , respectively . ( H ) MEFs transiently expressing mStr ‐ Rab12 were cultured under starved conditions and immunostained with the antibodies indicated . Scale bar , 20 μm . MEFs , mouse embryonic fibroblasts ; N , nutrient ‐ rich ; S , starved ; siRNA , short interfering RNA . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Quantification of the amount of LC3‐II protein in mouse Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1C online. Error bars represent the means and s.e.m. of three independent experiments.(B) Quantification of the amount of p62 protein in candidates Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1D online. Error bars represent the means and s.e.m. of three independent experiments.(C,D) Control and Rab12‐knockdown MEFs were cultured under N or S conditions, fixed and then immunostained with the antibodies indicated. Representative images are shown in supplementary Fig S2 online. The mean numbers of LC3‐positive (C) or Atg16L1‐positive (D) dots per cell are shown. Error bars represent the means and s.e.m. of representative data (n≥80) from three independent experiments.(E) MEFs that had been transfected with the control siRNA or Rab12 siRNA were cultured as in (C,D), and their lysates were analysed by immunoblotting with the antibodies indicated.(F) Quantification of (E).(G) Control and Rab12‐knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h. Cell lysates were analysed by immunoblotting with the antibodies indicated, and the intensity of the LC3‐II band was quantified. The normalized amount (arbitrary units) of LC3‐II in lanes 1, 2, 3, 4, 5 and 6 is 1.0, 2.3, 4.3, 0.9, 1.6 and 3.8, respectively.(H) MEFs transiently expressing mStr‐Rab12 were cultured under starved conditions and immunostained with the antibodies indicated. Scale bar, 20 μm. MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01; ***P0.005.


Figure 2 ( A ) Control and Rab12 ‐ knockdown MEFs were cultured under nutrient ‐ rich or starved conditions , fixed and then immunostained with the antibodies indicated . Scale bar , 20 μm . ( B ) Control and Rab12 ‐ knockdown MEFs were cultured as in ( A ) , and their lysates were analysed by immunoblotting with the antibodies indicated . ( C ) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated . ( D , E ) Quantification of the phospho ‐ S6K levels and phospho ‐ Akt levels shown in ( C ) . MEFs , mouse embryonic fibroblasts ; N , nutrient ‐ rich ; S , starved ; siRNA , short interfering RNA . * P0 . 05 ; * * P0 . 01 .

O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Control and Rab12‐knockdown MEFs were cultured under nutrient‐rich or starved conditions, fixed and then immunostained with the antibodies indicated. Scale bar, 20 μm.(B) Control and Rab12‐knockdown MEFs were cultured as in (A), and their lysates were analysed by immunoblotting with the antibodies indicated.(C) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated.(D,E) Quantification of the phospho‐S6K levels and phospho‐Akt levels shown in (C). MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01.


Figure 3 ( A ) MEFs transiently co ‐ expressing Myc ‐ PAT4 and mStr ‐ Rab12 were immunostained with anti ‐ Myc antibody . ( B ) MEFs transiently expressing Myc ‐ PAT4 were immunostained with the antibodies indicated . Scale bars , 20 μm . ( C ) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA ‐ PAT4 were analysed by immunoblotting with the antibodies indicated . ( D ) Lysates of MEFs stably expressing HA ‐ PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated . ( E ) Quantification of ( D ) . * * * P0 . 005 . ( F ) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse ‐ transcription PCR analyses . Gapdh was used as an internal control . ( G ) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA ‐ PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated . Note that the amounts of plasma ‐ membrane ‐ localized PAT4 protein and TfR protein were higher in Rab12 ‐ knockdown MEFs , whereas the total amounts of EGFR protein and plasma membrane ‐ localized EGFR protein in these cells were slightly lower , but the mechanisms responsible for these changes are unknown . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; MEFs , mouse embryonic fibroblasts ; PAT4 , proton ‐ coupled amino ‐ acid transporter 4 ; siRNA , short interfering RNA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) MEFs transiently co‐expressing Myc‐PAT4 and mStr‐Rab12 were immunostained with anti‐Myc antibody.(B) MEFs transiently expressing Myc‐PAT4 were immunostained with the antibodies indicated. Scale bars, 20 μm.(C) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 were analysed by immunoblotting with the antibodies indicated.(D) Lysates of MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated.(E) Quantification of (D). ***P0.005.(F) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse‐transcription PCR analyses. Gapdh was used as an internal control.(G) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated. Note that the amounts of plasma‐membrane‐localized PAT4 protein and TfR protein were higher in Rab12‐knockdown MEFs, whereas the total amounts of EGFR protein and plasma membrane‐localized EGFR protein in these cells were slightly lower, but the mechanisms responsible for these changes are unknown. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MEFs, mouse embryonic fibroblasts; PAT4, proton‐coupled amino‐acid transporter 4; siRNA, short interfering RNA.


Figure 4 ( A ) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated . ( B ) Quantification of the phospho ‐ S6K levels shown in ( A ) . ( C ) MEFs transfected with the siRNAs indicated were cultured under nutrient ‐ rich ( N ) or starved ( S ) conditions and were immunostained with anti ‐ LC3 antibody . The mean numbers of LC3 ‐ positive dots per cell are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated.(B) Quantification of the phospho‐S6K levels shown in (A).(C) MEFs transfected with the siRNAs indicated were cultured under nutrient‐rich (N) or starved (S) conditions and were immunostained with anti‐LC3 antibody. The mean numbers of LC3‐positive dots per cell are shown.


Figure 1 . ( A - C ) 10X phase contrast images of vero monolayers at 3 days post - inoculation . Panels shown are ( A ) mock , ( B ) NP , and ( C ) OP . ( D ) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids , seen as black dots .

O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) NP, and (C) OP.(D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.


Figure 2 . A ) Two SARS - CoV - 2 passage 4 stocks ( black and gray ) were quantified utilizing plaque assay at day two ( closed circles ) and day three ( open circles ) post infection of Vero E6 and Vero CCL81 . B ) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation . C ) Cell monolayers two days post infection in Vero E6 ( top ) and Vero CCL81 ( bottom row ) at multiple dilutions .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81.B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation.C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.


Figure 3 . Viral titer for SARS - CoV - 2 was quantitated by plaque assay on Vero E6 cells two days post inoculation . Protein lysates were probed by western blot with B ) anti - SARS nucleocapsid ( N ) antibody or C ) anti - SARS - CoV spike ( S ) antibody .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation.Protein lysates were probed by western blot with B) anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody.


Figure 1The VPR and DPR ( right ) were dissected from E10 . 5 Pdx1 - GFP embryos ( left ) . Scale bar : 500 µm . DPR , dorsal Pdx1 - expressing region ; VPR , ventral Pdx1 - expressing region ; Duo , duodenum bud . FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR . The ratios of GFPhigh cells to GFP + cells in the VPR and DPR . The data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * * * p - value < 0 . 001 . Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters ( a - d ) of genes ( p < 1 × 10 - 9 , cell cycle - related genes are excluded ) . Each column is a single - cell , and each row represents one gene . Cluster - specific TFs are listed on the right . The genes in bold are known to have roles in pancreatic development . L , GFPlow ; H , GFPhigh .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The VPR and DPR (right) were dissected from E10.5 Pdx1-GFP embryos (left). Scale bar: 500 µm. DPR, dorsal Pdx1-expressing region; VPR, ventral Pdx1-expressing region; Duo, duodenum bud.FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR.The ratios of GFPhigh cells to GFP+ cells in the VPR and DPR. The data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters (a-d) of genes (p < 1 ×10-9, cell cycle-related genes are excluded). Each column is a single-cell, and each row represents one gene. Cluster-specific TFs are listed on the right. The genes in bold are known to have roles in pancreatic development. L, GFPlow; H, GFPhigh.


Figure 2Expression levels of group - specific genes in individual cells . The gene expression values were projected onto the PCA ( left ) and box ( right ) plots . Each point represents a cell . Points were colored based on the hierarchical clustering in Fig 1E . The y - axes of the box plots represent the expression levels ( RPKM ) . Central line in the box represents median ; box limits represent the first and third quartile ; whiskers represent 1 . 5 × interquartile range . Unpaired Wilcoxon rank - sum test . * p - value < 1 x 10 - 3 ; * * p - value < 1 x 10 - 6 ; * * * p - value < 1 x 10 - 9 . Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR . Statistical analysis of BrdU + in different cell populations . The Data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * p - value < 0 . 05 ; * * * p - value < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Expression levels of group-specific genes in individual cells. The gene expression values were projected onto the PCA (left) and box (right) plots. Each point represents a cell. Points were colored based on the hierarchical clustering in Fig 1E. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. *p-value < 1 x 10-3; **p-value <1 x 10-6; ***p-value <1 x 10-9.Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR. Statistical analysis of BrdU+ in different cell populations. The Data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. *p-value < 0.05; ***p-value < 0.001.


Figure 3Flow cytometric analysis of endocrine progenitors and β - cells in the ventral pancreases ( V . P . ) and dorsal pancreases ( D . P . ) . ( A , D and G ) Dissected ventral and dorsal pancreatic tissues . Scale bar : 500 µm . ( B , E and H ) FACS gating for sorting Ngn3 - GFP + or Ins1 - RFP + cells from the V . P . and D . P . ( C and F ) statistical analysis of percentages of Ngn3 - GFPhigh cells among Ngn3 - GFP + cells in the V . P . and D . P . ( I ) Statistical analysis of percentages of Ins1 - RFP + cells among total pancreatic cells in the V . P . and D . P . Differentially expressed genes among cells from VPR , DPR , the V . P . and D . P . at various developmental stages . The numbers indicate significantly highly expressed genes in the ventral ( blue ) or dorsal ( orange ) regions . The dotted lines represent the two - fold change threshold .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Flow cytometric analysis of endocrine progenitors and β-cells in the ventral pancreases (V. P.) and dorsal pancreases (D. P.). (A, D and G) Dissected ventral and dorsal pancreatic tissues. Scale bar: 500 µm. (B, E and H) FACS gating for sorting Ngn3-GFP+ or Ins1-RFP+ cells from the V. P. and D. P. (C and F) statistical analysis of percentages of Ngn3-GFPhigh cells among Ngn3-GFP+ cells in the V. P. and D. P. (I) Statistical analysis of percentages of Ins1-RFP+ cells among total pancreatic cells in the V. P. and D. P.Differentially expressed genes among cells from VPR, DPR, the V. P. and D. P. at various developmental stages. The numbers indicate significantly highly expressed genes in the ventral (blue) or dorsal (orange) regions. The dotted lines represent the two-fold change threshold.


Figure 4Flow cytometric analyses of Liv2 + cells in the VPR , DPR and the liver buds ( LB ) . Liv2 + cells from the VPR - L ( brown arrow ) , Liv2 - cells from the VPR - L ( yellow arrow ) and Liv2 + cells from the LB ( black arrow ) were sorted for single - cell RNA - seq . Immunofluorescence of PDX1 and Liv2 on sagittal section of E10 . 5 mouse embryo . The white and yellow arrows denote PDX1low / Liv2 + cells in in the liver and EHBD regions , respectively . Scale bar : 50 µm . Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages ( p < 1 × 10 - 6 ) . Each column represents a single - cell and each row represents one gene . Cells were re - ordered according to the value of PC1 or PC2 . Genes in clusters ' a , b ' and ' c , d ' were re - ordered by correlations with PC2 and PC1 , respectively . The color of the cell type and cell source are indicated in ( C ) . Lineage - specific TFs are listed on the right . The gene expression values of lineage marker genes were projected onto the PCA ( E ) and box ( F ) plots . Each point represents a cell . The y - axes of the box plots represent the expression levels ( RPKM ) . Central line in the box represents median ; box limits represent the first and third quartile ; whiskers represent 1 . 5 × interquartile range . Unpaired Wilcoxon rank - sum test . * * p - value < 1 x 10 - 5 ; * * * p - value < 1 x 10 - 10 . Pdx1 and Sox17 expression levels in E9 . 5 VPR and DPR cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O

Figure 4Flow cytometric analyses of Liv2+ cells in the VPR, DPR and the liver buds (LB). Liv2+ cells from the VPR-L (brown arrow), Liv2- cells from the VPR-L (yellow arrow) and Liv2+ cells from the LB (black arrow) were sorted for single-cell RNA-seq.Immunofluorescence of PDX1 and Liv2 on sagittal section of E10.5 mouse embryo. The white and yellow arrows denote PDX1low/Liv2+ cells in in the liver and EHBD regions, respectively. Scale bar: 50 µm.Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages (p < 1 × 10-6). Each column represents a single-cell and each row represents one gene. Cells were re-ordered according to the value of PC1 or PC2. Genes in clusters 'a, b' and 'c, d' were re-ordered by correlations with PC2 and PC1, respectively. The color of the cell type and cell source are indicated in (C). Lineage-specific TFs are listed on the right.The gene expression values of lineage marker genes were projected onto the PCA (E) and box (F) plots. Each point represents a cell. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. **p-value <1 x 10-5; ***p-value <1 x 10-10.Pdx1 and Sox17 expression levels in E9.5 VPR and DPR cells.


Figure 5Sagittal section of E12 . 5 Pdx1 - Cre ; Rosa26 - LacZ mouse embryo counterstained for ALB ( brown ) . X - gal staining ( blue ) showing cells derived from Pdx1 - expressing cells . The arrowheads indicate cells co - stained with X - gal and ALB ( See Fig EV5D for more images of different sections ) . Scale bar : 50 µm ( left ) or 20 µm ( right ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Sagittal section of E12.5 Pdx1-Cre; Rosa26-LacZ mouse embryo counterstained for ALB (brown). X-gal staining (blue) showing cells derived from Pdx1-expressing cells. The arrowheads indicate cells co-stained with X-gal and ALB (See Fig EV5D for more images of different sections). Scale bar: 50 µm (left) or 20 µm (right).


Figure 1F . BAZ2A read coverage quantification at ± 1Kb from peak summit of BAZ2A - bound regions identified in PC3 cells and the corresponding reads for BAZ2A - BRD mutant ( Y1775F ) in PC3 cells . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Data information : Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range . G . Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac . Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells ( BAZ2Awt and BAZ2A - BRD mutant ( Y1775F ) ) at ± 1Kb from BAZ2A peak summits . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Data information : Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1F. BAZ2A read coverage quantification at ±1Kb from peak summit of BAZ2A-bound regions identified in PC3 cells and the corresponding reads for BAZ2A-BRD mutant (Y1775F) in PC3 cells. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.G. Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac. Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells (BAZ2Awt and BAZ2A-BRD mutant (Y1775F)) at ±1Kb from BAZ2A peak summits. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


Figure 2B . EP300 acetylates H3K14 in PC3 cells . Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A - 485 ( 10 μM ) . Histone H3 serves as protein loading control . F . qRT - PCR showing increased expression levels of genes with BAZ2A - bound promoter and downregulated in metastatic tumors ( AOX1 , MANB2 , DMPK , and DDB1 in PC3 cells treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 , * * * * < 0 . 0001 ) . Error bars represent SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A-485 (10 μM). Histone H3 serves as protein loading control.F. qRT-PCR showing increased expression levels of genes with BAZ2A-bound promoter and downregulated in metastatic tumors (AOX1, MANB2, DMPK, and DDB1 in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, ****<0.0001). Error bars represent SD.


Figure 3B . Inactive enhancers ( C2 ) are enriched in H3K14ac and BAZ2A levels . Read coverage at enhancer classes C1 and C2 for BAZ2A , H3K14ac , H3K27ac , H3K4me1 and H3K27me3 at ± 1Kb from annotated enhancer regions . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range . H . qRT - PCR showing increased expression levels of BAZ2A - bound C2 - enhancer genes in PC3 cells treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 , * * < 0 . 001 ) . Error bars represent SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Inactive enhancers (C2) are enriched in H3K14ac and BAZ2A levels. Read coverage at enhancer classes C1 and C2 for BAZ2A, H3K14ac, H3K27ac, H3K4me1 and H3K27me3 at ±1Kb from annotated enhancer regions. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.H. qRT-PCR showing increased expression levels of BAZ2A-bound C2-enhancer genes in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, **<0.001). Error bars represent SD.


Figure 4C . qRT - PCR of genes differentially expressed in PC3 - CSCs compared to PC3 cells showing the upregulation of CSC markers ( ALDH1A2 , LGR5 , CD24 ) and downregulation of genes related to developmental processes ( HOXA1 , HOXA4 , SOX7 ) . Data are from two independent experiments . mRNA levels were normalized to GAPDH . D . BAZ2A is required for the dedifferentiation of PC3 into PC3 - CSCs . Left panel : Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A . Right panel : BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA - BAZ2A were measured by qRT - PCR and normalized to GAPDH mRNA from 2 independent experiments . E . Left panel : qRT - PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A - BRD mutant ( BAZ2AY1775F ) . Values were normalized to MRPS7 mRNA . Data are from 3 independent experiments . Right panel : Quantification of tumorspheres from 3 independent experiments . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 ; * * < 0 . 01 ) . Error bars represent SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C. qRT-PCR of genes differentially expressed in PC3-CSCs compared to PC3 cells showing the upregulation of CSC markers (ALDH1A2, LGR5, CD24) and downregulation of genes related to developmental processes (HOXA1, HOXA4, SOX7). Data are from two independent experiments. mRNA levels were normalized to GAPDH.D. BAZ2A is required for the dedifferentiation of PC3 into PC3-CSCs. Left panel: Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A. Right panel: BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA-BAZ2A were measured by qRT-PCR and normalized to GAPDH mRNA from 2 independent experiments.E. Left panel: qRT-PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A-BRD mutant (BAZ2AY1775F). Values were normalized to MRPS7 mRNA. Data are from 3 independent experiments. Right panel: Quantification of tumorspheres from 3 independent experiments. Statistical significance (P-values) was calculated using two-tailed t-test (* <0.05; **< 0.01). Error bars represent SD.