Figure 5Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Five - day - old seedlings were immunostained with anti - KNOLLE ( green ) and counterstained with DAPI ( blue ) . In the pi4kβ1 pi4kβ2 double mutant , diffusion of KNOLLE occurred at the plasma membrane ( arrowheads ) . Scale bars , 10 μm . Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane ( PM ) from ( A ) . Data are mean ± SD from four independent experiments . * * , a significant difference ( p < 0 . 01 ) according to a two - tailed Student ' s t test ( untreated wild type , n = 97 cells , 25 roots ; untreated pi4kβ1 pi4kβ2 , n = 142 cells , 44 roots ; BFA treated wild type , n = 126 cells , 28 roots ; BFA treated pi4kβ1 pi4kβ2 , n = 183 cells , 43 roots ) . Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were immunostained with anti - KNOLLE antibodies ( green ) and counterstained with DAPI ( blue ) . Scale bars , 10 μm . Quantification of KNOLLE intensity at the cell plate from ( C ) , normalized to the signal intensity of intracellular compartments . * * * , a significant difference ( p < 0 . 0001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 26 cells , 20 roots ; pi4kβ1 pi4kβ2 , n = 41 cells , 37 roots ) . Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Enhanced accumulation of PIN2 - GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were stained with 2 μM FM 4 - 64 for 3 minutes at room temperature . Scale bars , 10 μm . Quantification of PIN2 - GFP signal at the cell plate from ( E ) , normalized to the intensity at the apical plasma membrane intensity , as indicated by the dashed lines . * * * , a significant difference ( p < 0 . 001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 28 cells , 16 roots ; pi4kβ1 pi4kβ2 , n = 45 cells , 26 roots ) . Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . panels g - j display data from the analysis of interphase cells . Internalization of PIN2 - GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min , then washed and incubated with 50 μM CHX and 25 μM BFA . Scale bars , 10 μm . Quantification of punctate signals from ( G ) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants . Data are mean ± SD . * , a significant difference ( p < 0 . 05 ) according to a two - tailed Student ' s t test ; ( wild type , n = 116 cells , 6 roots ; pi4kβ1 pi4kβ2 , n = 110 cells , 7 roots ) . Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . panels g - j display data from the analysis of interphase cells . Delayed internalization of FM 4 - 64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant . Four - day - old seedlings were pulsed with 2 μM FM 4 - 64 for 3 minutes on ice and then fluorescence was recorded for 20 min . Scale bars , 10 μm . Quantification of intracellular FM 4 - 64 signal from ( i ) , normalized to apical and basal plasma membrane intensities . * * * , a significant difference ( p < 0 . 0001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 116 cells , 6 roots ; pi4kβ1 pi4kβ2 , n = 66 cells , 9 roots ) .

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 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 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 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 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

Figure 5Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Five-day-old seedlings were immunostained with anti-KNOLLE (green) and counterstained with DAPI (blue). In the pi4kβ1 pi4kβ2 double mutant, diffusion of KNOLLE occurred at the plasma membrane (arrowheads). Scale bars, 10 μm. Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane (PM) from (A). Data are mean ± SD from four independent experiments. **, a significant difference (p<0.01) according to a two-tailed Student's t test (untreated wild type, n= 97 cells, 25 roots; untreated pi4kβ1 pi4kβ2, n = 142 cells, 44 roots; BFA treated wild type, n = 126 cells, 28 roots; BFA treated pi4kβ1 pi4kβ2, n = 183 cells, 43 roots).Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were immunostained with anti-KNOLLE antibodies (green) and counterstained with DAPI (blue). Scale bars, 10 μm. Quantification of KNOLLE intensity at the cell plate from (C), normalized to the signal intensity of intracellular compartments. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 26 cells, 20 roots; pi4kβ1 pi4kβ2, n = 41 cells, 37 roots).Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of PIN2-GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were stained with 2 μM FM 4-64 for 3 minutes at room temperature. Scale bars, 10 μm. Quantification of PIN2-GFP signal at the cell plate from (E), normalized to the intensity at the apical plasma membrane intensity, as indicated by the dashed lines. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 28 cells, 16 roots; pi4kβ1 pi4kβ2, n = 45 cells, 26 roots).Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Internalization of PIN2-GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min, then washed and incubated with 50 μM CHX and 25 μM BFA. Scale bars, 10 μm. Quantification of punctate signals from (G) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants. Data are mean ± SD. *, a significant difference (p<0.05) according to a two-tailed Student's t test; (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 110 cells, 7 roots).Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Delayed internalization of FM 4-64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant. Four-day-old seedlings were pulsed with 2 μM FM 4-64 for 3 minutes on ice and then fluorescence was recorded for 20 min. Scale bars, 10 μm. Quantification of intracellular FM 4-64 signal from (i), normalized to apical and basal plasma membrane intensities. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 66 cells, 9 roots).


Figure 6pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Immunostaining of meristem cells of five - day - old plants using anti - tubulin ( green ) counterstained with DAPI ( false colour red ) . Asterisks , multiple nuclei in one cell ; arrowheads , multiple phragmoplasts in one cell . Dashed lines indicate the outlines of exemplary multinucleated cells . Scale bars , 10 µm . pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Whole - mount immunostaining of root tips of five - day - old seedlings . Green , anti - KNOLLE ; red , anti - tubulin ; blue , DAPI . ( І ) , ( ІІ ) , magnifications of areas highlighted in dashed boxes . Arrowheads , solid phragmoplasts with thicker cell plates . Scale bars , 20 µm . pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Quantification of the incidences of multinucleated cells , multiple phragmoplasts and solid phragmoplasts during late cytokinesis ( multinucleated cells : wild type , n = 316 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 673 cells , 44 roots ; multiple phragmoplasts : wild type , n = 64 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 188 cells , 44 roots ; solid phragmoplast in late cytokinesis : wild type , n = 41 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 89 cells , 44 roots ) . Data are mean ± SD from three independent experiments . n . d . not detected .

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 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 6pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Immunostaining of meristem cells of five-day-old plants using anti-tubulin (green) counterstained with DAPI (false colour red). Asterisks, multiple nuclei in one cell; arrowheads, multiple phragmoplasts in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Whole-mount immunostaining of root tips of five-day-old seedlings. Green, anti-KNOLLE; red, anti-tubulin; blue, DAPI. (І), (ІІ), magnifications of areas highlighted in dashed boxes. Arrowheads, solid phragmoplasts with thicker cell plates. Scale bars, 20 µm.pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Quantification of the incidences of multinucleated cells, multiple phragmoplasts and solid phragmoplasts during late cytokinesis (multinucleated cells: wild type, n = 316 cells, 29 roots; pi4kβ1 pi4kβ2, n = 673 cells, 44 roots; multiple phragmoplasts: wild type, n = 64 cells, 29 roots; pi4kβ1 pi4kβ2, n = 188 cells, 44 roots; solid phragmoplast in late cytokinesis: wild type, n = 41 cells, 29 roots; pi4kβ1 pi4kβ2, n = 89 cells, 44 roots). Data are mean ± SD from three independent experiments. n.d. not detected.


Figure 7The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging . Five - day - old seedlings expressing mCherry - TUA5 were imaged at 1 frame per 2 min . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Cells are outlined by dashed lines in the first and last frames of each series . White arrowhead , ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant . Scale bars , 10 µm . Plot profiles obtained from dashed lines marked in ( A ) . Black arrowhead , ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant . The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging . 3D projections from time points selected from ( A ) , when the transition to a ring phragmoplast has occurred in wild type , but solid phragmoplast persisted in the double mutant . Scale bars , 10 µm . Duration of phragmoplast persistence during cytokinesis , determined from initiation to disbanding . * * * , a significant difference ( p < 0 . 001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 12 cells , 9 roots ; pi4kβ1 pi4kβ2 , n = 21 cells , 16 roots ) .

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 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

Figure 7The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging. Five-day-old seedlings expressing mCherry-TUA5 were imaged at 1 frame per 2 min. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Cells are outlined by dashed lines in the first and last frames of each series. White arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. Scale bars, 10 µm. Plot profiles obtained from dashed lines marked in (A). Black arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant.The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging. 3D projections from time points selected from (A), when the transition to a ring phragmoplast has occurred in wild type, but solid phragmoplast persisted in the double mutant. Scale bars, 10 µm. Duration of phragmoplast persistence during cytokinesis, determined from initiation to disbanding. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 12 cells, 9 roots; pi4kβ1 pi4kβ2, n = 21 cells, 16 roots).


Figure 8The dynamics of the microtubule bundling protein , MAP65 - 3 , were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants . Image series of 3D projections of GFP - MAP65 - 3 costained with FM 4 - 64 in root tips of four - day - old wild type seedlings . The best angle for visualization was obtained by rotation of the x - axis . Images are representative for 8 cells from 5 roots . Scale bar , 10 µm . Image series of 3D projections of GFP - MAP65 - 3 costained with FM 4 - 64 in root tips of four - day - old pi4kβ1 pi4kβ2 seedlings . The best angle for visualization was obtained by rotation of the x - axis . Images are representative for 11 cells from 10 roots . Arrowheads , persisting signal of GFP - MAP65 - 3 in the center of the cell division plane . Scale bars , 10 µm . Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts , marked by dashed lines in ( A ) and ( B ) . Arrowhead , persisting signal of GFP - MAP65 - 3 in the center of the cell division plane . Times in ( A - C ) are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Medial confocal planes from z - stacks from ( a ) and ( b ) demonstrating attachment of the FM 4 - 64 - stained cell plate with the peripheral plasma membrane , defining t0 . Scale bars , 10 µm . 3D reconstruction of ring and disc - shaped immunofluorescence patterns for GFP - MAP65 - 3 and microtubules , based on super resolution structured illumination microscopy ( SIM ) , costained with DAPI . Top panels , wild type ; bottom panels , pi4kβ1 pi4kβ2 double mutants . Scale bars , 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 8The dynamics of the microtubule bundling protein, MAP65-3, were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old wild type seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 8 cells from 5 roots. Scale bar, 10 µm. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old pi4kβ1 pi4kβ2 seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 11 cells from 10 roots. Arrowheads, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Scale bars, 10 µm. Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts, marked by dashed lines in (A) and (B). Arrowhead, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Times in (A-C) are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0.Medial confocal planes from z-stacks from (a) and (b) demonstrating attachment of the FM 4-64-stained cell plate with the peripheral plasma membrane, defining t0. Scale bars, 10 µm.3D reconstruction of ring and disc-shaped immunofluorescence patterns for GFP-MAP65-3 and microtubules, based on super resolution structured illumination microscopy (SIM), costained with DAPI. Top panels, wild type; bottom panels, pi4kβ1 pi4kβ2 double mutants. Scale bars, 10 µm.


Figure 9The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants . Crosses of pi4kβ1 pi4kβ2 and mpk4 - 2 mutants resulted in combined genotypes with increased growth defects , as indicated . 14 - day - old seedlings are shown . Scale bars , 1 cm . Top , The severe growth defects of the combined genotypes were accompanied by increased tissue distortion , as shown by 3D projections after propidium iodide staining of root tips of 14 - day - old seedlings . Scale bars , 100 µm . Bottom , Multinucleation of cells was increased in the combined genotypes , as evident from immunofluorescence of microtubules ( red ) counterstained with DAPI ( blue ) . Asterisks , multiple nuclei in one cell . Dashed lines indicate the outlines of exemplary multinucleated cells . Scale bars , 10 µm . Quantification of cytokinetic defects , as shownin the bottom panels of ( B ) . Cytokinetic defects were enhanced in pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) ( for multinucleated cells , including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells , both according to a Welch ANOVA with Games - Howell post - hoc test ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ( wild type , n = 1650 cells , 13 roots ; pi4kβ1 pi4kβ2 , n = 1441 cells , 19 roots ; mpk4 - 2 , n = 1429 cells , 16 roots ; pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) , n = 574 cells , 16 roots ) ; and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 05 , lowercase letters ( wild type , n = 1981 cells , 13 roots ; pi4kβ1 pi4kβ2 , n = 1758 cells , 19 roots ; mpk4 - 2 , n = 1721 cells , 16 roots , pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) , n = 660 cells , 16 roots ) .

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 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

Figure 9The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants. Crosses of pi4kβ1 pi4kβ2 and mpk4-2 mutants resulted in combined genotypes with increased growth defects, as indicated. 14-day-old seedlings are shown. Scale bars, 1 cm. Top, The severe growth defects of the combined genotypes were accompanied by increased tissue distortion, as shown by 3D projections after propidium iodide staining of root tips of 14-day-old seedlings. Scale bars, 100 µm. Bottom, Multinucleation of cells was increased in the combined genotypes, as evident from immunofluorescence of microtubules (red) counterstained with DAPI (blue). Asterisks, multiple nuclei in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.Quantification of cytokinetic defects, as shownin the bottom panels of (B). Cytokinetic defects were enhanced in pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-) (for multinucleated cells, including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells, both according to a Welch ANOVA with Games-Howell post-hoc test; ***, p < 0.001; **,p < 0.01 (wild type, n = 1650 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1441 cells, 19 roots; mpk4-2, n = 1429 cells, 16 roots; pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 574 cells, 16 roots); and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one-way ANOVA with post-hoc Tukey HSD; p < 0.05, lowercase letters (wild type, n = 1981 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1758 cells, 19 roots; mpk4-2, n = 1721 cells, 16 roots, pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 660 cells, 16 roots).


Figure 10Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . PI4Kβ1 interacts with MPK4 in yeast two hybrid tests . MPK4 was used as a bait , while PI4Kβ1 was divided into two fragments to be used as prey . The results are representative for three independent experiments . Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . In vivo co - immunoprecipitation of MPK4 and PI4Kβ1 from 14 - day - old plants . PI4Kβ1 was specifically co - precipitated with MPK4 - myc using anti - myc antibodies . Arrowhead , Migration of PI4Kβ1 . The results are representative for three independent experiments . Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . Coordination of MPK4 - YFP and mCherry - PI4Kβ1 during cytokinesis in five - day - old root meristem cells . MPK4 co - localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates . Images were obtained with an LSM880 in Airyscan super - resolution ( SR ) mode . Scale bars , 10 µm . Live cell time lapse of MPK4 - YFP and mRFPFAPP1 - PH during cytokinesis , obtained with an LSM880 in Airyscan resolution versus sensitivity ( R - S ) mode . The time series is representative for 6 cells from 5 roots . Arrowheads indicate the initiation and leading edges of the growing cell plate . Scale bar , 10 µm . Left panels , The distribution of mRFPFAPP1 - PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls ( top ) and mpk4 - 2 mutants ( bottom ) . Intensity profiles were recorded as indicated by the dashed lines . Scale bars , 10 µm . Right top , intensity of mRFPFAPP1 - PH at the cell plate normalized vs . the intensity at the apical plasma membrane ( wild type , n = 43 cells , 34 roots ; mpk4 - 2 , n = 32 cells , 28 roots ) . Right bottom , intensity of mRFPFAPP1 - PH at the TGN normalized vs . the intensity at the apical plasma membrane ( wild type , n = 40 cells , 29 roots ; mpk4 - 2 , n = 26 cells , 23 roots ) . * , a significant difference ( p < 0 . 05 ) according to a two - tailed Student ' s test .

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Figure 10Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. PI4Kβ1 interacts with MPK4 in yeast two hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments.Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. In vivo co-immunoprecipitation of MPK4 and PI4Kβ1 from 14-day-old plants. PI4Kβ1 was specifically co-precipitated with MPK4-myc using anti-myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments.Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. Coordination of MPK4-YFP and mCherry-PI4Kβ1 during cytokinesis in five-day-old root meristem cells. MPK4 co-localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super-resolution (SR) mode. Scale bars, 10 µm.Live cell time lapse of MPK4-YFP and mRFPFAPP1-PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R-S) mode. The time series is representative for 6 cells from 5 roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 µm.Left panels, The distribution of mRFPFAPP1-PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls (top) and mpk4-2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 µm. Right top, intensity of mRFPFAPP1-PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4-2, n = 32 cells, 28 roots). Right bottom, intensity of mRFPFAPP1-PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots ; mpk4-2, n = 26 cells, 23 roots). *, a significant difference (p<0.05) according to a two-tailed Student's test.


Figure 2A . Growth curves of the library ( black ) compared to two different controls under increasing selective pressures . DSB ( double - stranded break ) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK . n = 3 for each

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Figure 2A. Growth curves of the library (black) compared to two different controls under increasing selective pressures. DSB (double-stranded break) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK. n=3 for each curve


Figure 3B . Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non - Target gRNA . n = 3 . C . Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non - Target gRNA . n =

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Figure 3B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3


Figure 4D . Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants . Quantification was performed using LC - MS , as described in the methods section . n = 3 . A two - sample student t - test assuming unequal variances was performed to calculate statistical significance . Concentrations are reported as fold - change relative to the wild - type control samplesE . DapF assay showing kinetics of the wild type , G210D and M260Y mutants . Assay was performed as described in the methods section . n = 5F . Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT , DapF G210D and DapF M260Y

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Figure 4D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samplesE. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds


Figure 5C . Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control samplesF . Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control

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Figure 5C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samplesF. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples


Figure 1 ( G ) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group ( numbered 1 - 4 ) was detected by anti - IgG secondary antibody .

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Figure 1(G) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group (numbered 1-4) was detected by anti-IgG secondary antibody .


Figure 3Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . After 2 weeks , 7 μm spleen sections were stained with Red : Alexa - Fluor - 594 - CD4 and Green : FITC - B220 ( D ) or FITC - GL7 ( E ) . Scale bar represent 100 nm . Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . Serum was assessed for level of IgG antibodies binding NP - 31 and NP - 4 ( F ) .

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Figure 3Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm.Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).


Figure 8 ( A ) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3 + CD4 + Th cells against CD127 ( left ) and intracellular Foxp3 ( right ) NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . Representative staining is shown for CD4 + CD3 + Th cells ( B ) , and summary data for CD3 + CD4 + Th cells ( C ) Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test . NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . summary data for CD3 + CD4 + CD127hi conventional cells ( D ) . Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test . ( E ) Individuals were divided into NRP1 - high subjects ( NRP1 + cells > 1 . 3 % in Th cells ) and NRP1 - low subjects ( NRP1 + cells ≤ 1 . 35 % in Th cells ) . Numbers of HC and SLE falling into each group are shown . * * * p < 0 . 001 Chi squared test with Yates ' correction . ( F , G ) NRP1 - expressing cells amongst CD3 + CD4 + CD45RO - cells were measured , representative staining ( F ) and summary data ( G ) . The plot shows individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * * p < 0 . 0001 Unpaired Student ' s T test .

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Figure 8(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. summary data for CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.


Figure 2The violin plot depicting the smaller genome size of HAC isolates ( n = 820 ) than that of DPC ( n = 407 ) and HPC ( n = 262 ) . Inside the violins are the box and whisker plots , with the dot showing the median , the box showing the quartiles , and the whisker showing the 95 % percentiles . P - values were calculated using unpaired t - test ( * * , P < 0 . 05 ) . The survival curve for zebrafish inoculated with representative S . suis isolates from HAC and HPC . In total twenty - five representative isolates were tested , including fourteen isolates from HAC and ten healthy - pig isolates from HPC , as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control . The cumulative mortalities were calculated using the combined data from the isolates in each group . As expected , all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates ( P < 0 . 05 ) . Survival curve and statistical analysis were created using Kaplan - Meier and SPSS 23 . 0 . P - values were calculated using Log rank test . P < 0 . 05 was considered significant ( * * , P < 0 . 05 ; NS , P > 0 . 05 ) . No significant difference ( NS , P = 0 . 072 ) was found between HAC isolates and SC19 .

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Figure 2The violin plot depicting the smaller genome size of HAC isolates (n=820) than that of DPC (n=407) and HPC (n=262). Inside the violins are the box and whisker plots, with the dot showing the median, the box showing the quartiles, and the whisker showing the 95% percentiles. P-values were calculated using unpaired t-test (**, P<0.05).The survival curve for zebrafish inoculated with representative S. suis isolates from HAC and HPC. In total twenty-five representative isolates were tested, including fourteen isolates from HAC and ten healthy-pig isolates from HPC, as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control. The cumulative mortalities were calculated using the combined data from the isolates in each group. As expected, all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates (P<0.05). Survival curve and statistical analysis were created using Kaplan-Meier and SPSS 23.0. P-values were calculated using Log rank test. P<0.05 was considered significant (**, P<0.05; NS, P>0.05). No significant difference (NS, P=0.072) was found between HAC isolates and SC19.


Figure 1 ( C ) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype ( left ) . Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting ( right ) . Cropped blots are

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Figure 1(C) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype (left). Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting (right). Cropped blots are shown


Figure 3 ( F ) Representative PCR amplification of MYC , TP53R249S , KRASG12D , NRASG12D , CTNNB1S45F , BRAFV600E , AXIN1G652S , IL6 , PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC - PHHs ( Tumor ) and their corresponding overexpression vector plasmids as positive controls ( OCs ) ( I ) Expression of MYC , p53 , and KRASG12D was detected in the iHCC samples ( n = 3 , biological replicates ) by Western blotting . Cropped blots are

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Figure 3(F) Representative PCR amplification of MYC, TP53R249S, KRASG12D, NRASG12D, CTNNB1S45F, BRAFV600E, AXIN1G652S, IL6, PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC-PHHs (Tumor) and their corresponding overexpression vector plasmids as positive controls (OCs)(I) Expression of MYC, p53, and KRASG12D was detected in the iHCC samples (n=3, biological replicates) by Western blotting. Cropped blots are shown


Figure 4 ( B ) Genomic DNA of colonies that were derived from single cells of iHCC1 - 1 and iHCC2 - 1 tumor samples were extracted for PCR amplification of MYC , TP53R249S , and

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Figure 4(B) Genomic DNA of colonies that were derived from single cells of iHCC1-1 and iHCC2-1 tumor samples were extracted for PCR amplification of MYC, TP53R249S, and KRASG12D


Figure 5 ( B ) Volcano plot of RNA - seq data showing genes with differential expression ( 1 , 591 upregulated and 2 , 243 downregulated genes , fold change ≥ 2 and P value ≤ 0 . 05 ) in iHCC cells compared to PHHs . TP53 , KRAS , MYC , MUC1 , and AFP were highly expressed in iHCC cells , whereas hepatocyte signature markers , including FOXA2 , FOXA3 , ATF5 , HNF4A , and ALB were highly expressed in PHHs ( D ) Heat map analysis of the log2 ( RPKM ) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells . These genes were classified into three categories based on their

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Figure 5(B) Volcano plot of RNA-seq data showing genes with differential expression (1,591 upregulated and 2,243 downregulated genes, fold change≥2 and P value≤0.05) in iHCC cells compared to PHHs. TP53, KRAS, MYC, MUC1, and AFP were highly expressed in iHCC cells, whereas hepatocyte signature markers, including FOXA2, FOXA3, ATF5, HNF4A, and ALB were highly expressed in PHHs(D) Heat map analysis of the log2(RPKM) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells. These genes were classified into three categories based on their functions


Figure 6 ( A ) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs . Within these 20 genes , seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA - HCC ( TCGA - LIHC ) analysis ( B ) Representative cytotoxicity CARFAP - T and CARMUC1 - T cells assessed by coincubation with iHCC2 - 1 cells . P = 0 . 0038 ( CARFAP - T vs . mock T ) , and P < 0 . 0478 ( CARMUC1 - T vs . mock - T ) . P values were determined by paired t - test , * * P ≤ 0 . 05 . Cells were seeded at E : T ratios ranging from 4 : 1 to 1 : 4 ; data are presented as the mean ± SD . N - numbers refer to biological replicates ( n = 3 ) ( D ) Clonogenic assay of iHCC1 - 1 cells treated with a MEK inhibitor ( trametinib ) and / or a CDK4 / 6 inhibitor ( palbociclib ) ; Representative images were shown based on three biological replicates ( F ) Clonogenic assays ( Top ) of iHCC1 - 1 cells treated with XL413 . The data are the representative of three biological replications . ( Bottom ) Growth curves of iHCC in xenografts ( n = 5 ) treated with PBS or XL413 at 50 mm3 tumor volume . P = 0 . 0486 ( XL413 vs . PBS group ) by two - way ANOVA with Sidak ' s multiple comparison test at Day24 , * P ≤ 0 . 05 . Data are presented as the mean ± SD . N - numbers refer to biological replicates .

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Figure 6(A) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs. Within these 20 genes, seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA-HCC (TCGA-LIHC) analysis(B) Representative cytotoxicity CARFAP-T and CARMUC1-T cells assessed by coincubation with iHCC2-1 cells. P=0.0038 (CARFAP-T vs. mock T), and P<0.0478 (CARMUC1-T vs. mock-T). P values were determined by paired t-test, **P≤0.05. Cells were seeded at E: T ratios ranging from 4:1 to 1:4; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3)(D) Clonogenic assay of iHCC1-1 cells treated with a MEK inhibitor (trametinib) and/or a CDK4/6 inhibitor (palbociclib); Representative images were shown based on three biological replicates(F) Clonogenic assays (Top) of iHCC1-1 cells treated with XL413. The data are the representative of three biological replications. (Bottom) Growth curves of iHCC in xenografts (n=5) treated with PBS or XL413 at 50 mm3 tumor volume. P=0.0486 (XL413 vs. PBS group) by two-way ANOVA with Sidak's multiple comparison test at Day24, *P≤0.05. Data are presented as the mean ± SD. N-numbers refer to biological replicates.


Figure 1 ( a ) Silver - stained gel image of the affinity - purified complexes of PSPC1 , SFPQ and NONO . The respective proteins are marked along with AcTEV protease used for the elution of purified proteins . ( f ) Western blotting of affinity purified complexes of PSPC1 , SFPQ and NONO with SWI / SNF antibodies . cBAF , PBAF , ncBAF specific , as well as common subunits , are marked .

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Figure 1(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.(f) Western blotting of affinity purified complexes of PSPC1, SFPQ and NONO with SWI/SNF antibodies. cBAF, PBAF, ncBAF specific, as well as common subunits, are marked.


Figure 2 ( a ) Silver - stained gel image of the affinity - purified complexes of ARID1A and ARID1B . The bait and a few other SWI / SNF subunits are marked as per their molecular weight . Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( c ) SWI / SNF subunitsHeat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( e ) PSPs . ( f ) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components . SMARCA4 was used as a positive control for purification .

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Figure 2(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (c) SWI/SNF subunitsHeat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (e) PSPs.(f) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components. SMARCA4 was used as a positive control for purification.


Figure 3 ( a ) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo - FLAG - tagged ARID1B . Input and eluted samples were resolved on a gel followed by western blotting with various antibodies . Western with SWI / SNF subunits - SMARCA4 and SMARCD1 were used as the control for purification , while beta - actin was used as a loading control . ( e , f ) ( e ) RIP qPCR and ( f ) PCR analysis performed using an antibody against endogenous ARID1B . for monitoring NEAT1 _ 2 levels . RIP with NONO and IgG was used as a positive and negative control respectively . GAPDH levels were also assessed in the assay . Data are expressed as a percent of Input .

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Figure 3(a) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo-FLAG-tagged ARID1B. Input and eluted samples were resolved on a gel followed by western blotting with various antibodies. Western with SWI/SNF subunits- SMARCA4 and SMARCD1 were used as the control for purification, while beta-actin was used as a loading control.(e, f) (e) RIP qPCR and (f) PCR analysis performed using an antibody against endogenous ARID1B. for monitoring NEAT1_2 levels. RIP with NONO and IgG was used as a positive and negative control respectively. GAPDH levels were also assessed in the assay. Data are expressed as a percent of Input.


Figure 4 ( c ) smFISH analysis was performed to detect NEAT1 signal in the nucleus . The scale bar corresponds to 10 µm . Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control . The number of nuclei ( n ) used for quantification is mentioned .

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Figure 4(c) smFISH analysis was performed to detect NEAT1 signal in the nucleus. The scale bar corresponds to 10 µm. Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control. The number of nuclei (n) used for quantification is mentioned.


Figure 1A Determination of a time window for 2a2iL treatment needed to induce a naïve - like pluripotency state in primed hPSCs . One day treatment with 2a followed by the addition of 2a2iL for up to 14 days . Scale bars : 200 µm . C Characteristics of 2a2iL - RH6 cells represented by dome - shaped colonies , ALP activity , and a normal male karyotype ( 46 , XY ) . 2a2iL - RH6 expressed the common pluripotency markers OCT4 , NANOG , SOX2 , and TRA - 1 - 81 , but not SSEA1 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) and Propidium iodide ( PI ) . Scale bars : 200 , 100 , and 50 µm in the inset .

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Figure 1A Determination of a time window for 2a2iL treatment needed to induce a naïve-like pluripotency state in primed hPSCs. One day treatment with 2a followed by the addition of 2a2iL for up to 14 days. Scale bars: 200 µm.C Characteristics of 2a2iL-RH6 cells represented by dome-shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL-RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA-1-81, but not SSEA1. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI). Scale bars: 200, 100, and 50 µm in the inset.


Figure 2A Doubling time in pRH6 versus 2a2iL - RH6 . * * P < 0 . 01 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . B Cloning efficiency in the presence ( + ) and absence ( − ) of ROCK inhibitor ( ROCKi ) in pRH6 versus 2a2iL - RH6 . ROCKi ( − ) * P < 0 . 05 , ROCKi ( + ) * P < 0 . 01 ( t - test ) , data presented as mean ± SD ( n = 3 ) . C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL - RH6 . The ratio of firefly luciferase expression to Renilla luciferase expression ( expressed from co - transfected plasmid ) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL - RH6 . OCT4 PE - Luc * * * P < 0 . 001 , OCT4 DE - Luc * P < 0 . 05 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . D qRT - PCR analysis of pluripotency - related genes in pRH6 and 2a2iL - RH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . E Immunofluorescence staining for several naïve specific markers in 2a2iL - RH6 . Scale bar : 100 µm . F Western blot analysis of naïve - related pluripotency markers in pRH6 and 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . G Immunofluorescence staining against pRH5 and 2a2iL - RH5 ( 46 , XX ) for H3K27me3 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Reactivation of inactive X - chromosome in female 2a2iL - RH5 was confirmed by the lack of H3K27me3 nuclear foci . Scale bar : 50 µm .

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Figure 2A Doubling time in pRH6 versus 2a2iL-RH6. **P<0.01 (t-test). Data presented as mean ± SD (n=3).B Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL-RH6. ROCKi (−)*P<0.05, ROCKi (+) *P<0.01 (t-test), data presented as mean ± SD (n=3).C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P<0.001, OCT4 DE-Luc *P<0.05 (t-test). Data presented as mean ± SD (n=3).D qRT-PCR analysis of pluripotency-related genes in pRH6 and 2a2iL-RH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm.F Western blot analysis of naïve-related pluripotency markers in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.


Figure 3A Phase contrast images of embryoid bodies ( EBs ) from 2a2iL - RH6 cells that differentiated into neurons . Scale bars : 200 and 100 µm in the inset . B qRT - PCR analysis of 20 day - EBs from pRH6 and 2a2iL - RH6 for markers of different embryonic germ layer derivatives . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . C Immunofluorescence images of differentiated 2a2iL - RH6 cells that depict expression of markers for neuron - like cells ( TUJ - 1 ) , cardiac - muscle - like cells ( CTNT ) , and hepatocyte - like cells ( SOX17 ) . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm . D 2a2iL - RH6 cells form teratomas that consist of three embryonic germ layer - derived tissues . ( i ) Rosette structures ( ectoderm ) , ( ii ) cartilage ( mesoderm ) , and ( iii ) primitive gut ( endoderm ; scale bar 50 µm ) . E From left to right : Phase contrast and GFP representation in 2a2iL - RH6 - eGFP ; presence of injected 2a2iL - RH6 - eGFP in the mouse blastocyst and integration into the inner cell mass ( ICM ) after 3 , 24 and 48 h incubation periods . Scale bars : 200 , 100 , and 50 µm in the inset . F Fluorescence images of tissue - sections from human - mouse chimeras in E10 . 5 showed cells positive for GFP ( top ) and human nuclear antigen ( HNA ) ( bottom ) in different parts of the embryos , including the head ( i ) , trunk ( ii ) , and tail ( iii ) with the higher magnification in the right panels . Scale bars : 200 and 100 µm in the inset . G Genomic PCR analysis for a human specific mitochondrial sequence ( 391 bp ) in human - mouse chimeras . H Immunofluorescence staining against NANOG shows lack of expression in E10 . 5 human - mouse chimera tissue sections . Scale bars : 200 and 100 µm in the inset . I Immunofluorescence staining against TUJ1 , BRACHYURY ( BRA ) , and SOX17 in E10 . 5 human - mouse chimera tissue sections represent differentiation - derivatives of 2a2iL - RH6 . Scale bars : 200 and 100 µm in the inset .

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 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 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 O O O O O O O O O O O O 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 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 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 3A Phase contrast images of embryoid bodies (EBs) from 2a2iL-RH6 cells that differentiated into neurons. Scale bars: 200 and 100 µm in the inset.B qRT-PCR analysis of 20 day-EBs from pRH6 and 2a2iL-RH6 for markers of different embryonic germ layer derivatives. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.D 2a2iL-RH6 cells form teratomas that consist of three embryonic germ layer-derived tissues. (i) Rosette structures (ectoderm), (ii) cartilage (mesoderm), and (iii) primitive gut (endoderm; scale bar 50 µm).E From left to right: Phase contrast and GFP representation in 2a2iL-RH6-eGFP; presence of injected 2a2iL-RH6-eGFP in the mouse blastocyst and integration into the inner cell mass (ICM) after 3, 24 and 48 h incubation periods. Scale bars: 200, 100, and 50 µm in the inset.F Fluorescence images of tissue-sections from human-mouse chimeras in E10.5 showed cells positive for GFP (top) and human nuclear antigen (HNA) (bottom) in different parts of the embryos, including the head (i), trunk (ii), and tail (iii) with the higher magnification in the right panels. Scale bars: 200 and 100 µm in the inset.G Genomic PCR analysis for a human specific mitochondrial sequence (391 bp) in human-mouse chimeras.H Immunofluorescence staining against NANOG shows lack of expression in E10.5 human-mouse chimera tissue sections. Scale bars: 200 and 100 µm in the inset.I Immunofluorescence staining against TUJ1, BRACHYURY (BRA), and SOX17 in E10.5 human-mouse chimera tissue sections represent differentiation-derivatives of 2a2iL-RH6. Scale bars: 200 and 100 µm in the inset.


Figure 4B Morphology of ICM - outgrowths by protocols 1 and 2 during the first 4 days after seeding . nRH11 - 14 ( naïve Royan H11 , 12 , 13 , and 14 ) : naïve hESC lines produced from human embryos using 2a2iL condition . Scale bar : 100 µm . C Comparison of the efficiency of naïve - like hESCs derivation from human blastocysts by two protocols . D Representative example of naïve - like hESCs ( nRH13 ) at passage 33 . Dome - shaped colonies were stained for ALP activity and karyotyped . Scale bars : 200 and 100 µm in the inset . E Expression of the common pluripotency markers OCT4 , SOX2 , and TRA - 1 - 81 , and naïve - related pluripotency markers NANOG , KLF17 , and TFCP2L1 in nRH13 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm . F qRT - PCR analysis for pluripotency markers in nRH13 in comparison to a primed hESC line , pRH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . G Expression of lineage markers after embryoid body ( EB ) formation and spontaneous differentiation in nRH13 cells . * P < 0 . 05 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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Figure 4B Morphology of ICM-outgrowths by protocols 1 and 2 during the first 4 days after seeding. nRH11-14 (naïve Royan H11, 12, 13, and 14): naïve hESC lines produced from human embryos using 2a2iL condition. Scale bar: 100 µm.C Comparison of the efficiency of naïve-like hESCs derivation from human blastocysts by two protocols.D Representative example of naïve-like hESCs (nRH13) at passage 33. Dome-shaped colonies were stained for ALP activity and karyotyped. Scale bars: 200 and 100 µm in the inset.E Expression of the common pluripotency markers OCT4, SOX2, and TRA-1-81, and naïve-related pluripotency markers NANOG, KLF17, and TFCP2L1 in nRH13. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.F qRT-PCR analysis for pluripotency markers in nRH13 in comparison to a primed hESC line, pRH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).G Expression of lineage markers after embryoid body (EB) formation and spontaneous differentiation in nRH13 cells. *P<0.05, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


Figure 5A Pairwise Pearson correlation coefficient heatmap of transcriptome profiles ( RNA - seq ) from 2a2iL - , pRH5 , and pRH6 cell lines . Each cell of the heatmap shows the pairwise Pearson correlation coefficient heatmap of genes with the largest expression variances in a pair of samples . C The volcano plot shows a differential expression between 2a2iL - versus primed hPSCs lines . X - axis shows the log2 fold - change , and the y - axis shows the negative log10 scaled adjusted P - value ( Benjamini - Hochberg ) . Red and blue points represent up - regulated genes in 2a2iL - and primed cells , respectively ( absolute log - 2 fold - change > 1 , adjusted P - value < 0 . 05 ) . Gray points indicate non - significant genes between these states .

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Figure 5A Pairwise Pearson correlation coefficient heatmap of transcriptome profiles (RNA-seq) from 2a2iL-, pRH5, and pRH6 cell lines. Each cell of the heatmap shows the pairwise Pearson correlation coefficient heatmap of genes with the largest expression variances in a pair of samples.C The volcano plot shows a differential expression between 2a2iL- versus primed hPSCs lines. X-axis shows the log2 fold-change, and the y-axis shows the negative log10 scaled adjusted P-value (Benjamini-Hochberg). Red and blue points represent up-regulated genes in 2a2iL- and primed cells, respectively (absolute log-2 fold-change >1, adjusted P-value <0.05). Gray points indicate non-significant genes between these states.


Figure 6A Position of TGF - β family members in the volcano plot of differentially expressed genes in 2a2iL - and primed hPSCs . X - axis shows log2 fold - change and y - axis shows negative log10 adjusted P - value < 0 . 05 ( Adjusted P - value was obtained using the Benjamini and Hochberg correction to determine the false discovery rate ) . Red and blue points represent up - regulated genes in 2a2iL - and primed hPSCs , respectively . Genes with no significant expression change are shown in gray . B qRT - PCR analysis of several TGF - β - related genes in pRH6 and 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . C Inhibition of the TGF - β pathway by two small molecules , SB43 and A83 . Left panel : morphology of naïve - like cells three days after treatment . Right panel : morphology of treated cells after passaging . Scale bar : 100 µm . D Heatmap shows expression of putative LRH - 1 target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs , with a range from dark blue for lower expression to dark red for higher expression . E Heatmap shows expression of putative RARγ target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs , with a range from dark blue for lower expression to dark red for higher expression .

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Figure 6A Position of TGF-β family members in the volcano plot of differentially expressed genes in 2a2iL- and primed hPSCs. X-axis shows log2 fold-change and y-axis shows negative log10 adjusted P-value <0.05 (Adjusted P-value was obtained using the Benjamini and Hochberg correction to determine the false discovery rate). Red and blue points represent up-regulated genes in 2a2iL- and primed hPSCs, respectively. Genes with no significant expression change are shown in gray.B qRT-PCR analysis of several TGF-β-related genes in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).C Inhibition of the TGF-β pathway by two small molecules, SB43 and A83. Left panel: morphology of naïve-like cells three days after treatment. Right panel: morphology of treated cells after passaging. Scale bar: 100 µm.D Heatmap shows expression of putative LRH-1 target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression. E Heatmap shows expression of putative RARγ target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression.


Figure 7A qRT - PCR analysis of ligands associated with TGF - β signaling during conversion of primed to naïve - like pluripotency by 2a2iL induction protocol at days 1 , 3 , and 5 . 2iL was selected as a control group . * * * P < 0 . 0001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) . B Inhibition of TGF - β pathway using SB43 during conversion of primed to naïve pluripotency . Scale bar : 100 µm . C Morphology of TGFβ2iL - treated RH6 cells along with ALP activity . Scale bar : 200 µm . D qRT - PCR analysis of naïve related genes in pRH6 , 2a2iL - , and TGFβ2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) . E Bar plot representing cell proliferation rates of 2a2iL - and TGFβ2iL - RH6 by cell counts . * * P < 0 . 01 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL - RH6 based on the number of ALP positive colonies . * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) . G Immunofluorescence images show the expression of naïve pluripotency markers in TGF - β2iL - RH6 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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Figure 7A qRT-PCR analysis of ligands associated with TGF-β signaling during conversion of primed to naïve-like pluripotency by 2a2iL induction protocol at days 1, 3, and 5. 2iL was selected as a control group. ***P<0.0001. One-way ANOVA. Data presented as mean ± SD (n=3).B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm.C Morphology of TGFβ2iL-treated RH6 cells along with ALP activity. Scale bar: 200 µm.D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3).E Bar plot representing cell proliferation rates of 2a2iL- and TGFβ2iL-RH6 by cell counts. **P<0.01 (t-test). Data presented as mean ± SD (n=3).F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL-RH6 based on the number of ALP positive colonies. ***P<0.001 (t-test). Data presented as mean ± SD (n=3).G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


Figure 1A . In the Discovery cohort , Kaplan - Meier estimates of disease - free survival ( DFS ) in years ( y ) in patients with low ( red ) versus high ( blue ) nuclear PRMT5 expression ( upper panel ) and in 2 groups of patients according to their treatment . Patients treated with tamoxifen ( Tam , middle panel ) and with aromatase inhibitors ( AIs , lower panel ) . P - values are calculated with the logrank test . B . The same analyses were performed in the Validation cohort .

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Figure 1A. In the Discovery cohort, Kaplan-Meier estimates of disease-free survival (DFS) in years (y) in patients with low (red) versus high (blue) nuclear PRMT5 expression (upper panel) and in 2 groups of patients according to their treatment. Patients treated with tamoxifen (Tam, middle panel) and with aromatase inhibitors (AIs, lower panel). P-values are calculated with the logrank test. B. The same analyses were performed in the Validation cohort.


Figure 2A . GST and GST - PRMT5 fusion proteins were incubated with in vitro - translated ERα , the interaction was then visualized by Western blotting using an anti - ERα antibody . The corresponding Coomassie - stained gel is shown in the right panel . B . ERα / PRMT5 interaction was assessed by co - immunoprecipitation of MCF7 cell extracts treated or not with E2 for the indicated times , using an anti - PRMT5 antibody . The presence of PRMT5 , ERα and β - actin was evaluated by Western blot analysis . C . The same experiment was performed for MCF7 cells treated or not with tamoxifen ( Tam ) for the indicated times . The scale bar is 10µM . D . Proximity Ligation Assay ( PLA ) to detect interactions between ERα and PRMT5 in MCF7 cells treated with E2 , Tam 1 µM or both for 6 h . After fixation , PLA experiments were performed to evaluate the interactions between ERα / PRMT5 using specific antibodies . The detected dimers are represented by red dots . The nuclei were counterstained with mounting medium containing DAPI ( blue ) ( Obj : X60 ) . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test . F . Paraffin - embedded sections from a fresh tumor were incubated with or without G595 , and PRMT5 and SDMA expression were assessed by IHC .

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Figure 2A. GST and GST-PRMT5 fusion proteins were incubated with in vitro-translated ERα, the interaction was then visualized by Western blotting using an anti-ERα antibody. The corresponding Coomassie-stained gel is shown in the right panel.B. ERα/PRMT5 interaction was assessed by co-immunoprecipitation of MCF7 cell extracts treated or not with E2 for the indicated times, using an anti-PRMT5 antibody. The presence of PRMT5, ERα and β-actin was evaluated by Western blot analysis.C. The same experiment was performed for MCF7 cells treated or not with tamoxifen (Tam) for the indicated times. The scale bar is 10µM.D. Proximity Ligation Assay (PLA) to detect interactions between ERα and PRMT5 in MCF7 cells treated with E2, Tam 1 µM or both for 6 h. After fixation, PLA experiments were performed to evaluate the interactions between ERα/PRMT5 using specific antibodies. The detected dimers are represented by red dots. The nuclei were counterstained with mounting medium containing DAPI (blue) (Obj:X60). The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.F. Paraffin-embedded sections from a fresh tumor were incubated with or without G595, and PRMT5 and SDMA expression were assessed by IHC.


Figure 3C . MCF7 cells were treated with or without E2 , Tam or both for 6 h . ERα methylation was determined by PLA using an anti - ERα and a pan methyl recognizing symmetrical dimethylation ( SDMA ) . The scale bar is 10µM . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test . D . The same experiment was performed with G595 to verify that ERα methylation was PRMT5 - dependent . Red dots reflect ERα methylation . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test . The sale bar is 40µM . Western blotting was performed to assess ERα , PRMT5 expression and the global profile of methylation using a pan SDMA antibody . E . MCF7 cells were treated as in C and lysates were immunoprecipitated with a pan SDMA antibody and immunoblotted with an anti - ERα antibody . ERα , SDMA and GAPDH expression was evaluated in the input .

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Figure 3C. MCF7 cells were treated with or without E2, Tam or both for 6 h. ERα methylation was determined by PLA using an anti-ERα and a pan methyl recognizing symmetrical dimethylation (SDMA). The scale bar is 10µM. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.D. The same experiment was performed with G595 to verify that ERα methylation was PRMT5-dependent. Red dots reflect ERα methylation. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. The sale bar is 40µM. Western blotting was performed to assess ERα, PRMT5 expression and the global profile of methylation using a pan SDMA antibody.E. MCF7 cells were treated as in C and lysates were immunoprecipitated with a pan SDMA antibody and immunoblotted with an anti-ERα antibody. ERα, SDMA and GAPDH expression was evaluated in the input.


Figure 4B . MCF7 cells were treated with Tam for 6 h in the presence or absence of G595 , cell extracts were then subjected to ChIP assay using anti - ERα or anti - SMRT antibodies . The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters . The results are expressed relative to the signal obtained from input chromatin . The mean ± SEM of at least three experiments is shown . P - values were determined using a Student t - test . C . MCF7 cells were treated with Tam for 6 h and total RNA was prepared and cDNAs were analyzed by RT - QPCR with specific primers for GREB1 . The values were normalized against 28S mRNA and represent the ± SEM of three experiments .

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Figure 4B. MCF7 cells were treated with Tam for 6 h in the presence or absence of G595, cell extracts were then subjected to ChIP assay using anti-ERα or anti-SMRT antibodies. The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters. The results are expressed relative to the signal obtained from input chromatin. The mean ± SEM of at least three experiments is shown. P-values were determined using a Student t-test.C. MCF7 cells were treated with Tam for 6 h and total RNA was prepared and cDNAs were analyzed by RT-QPCR with specific primers for GREB1. The values were normalized against 28S mRNA and represent the ± SEM of three experiments.


Figure 5B . MCF7 cells were treated with Tam for 6h in the presence or in absence of G595 , then ERα interaction with SIRT2 and SAP18 was determined by PLA using specific antibodies against both proteins . The scale bar is 10µM . Quantification was performed as in Figure 2 . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test . Western blotting was performed to assess ERα , SIRT2 , SAP18 and GAPDH expression and the global profile of SDMA .

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Figure 5B. MCF7 cells were treated with Tam for 6h in the presence or in absence of G595, then ERα interaction with SIRT2 and SAP18 was determined by PLA using specific antibodies against both proteins. The scale bar is 10µM. Quantification was performed as in Figure 2. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. Western blotting was performed to assess ERα, SIRT2, SAP18 and GAPDH expression and the global profile of SDMA.


Figure 6A . ERα methylation SDMA was studied by PLA , and PRMT5 and MEP50 expression were analyzed by IHC on formalin - fixed MCF7 xenografts treated or not with Tam ( Obj : X40 ) . The scale bar is 50 µM . Histogram quantifying the number of dots / cells and the H - scores for nuclear PRMT5 and MEP50 are presented on the right . The mean ± SEM of 7 tumors is shown . P - values were determined using a Student t - test . B . ERα methylation SDMA , PRMT5 and MEP50 expression were studied as in A on a Tam - resistant formalin - fixed PDX model HBCx - 34 Tam R treated or not with Tam . ERα methylation SDMA was analyzed as described above and the H - score for nuclear PRMT5 and MEP50 was evaluated as in A . The scale bar is 50 µM . The mean ± SEM of 3 tumors is shown . P - values were determined using a Student t - test . C . ERα methylation SDMA , PRMT5 and MEP50 expression were studied as in A on a Tam - resistant formalin - fixed the PDX model HBCx - 22 Tam R treated or not with Tam . ERα methylation SDMA was analyzed as described above and the H - score for nuclear PRMT5 and MEP50 was evaluated as in A . The scale bar is 50 µM . The mean ± SEM of 7 tumors is shown .

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Figure 6A. ERα methylation SDMA was studied by PLA, and PRMT5 and MEP50 expression were analyzed by IHC on formalin-fixed MCF7 xenografts treated or not with Tam (Obj: X40). The scale bar is 50 µM. Histogram quantifying the number of dots/cells and the H-scores for nuclear PRMT5 and MEP50 are presented on the right. The mean ± SEM of 7 tumors is shown. P-values were determined using a Student t-test.B. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed PDX model HBCx-34 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 3 tumors is shown. P-values were determined using a Student t-test.C. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed the PDX model HBCx-22 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 7 tumors is shown.


Figure 7A . For each tumor , we analyzed the level of ERα SDMA methylation by PLA . The H - score for nuclear PRMT5 expression and the number of dots / cell are listed under the figures . The scale bar is 20µM .

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Figure 7A. For each tumor, we analyzed the level of ERα SDMA methylation by PLA. The H-score for nuclear PRMT5 expression and the number of dots/cell are listed under the figures. The scale bar is 20µM.


Figure 1RFP + ( G1 phase ) , GFP + ( G2 phase ) , and GFP + / RFP + ( S phase ) RPE - FUCCI cells were sorted by flow cytometry , RNA was extracted and analyzed using qRT - PCR ( n = 3 ) . Expression is normalized to GAPDH mRNA . Bars show mean ± SD .

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Figure 1RFP+ (G1 phase), GFP+ (G2 phase), and GFP+/RFP+ (S phase) RPE-FUCCI cells were sorted by flow cytometry, RNA was extracted and analyzed using qRT-PCR (n=3). Expression is normalized to GAPDH mRNA. Bars show mean ± SD.


Figure 2RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS . Nocodazole was added after 4 h to arrest cells in mitosis . Alternatively , RO3306 inhibitor was added to prevent mitotic entry . Where indicated sample was collected by mitotic shake - off ( MS ) to obtain pure mitotic population . Samples were analyzed by immunoblotting ( upper panel ) . Staining for pS10 - H3 was used as a marker of mitosis , TFIIH as a loading control . In parallel , cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining . Representative plot is shown , M indicates percentage of mitotic cells ( middle panel ) . Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample ( lower panel ) . Bars show median ± SD . RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA , probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ - tubulin , and were analyzed by confocal microscopy . Shown is maximal projection of representative mitotic ( upper panel ) and interphase ( lower panel ) cells . Scale bars indicate 10 μm .

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Figure 2RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS. Nocodazole was added after 4 h to arrest cells in mitosis. Alternatively, RO3306 inhibitor was added to prevent mitotic entry. Where indicated sample was collected by mitotic shake-off (MS) to obtain pure mitotic population. Samples were analyzed by immunoblotting (upper panel). Staining for pS10-H3 was used as a marker of mitosis, TFIIH as a loading control. In parallel, cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining. Representative plot is shown, M indicates percentage of mitotic cells (middle panel). Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample (lower panel). Bars show median ± SD.RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA, probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ-tubulin, and were analyzed by confocal microscopy. Shown is maximal projection of representative mitotic (upper panel) and interphase (lower panel) cells. Scale bars indicate 10 μm.


Figure 3Quantification of the mitotic index in three experiments from ( A ) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel . Shown is median ± SD ( n = 3 ) . Statistical significance was determined by ANOVA ( * p < 0 . 05 , * * p < 0 . 005 ) . RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase . Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown . Scale bars indicate 20 μm , arrowheads point to misaligned chromosomes . More than 30 metaphases were evaluated per condition ( n = 3 ) . Bars indicate median ± SD . Statistical significance was determined by two - tailed t - test , ( * * * p < 0 . 001 , * p < 0 . 05 ) . Quantification of lagging chromosomes in anaphase cells from ( E ) . More than 30 anaphases were evaluated per condition ( n = 3 ) . Representative images of abnormal anaphase are shown , scale bars indicate 20 μm , arrowheads point to incorrectly segregating chromosomes . Statistical significance was determined by two - tailed t - test . Bars indicate median ± SD ( * * * p < 0 . 001 ) .

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Figure 3Quantification of the mitotic index in three experiments from (A) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel. Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (* p<0.05, ** p<0.005).RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase. Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown. Scale bars indicate 20 μm, arrowheads point to misaligned chromosomes. More than 30 metaphases were evaluated per condition (n=3). Bars indicate median ±SD. Statistical significance was determined by two-tailed t-test, (*** p<0.001, * p<0.05).   Quantification of lagging chromosomes in anaphase cells from (E). More than 30 anaphases were evaluated per condition (n=3). Representative images of abnormal anaphase are shown, scale bars indicate 20 μm, arrowheads point to incorrectly segregating chromosomes. Statistical significance was determined by two-tailed t-test. Bars indicate median ±SD (*** p<0.001).


Figure 4U2OS cells stably expressing H2B - EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals . Time frame closest to the nuclear envelope breakdown ( NEB ) was set as 0 min . Representative examples are shown ( n = 3 ) . Black bottom line indicates the time frames from NEB to the metaphase - to - anaphase transition . Scale bars indicate 10 μm . Progression through mitosis was categorized in cells from ( A ) ( 100 / condition ) as follows : nuclear envelope breakdown to the metaphase - to - anaphase transition ( MAT ) , anaphase , telophase and interphase . Data from one of three experiments are shown , each bar indicates one cell . Cells that died during imaging are shown in grey . Time from the nuclear envelope breakdown to MAT was quantified in cells from ( B ) . Shown is median ± SD ( n = 3 ) . Statistical significance was determined by ANOVA ( * * * * p < 0 . 0001 ) .

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Figure 4U2OS cells stably expressing H2B-EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals. Time frame closest to the nuclear envelope breakdown (NEB) was set as 0 min. Representative examples are shown (n=3). Black bottom line indicates the time frames from NEB to the metaphase-to-anaphase transition. Scale bars indicate 10 μm.Progression through mitosis was categorized in cells from (A) (100/condition) as follows: nuclear envelope breakdown to the metaphase-to-anaphase transition (MAT), anaphase, telophase and interphase. Data from one of three experiments are shown, each bar indicates one cell. Cells that died during imaging are shown in grey.Time from the nuclear envelope breakdown to MAT was quantified in cells from (B). Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (****p<0.0001).


Figure 5Cell extracts from asynchronously growing or mitotic RPE - EGFP or RPE - EGFP - FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting . Antibody against pS10H3 was used as a marker of mitosis .

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Figure 5Cell extracts from asynchronously growing or mitotic RPE-EGFP or RPE-EGFP-FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting. Antibody against pS10H3 was used as a marker of mitosis.