f10 ( a ) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co - immunoprecipitation of human NDP52 ( hNDP52 ) using a Tau5 , 12E8 or PHF1 antibody . ( b ) Homogenates used for co - immunoprecipitation in a were analysed by immunoblotting using anti - NDP52 , Tau5 , 12E8 or PHF1 antibody . ( c ) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl - soluble and insoluble fractions . The levels of total tau and tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 in the sarkosyl - insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies , respectively . Samples were also immunoblotted with a polyclonal to total tau ( Tau ) antibody . The levels of hNDP52 , p62 / SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( d - f ) The regression graphs show the relative amount of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( d ) , at Ser396 / Ser404 ( PHF1 ) ( e ) or total tau ( f ) compared to hNDP52 present in the sarkosyl - insoluble fractions .

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 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

f10(a) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co-immunoprecipitation of human NDP52 (hNDP52) using a Tau5, 12E8 or PHF1 antibody.(b) Homogenates used for co-immunoprecipitation in a were analysed by immunoblotting using anti-NDP52, Tau5, 12E8 or PHF1 antibody.(c) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl-soluble and insoluble fractions. The levels of total tau and tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 in the sarkosyl-insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Samples were also immunoblotted with a polyclonal to total tau (Tau) antibody. The levels of hNDP52, p62/SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting. The relative molecular masses (kDa) are indicated to the left of each blot. (d-f) The regression graphs show the relative amount of tau phosphorylated at Ser262/Ser356 (12E8) (d), at Ser396/Ser404 (PHF1) (e) or total tau (f) compared to hNDP52 present in the sarkosyl-insoluble fractions.


Figure 1The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects . The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2 , or by mCherry - tagged PI4Kβ1 expressed from a genomic fragment . Plants shown are one month old . Scale bar , 10 cm . The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants , as shown by propidium iodide ( PI ) staining of five - day - old roots . Arrowhead , oblique cell wall . Insets , magnifications of areas showing cell wall stubs . Scale bars , 50 µm . Quantifications of cell wall stubs , oblique cell walls and root meristem size ( wild type , n = 15 roots ; pi4kβ1 , n = 15 roots ; pi4kβ2 , n = 14 roots ; pi4kβ1 pi4kβ2 , n = 18 roots ; pPI4Kβ1 : PI4Kβ1 , n = 14 roots , pPI4Kβ2 : PI4Kβ2 , n = 15 roots ) . n . d . , not detected . Data are mean ± SD . Lowercase letters indicate a significant increase of oblique cell walls ( Welch ANOVA with Games - Howell post - hoc test ; p < 0 . 0001 ) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant ( one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 001 ) . Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG - PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters , as indicated , shown for one - month - old plants . Scale bar , 10 cm . Complementation with the pKNOLLE - driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2 / M phase . PI staining of pKNOLLE : FLAG - PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects . Arrowhead , cell wall stub in the pi4kβ1 pi4kβ2 double mutant . Scale bars , 50 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 1The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2, or by mCherry-tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are one month old. Scale bar, 10 cm.The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of five-day-old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 µm.Quantifications of cell wall stubs, oblique cell walls and root meristem size (wild type, n = 15 roots; pi4kβ1, n = 15 roots; pi4kβ2, n = 14 roots; pi4kβ1 pi4kβ2, n = 18 roots; pPI4Kβ1:PI4Kβ1, n = 14 roots, pPI4Kβ2:PI4Kβ2, n = 15 roots). n.d., not detected. Data are mean ± SD. Lowercase letters indicate a significant increase of oblique cell walls (Welch ANOVA with Games-Howell post-hoc test; p < 0.0001) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant (one-way ANOVA with post-hoc Tukey HSD; p < 0.001).Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG-PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters, as indicated, shown for one-month-old plants. Scale bar, 10 cm. Complementation with the pKNOLLE-driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2/M phase.PI staining of pKNOLLE:FLAG-PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects. Arrowhead, cell wall stub in the pi4kβ1 pi4kβ2 double mutant. Scale bars, 50 µm.


Figure 2In vivo localization of mCherry - PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five - day - old complemented pi4kβ1 pi4kβ2 plants . The mCherry - PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole ( VP ) mode with the pinhole set to 2 . Arrowheads , nascent cell plates decorated by mCherry - PI4Kβ1 . Scale bar , 20 µm . Whole - mount immunostaining of five - day - old seedlings expressing mCherry - PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti - tubulin ( red ) and anti - mCherry ( green ) antibodies , and DAPI ( blue ) . ( І ) , ( ІІ ) , magnifications of regions marked in b , representing early and late cytokinetic stages . Scale bars , 20 µm . Relative localization of mCherry - PI4Kβ1 and the TGN marker , ARF1 , during cytokinesis . Four - day - old seedlings were immunostainned with anti - mCherry ( green ) and anti - ARF1 ( red ) antibodies . Scale bars are 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 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 2In vivo localization of mCherry-PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five-day-old complemented pi4kβ1 pi4kβ2 plants. The mCherry-PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry-PI4Kβ1. Scale bar, 20 µm. Whole-mount immunostaining of five-day-old seedlings expressing mCherry-PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti-tubulin (red) and anti-mCherry (green) antibodies, and DAPI (blue). (І), (ІІ), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 µm.Relative localization of mCherry-PI4Kβ1 and the TGN marker, ARF1, during cytokinesis. Four-day-old seedlings were immunostainned with anti-mCherry (green) and anti-ARF1 (red) antibodies. Scale bars are 10 µm.


Figure 3The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five - day - old root tip meristems . Images shown are from 3 roots ( wild type ) and 7 roots ( pi4kβ1 pi4kβ2 double mutant ) . In wild type controls ( A ) , vesicles fused at the cell division plane to give rise to tubular network ( TN ) of a nascent cell plate . White arrowheads , fused vesicles at the division plane . n = 8 cells . In the pi4kβ1 pi4kβ2 double mutant ( B ) , vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse . The nuclei of both wild type and double mutant cells were at telophase or interphase , as judged by the presence of decondensed chromatin and a nuclear envelope . At this stage , failed cell plate formation was never seen in wild type ( A ) . Black arrowheads , unfused vesicles . n = 6 cells . Unfused vesicles clustered around a cell wall stub ( arrow ) . White arrowheads , light vesicles ; black arrowheads , dark vesicles ; black arrow , cell wall stub . n = 8 cells . No cross wall was seen in this cell with two nuclei . n = 6 cells . n , nucleus ; ne , nuclear envelope . Scale bars in magnified figures , 2 μm ; in insets , 1 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

Figure 3The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five-day-old root tip meristems. Images shown are from 3 roots (wild type) and 7 roots (pi4kβ1 pi4kβ2 double mutant). In wild type controls (A), vesicles fused at the cell division plane to give rise to tubular network (TN) of a nascent cell plate. White arrowheads, fused vesicles at the division plane. n = 8 cells. In the pi4kβ1 pi4kβ2 double mutant (B), vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse. The nuclei of both wild type and double mutant cells were at telophase or interphase, as judged by the presence of decondensed chromatin and a nuclear envelope. At this stage, failed cell plate formation was never seen in wild type (A). Black arrowheads, unfused vesicles. n = 6 cells. Unfused vesicles clustered around a cell wall stub (arrow). White arrowheads, light vesicles; black arrowheads, dark vesicles; black arrow, cell wall stub. n = 8 cells. No cross wall was seen in this cell with two nuclei. n = 6 cells. n, nucleus; ne, nuclear envelope. Scale bars in magnified figures, 2 μm; in insets, 1 μm.


Figure 4The effects of reduced association of PtdIns ( 4 ) P with the cell plate on the recruitment of CLC2 - GFP were analyzed in root meristem cells of five - day - old plants . Top , Cell plate - associated fluorescence of the PtdIns ( 4 ) P reporter , 2 × mCherryFAPP1 - PH , was monitored at the end of cytokinesis in five - day - old wild type controls or pi4kβ1 pi4kβ2 double mutants , as indicated . Scale bars , 10 µm . Bottom , The intensity of 2 × mCherryFAPP1 - PH signals was quantified at the end of cytokinesis at the cell plate ( left plot ) and at the TGN ( right plot ) . Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane ( wild type , n = 37 cells , 24 roots ; pi4kβ1 pi4kβ2 , n = 55 cells , 41 roots ) . TGN intensities were recorded using the Airyscan VP mode , and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity ( wild type , n = 30 cells , 22 roots ; pi4kβ1 pi4kβ2 , n = 37 cells , 31 roots ) . * * * , significant differences ( p < 0 . 0001 ) according to two - tailed Mann - Whitney U - tests . Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of wild type seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks . Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks . Left , quantification of the time of CLC2 - GFP appearance at the cell plate in wild type ( B ) or the pi4kβ1 pi4kβ2 double mutant ( C ) ( wild type , n = 10 cells , 7 roots ; pi4kβ1 pi4kβ2 , n = 24 cells , 19 roots ) . Right , quantification of CLC2 - GFP at the TGN in interphase cells . Five - day - old roots expressing CLC2 - GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity ( wild type , n = 14 cells , 2 roots ; pi4kβ1 pi4kβ2 , n = 15 cells , 2 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 4The effects of reduced association of PtdIns(4)P with the cell plate on the recruitment of CLC2-GFP were analyzed in root meristem cells of five-day-old plants. Top, Cell plate-associated fluorescence of the PtdIns(4)P reporter, 2×mCherryFAPP1-PH, was monitored at the end of cytokinesis in five-day-old wild type controls or pi4kβ1 pi4kβ2 double mutants, as indicated. Scale bars, 10 µm. Bottom, The intensity of 2×mCherryFAPP1-PH signals was quantified at the end of cytokinesis at the cell plate (left plot) and at the TGN (right plot). Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane (wild type, n = 37 cells, 24 roots; pi4kβ1 pi4kβ2, n = 55 cells, 41 roots). TGN intensities were recorded using the Airyscan VP mode, and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity (wild type, n=30 cells, 22 roots; pi4kβ1 pi4kβ2, n=37 cells, 31 roots). ***, significant differences (p<0.0001) according to two-tailed Mann-Whitney U-tests.Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of wild type seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.Left, quantification of the time of CLC2-GFP appearance at the cell plate in wild type (B) or the pi4kβ1 pi4kβ2 double mutant (C) (wild type, n = 10 cells, 7 roots; pi4kβ1 pi4kβ2, n = 24 cells, 19 roots). Right, quantification of CLC2-GFP at the TGN in interphase cells. Five-day-old roots expressing CLC2-GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity (wild type, n = 14 cells, 2 roots; pi4kβ1 pi4kβ2, n = 15 cells, 2 roots).


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 .

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 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 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 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 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

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 1 ( A ) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients . ( B ) RT - PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples . ( C ) RT - PCR analysis of the expression of IFN - γ and TNF - α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients . ( D ) RT - PCR analysis of the expression of miR - 125b - 5p and miR - 99a - 5p in γδ T cells isolated from indicated samples . ( E ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from indicated samples .

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 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

Figure 1(A) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients.(B) RT-PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples.(C) RT-PCR analysis of the expression of IFN-γ and TNF-α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients. (D) RT-PCR analysis of the expression of miR-125b-5p and miR-99a-5p in γδ T cells isolated from indicated samples. (E) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from indicated samples.


Figure 2 ( A ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from thymic biopsies , cultured for 11 days with IL - 7 or IL - 2 ( n = 12 - 15 independent biological samples ) . ( B ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells freshly isolated from thymic biopsies ( Thymus ) and PBLs ( n = 6 - 14 independent biological samples ) . ( C - D ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from thymus ( C ) or from PBLs ( D ) and cultured with the indicated cytokines , respectively for 11 days ( C ) or for 4 - 6 days ( D ) . When indicated , γδ PBLs were also co - cultured with an anti - TGF - β blocking antibody . Data are normalized to the value obtained in IL - 7 cultures ( n = 4 - 10 independent biological samples ) . ( E - F ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) versus FACS analysis of the expression of TNF - α ( left panel ) , IFN - γ ( middle panel ) and NKGD2 ( right panel ) in γδ T cells isolated from thymus ( E ) and PBLs ( F ) . Samples were either freshly isolated or cultured for 11 days ( γδ thymocytes ) or for 6 days ( γδ PBLs ) with the indicated cytokines ( n = 5 independent biological samples ) .

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 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

Figure 2(A) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymic biopsies, cultured for 11 days with IL-7 or IL-2 (n=12-15 independent biological samples). (B) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells freshly isolated from thymic biopsies (Thymus) and PBLs (n=6-14 independent biological samples).(C-D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4-6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n=4-10 independent biological samples).(E-F) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel) and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n=5 independent biological samples).


Figure 3 ( D ) FACS analysis of the expression of Annexin V in miR - 181a versus empty transduced ( GFP + ) and untransduced ( GFP - ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) . ( E ) RT - PCR analysis of the expression of Bcl2 and Bcl - xL ( normalized to the values obtained with the empty virus ) in transduced ( GFP + ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) . ( F ) FACS - derived expression of indicated surface and intracellular markers in miR - 181a versus empty transduced ( GFP + ) and untransduced ( GFP - ) γδ T thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 7 - 11 independent biological samples ) .

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 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 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

Figure 3(D) FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).(E) RT-PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).(F) FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=7-11 independent biological samples).


Figure 4 ( B ) RT - PCR analysis of the expression of the indicated genes associated with Mapk , Notch , Irf4 or Stat1 signaling in sorted GFP + γδ thymocytes transduced with empty or miR - 181a viral vector . Data are normalized to the value obtained with the empty virus . Samples were cultured with IL - 7 plus IL - 2 for 11 days ( n = 3 - 5 independent biological samples ) . ( C ) Dual luciferase reporter assay performed to verify binding between miR - 181a and Map3k2 , Notch2 , Irf4 or Stat1 mRNA . HEK293 T cells were co - transfected with a pmirGLO Dual - Luciferase miRNA Target Expression Vector containing either the WT or mutated ( Mut ) 3 ′ UTR target sites plus miR - 181a . A negative construct ( without miR - 181a binding sites ) was included . Firefly / Renilla ratios were normalized to those obtained for the empty vector ( n = 4 independent biological samples ) . ( E - G ) RT - PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression ( E ) , Nkg2d or Tnfa expression ( F ) , and NKG2D or TNF - α protein expression ( G ) after siRNA - mediated knockdown of Map3k2 or Notch2 . In each experiment , a control condition was also used where a nontargeting negative control siRNA was transfected . γδ were cultured with IL - 7 for 4 days ( n = 6 independent biological samples ) .

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 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 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

Figure 4(B) RT-PCR analysis of the expression of the indicated genes associated with Mapk, Notch, Irf4 or Stat1 signaling in sorted GFP+ γδ thymocytes transduced with empty or miR-181a viral vector. Data are normalized to the value obtained with the empty virus. Samples were cultured with IL-7 plus IL-2 for 11 days (n=3-5 independent biological samples).(C) Dual luciferase reporter assay performed to verify binding between miR-181a and Map3k2, Notch2, Irf4 or Stat1 mRNA. HEK293 T cells were co-transfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing either the WT or mutated (Mut) 3′ UTR target sites plus miR-181a. A negative construct (without miR-181a binding sites) was included. Firefly/Renilla ratios were normalized to those obtained for the empty vector (n=4 independent biological samples).(E-G) RT-PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression (E), Nkg2d or Tnfa expression (F), and NKG2D or TNF-α protein expression (G) after siRNA-mediated knockdown of Map3k2 or Notch2. In each experiment, a control condition was also used where a nontargeting negative control siRNA was transfected. γδ were cultured with IL-7 for 4 days (n=6 independent biological samples).


Figure 1 ( B ) Cytoplasmic TDP - 43 immunostaining is elevated not only in poly - GA transduced neurons , but also in the non - transduced receiver cells . White and red arrows indicate cells with cytoplasmic TDP - 43 in GFP positive and negative cells , respectively . ( C ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . Cells with and without GFP signal were counted separately ( indicated by + / − ) . Two groups ( GFP negative donor , GFP positive receiver ) were excluded due to very high GFP transduction rate and very low GFP transmission rate . n = 4 biological replicates . In total 283 donor GFP , 273 donor GA175 - GFP , 284 receiver GFP and 266 receiver GA175 - GFP cells were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . Immunofluorescence staining of TDP - 43 ∆ NLS aggregate number per cell Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . automatic quantification of TDP - 43 ∆ NLS aggregate number per cell In ( E ) n = 3 biological replicates with 368 donor iRFP , 251 donor GA175 - iRFP , 430 receiver iRFP and 328 GA175 - iRFP cells were analyzed . Cells with and without GFP signal were analyzed separately ( indicated by + / − ) . White and red arrows indicate cells with cytoplasmic TDP - 43 in GFP positive and negative cells , respectively . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . ( F ) in addition to filter trap assay of SDS - insoluble TDP - 43ΔNLS - GFP aggregates compared in iRFP or GA175 - iRFP transfected cells . ( G ) GFP mRNA expression levels were measured by qPCR . RNA levels were normalized to GAPDH , β - actin and β2 - microglobulin mRNA . Bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction . * denotes p < 0 . 05 , and * * denotes p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) Cytoplasmic TDP-43 immunostaining is elevated not only in poly-GA transduced neurons, but also in the non-transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively.  (C) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP negative donor, GFP positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n=4 biological replicates. In total 283 donor GFP, 273 donor GA175-GFP, 284 receiver GFP and 266 receiver GA175-GFP cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.  Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. Immunofluorescence staining of TDP-43∆NLS aggregate number per cell  Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. automatic quantification of TDP-43∆NLS aggregate number per cell In (E) n=3 biological replicates with 368 donor iRFP, 251 donor GA175-iRFP, 430 receiver iRFP and 328 GA175-iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/−). White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  (F) in addition to filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates compared in iRFP or GA175-iRFP transfected cells.  (G) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. * denotes p<0.05, and ** denotes p<0.01.


Figure 2 ( A ) Confocal imaging showed anti - GA antibody treatment reduces poly - GA aggregates and TDP - 43 mislocalization in hippocampal neurons . White and red arrows show cells with cytoplasmic TDP - 43 in GFP - positive and - negative cells , respectively . Scale bar denotes 30 µm . ( B ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . Four groups were excluded due to very high GFP transduction rate ( GFP - negative donor ) and very low GFP transmission rate ( GFP - positive receiver with IgG and anti - GA ) and complete prevention of GA - RFP transmission of anti - GA immunodepletion ( GA - GFP receiver with anti - GA ) . n = 3 biological replicates . In total 280 donor GFP , 284 receiver GFP with IgG , 317 receiver GFP with anti - GA , 277 donor GA175 - GFP , 294 receiver GA175 - GFP with IgG , 311 receiver GA175 - GFP with anti - GA cells were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . ( C ) Immunoblotting of poly - GA immunoprecipitated from conditioned media using antibody - coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media . ( D ) Poly - GA levels in conditioned media before and after immunodepletion with control IgG and anti - GA antibody were determined by immunoassay . n = 3 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * denotes p < 0 . 01 , and * * * denotes p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal imaging showed anti-GA antibody treatment reduces poly-GA aggregates and TDP-43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP-43 in GFP-positive and -negative cells, respectively. Scale bar denotes 30 µm.  (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP-negative donor) and very low GFP transmission rate (GFP-positive receiver with IgG and anti-GA) and complete prevention of GA-RFP transmission of anti-GA immunodepletion (GA-GFP receiver with anti-GA). n=3 biological replicates. In total 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti-GA, 277 donor GA175-GFP, 294 receiver GA175-GFP with IgG, 311 receiver GA175-GFP with anti-GA cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.  (C) Immunoblotting of poly-GA immunoprecipitated from conditioned media using antibody-coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media.  (D) Poly-GA levels in conditioned media before and after immunodepletion with control IgG and anti-GA antibody were determined by immunoassay. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ** denotes p<0.01, and *** denotes p<0.001.


Figure 3 Double immunofluorescence of the proteasome subunit PSMC4 and poly - GA inclusions in spinal cord of GA149 - CFP transgenic mouse Scale bar denotes 20 µm . Double immunofluorescence of the proteasome subunit PSMC4 and poly - GA inclusions in cortex of C9orf72 patients compared to controls . Scale bar denotes 20 µm . Co - culture model of HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and an UbG76V - GFP proteostasis reporter in donor and receiver compartment ( 48 h ) . ( C ) Separate analysis of both compartments by immunoblot and ( D ) immunoblot quantification . For quantitative analysis of immunoblots UbG76V - GFP was normalized to calnexin . n = 3 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Red dashed line indicates the control ' s expression level . * denotes p < 0 . 05 , * * * denotes p < 0 . 001 . ( E ) GFP mRNA expression levels were measured by qPCR . RNA levels were normalized to GAPDH , β - actin and β2 - microglobulin mRNA . Bar - graphs of mean ± SD . n = 3 biological replicates . Unpaired two - tailed t - test with Welch ' s correction . ( F , G ) Flow cytometry analysis of a UbG76V - GFP reporter cell line incubated 48h with conditioned media from RFP or GA175 - RFP transfected cells upon immunodepletion of poly - GA or control depletion using unspecific IgG . n = 3 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * denotes p < 0 . 05 . ( G ) Comparisons of the corresponding histograms for compensated RFP and UbG76V - GFP fluorescence from one representative experiment that shows specific transmission of GA175 - RFP associated with accumulation of UbG76V - GFP in cells incubated with GA175 - RFP conditioned media .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in spinal cord of GA149-CFP transgenic mouse Scale bar denotes 20 µm.  Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in cortex of C9orf72 patients compared to controls. Scale bar denotes 20 µm.  Co-culture model of HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and an UbG76V-GFP proteostasis reporter in donor and receiver compartment (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots UbG76V-GFP was normalized to calnexin. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * denotes p<0.05, *** denotes p<0.001.  (E) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. n=3 biological replicates. Unpaired two-tailed t-test with Welch's correction.  (F,G) Flow cytometry analysis of a UbG76V-GFP reporter cell line incubated 48h with conditioned media from RFP or GA175-RFP transfected cells upon immunodepletion of poly-GA or control depletion using unspecific IgG. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05. (G) Comparisons of the corresponding histograms for compensated RFP and UbG76V-GFP fluorescence from one representative experiment that shows specific transmission of GA175-RFP associated with accumulation of UbG76V-GFP in cells incubated with GA175-RFP conditioned media.


Figure 4 Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . ( A ) Immunofluorescence reveals enhanced cytoplasmic TDP - 43 levels in neurons with poly - GA aggregates or treated with MG132 . Arrows mark punctate TDP - 43 staining . Rolipram treatment reduced cytoplasmic TDP - 43 in GA175 - GFP neurons . Scale bar denotes 20 µm . ( B ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced neurons . n = 4 biological replicates . In total 462 GFP and 371 GA175 - GFP cells treated with vehicle , and 386 GFP and 529 GA175 - GFP cells treated with MG132 , 513 GFP and 434 GA175 - GFP cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . ( C ) Immunoblot to show effects of MG132 and rolipram on GA175 - GFP and GFP expression . Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . Filter trap assay with quantification of SDS - insoluble aggregated GA175 - GFP . n = 5 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . ( F , G ) HeLa cells were co - transfected with RFP - TDP - CTF and GFP or GA175 - GFP for 2 days . For the final 16h , cells were treated with rolipram ( 30 µM ) or MG132 ( 10 µM ) . Filter trap assay of SDS - insoluble TDP - CTF aggregates quantified by densitometry . n = 4 biological replicates . Scatter dot plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . ( H , I ) HeLa cells were co - transfected with TDP - 43ΔNLS - GFP and iRFP or GA175 - iRFP for 2 days . For the final 16h , cells were treated with either vehicle or rolipram ( 30 µM ) . Filter trap assay of SDS - insoluble TDP - 43ΔNLS - GFP aggregates quantified by densitometry . n = 3 biological replicates . Scatter dot plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP-43 levels in neurons with poly-GA aggregates or treated with MG132. Arrows mark punctate TDP-43 staining. Rolipram treatment reduced cytoplasmic TDP-43 in GA175-GFP neurons. Scale bar denotes 20 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced neurons. n=4 biological replicates. In total 462 GFP and 371 GA175-GFP cells treated with vehicle, and 386 GFP and 529 GA175-GFP cells treated with MG132, 513 GFP and 434 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (C) Immunoblot to show effects of MG132 and rolipram on GA175-GFP and GFP expression.  Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. Filter trap assay with quantification of SDS-insoluble aggregated GA175-GFP. n=5 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  (F,G) HeLa cells were co-transfected with RFP-TDP-CTF and GFP or GA175-GFP for 2 days. For the final 16h, cells were treated with rolipram (30 µM) or MG132 (10 µM). Filter trap assay of SDS-insoluble TDP-CTF aggregates quantified by densitometry. n=4 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  (H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Figure 5 Hela cells were co - transfected with an RFP - based TDP - NLS reporter and GFP or GA175 - GFP . 24 h after transfection , cells were treated with rolipram ( 30 µM ) for 16h . In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP - reporter . White arrows indicate cells with cytoplasmic TDP - 43 . ( B ) Automated quantification of cells with cytoplasmic TDP - NLS reporter in GFP and GA175 - GFP positive cells . n = 4 biological replicates . In total 345 GFP and 386 GA175 - GFP cells treated with vehicle , 371 GFP and 404 GA175 - GFP cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Hela cells were co - transfected with an RFP - based TDP - NLS reporter and GFP or GA175 - GFP . 24 h after transfection , cells were treated with rolipram ( 30 µM ) for 16h . ( C ) RFP - NLSTDPWT mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Hela cells were co - transfected with the RFP - based TDP - NLS reporter , GFP or GA175 - GFP , as well as PSMD11 or empty vector . Image analysis as in ( A ) . n = 4 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . 354 GFP and 330 GA175 - GFP cells with vector , 367 GFP and 369 GA175 - GFP cells with PSMD11 in total were analyzed . Hela cells were co - transfected with the RFP - based TDP - NLS reporter , GFP or GA175 - GFP , as well as PSMD11 or empty vector . F ) RFP - NLSTDPWT mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Immunofluorescence of Hela cells transfected with GFP or GA175 - GFP showing reduced poly - GA aggregation upon rolipram treatment ( 30 µM , 16 h ) . ( H ) Automated quantification of poly - GA aggregate number per cell . n = 3 biological replicates . In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction . ( I ) GA - GFP mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5 Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP-reporter. White arrows indicate cells with cytoplasmic TDP-43. (B) Automated quantification of cells with cytoplasmic TDP-NLS reporter in GFP and GA175-GFP positive cells. n=4 biological replicates. In total 345 GFP and 386 GA175-GFP cells treated with vehicle, 371 GFP and 404 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. (C) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. Image analysis as in (A). n=4 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA175-GFP cells with vector, 367 GFP and 369 GA175-GFP cells with PSMD11 in total were analyzed.  Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. F) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.  Immunofluorescence of Hela cells transfected with GFP or GA175-GFP showing reduced poly-GA aggregation upon rolipram treatment (30 µM, 16 h). (H) Automated quantification of poly-GA aggregate number per cell. n=3 biological replicates. In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.  (I) GA-GFP mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.


Figure 6 ( B ) Immunoblot of HeLa cells transfected with RFP - based TDP - NLS wild - type ( WT ) or mutants ( K84A , K84R , K95A , K95R ) . ( C HeLa cells were co - transfected with the indicated TDP - NLS reporters as well as GFP or GA175 - GFP , and treated with MG132 ( 10µM ) or vehicle for 16h . ( D ) Automated quantification of RFP - NLS reporters in GFP positive cells . Note that K84A and K84R block overall import , while K95A and K95R allow import but are resistant to inhibition by poly - GA . n = 4 biological replicates . The total number of cells analyzed per group was ( from left to right ) : 667 , 581 , 789 , 783 , 809 , 708 , 628 , 721 , 938 , 557 , 857 , 861 , 886 , 699 , 789 , 539 , 636 , 577 , 638 and 870 . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . Scale bar denotes 20 µm . ( E , F ) HeLa cells were co - transfected with the indicated GFP - TDP NLS reporters and iRFP or GA175 - iRFP . Cell lysates were immunoprecipitated with anti - GFP and immunoblotted with indicated antibodies to detect co - immunoprecipitation of the TDP - 43 NLS with importin - α5 / KPNA1 nuclear import receptor . ( F ) Quantification of KPNA1 levels normalized to total GFP - NLSTDP reporter levels in anti - GFP immunoprecipitates . n = 3 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . See also Figure EV4 . Red dashed line indicates the control ' s expression level .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6 (B) Immunoblot of HeLa cells transfected with RFP-based TDP-NLS wild-type (WT) or mutants (K84A, K84R, K95A, K95R).  (C HeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10µM) or vehicle for 16h.  (D) Automated quantification of RFP-NLS reporters in GFP positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly-GA. n=4 biological replicates. The total number of cells analyzed per group was (from left to right): 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638 and 870. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. Scale bar denotes 20 µm.  (E,F) HeLa cells were co-transfected with the indicated GFP-TDP NLS reporters and iRFP or GA175-iRFP. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with indicated antibodies to detect co-immunoprecipitation of the TDP-43 NLS with importin-α5/KPNA1 nuclear import receptor. (F) Quantification of KPNA1 levels normalized to total GFP-NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. See also Figure EV4. Red dashed line indicates the control's expression level.


Figure 7 HeLa cells were co - transfected with wildtype or K95A GFP - TDP NLS , HA - ubiquitin and iRFP or GA175 - iRFP . 24h after transfection , cells were treated with rolipram ( 30 µM ) , MG132 ( 10 µM ) or DMSO ( vehicle ) for 16h . Lysates were immunoprecipitated with Protein G beads coupled with anti - GFP antibody . ( A ) Immunoblotting of input ( left panels ) and anti - GFP immunoprecipitates ( right panels ) to show GFP - reporter levels and poly - ubiquitination . ( B ) Quantification of HA - ubiquitin levels normalized to total GFP - NLSTDP reporter levels in anti - GFP immunoprecipitates . n = 3 biological replicates . Scatter plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * denotes p < 0 . 05 , and * * * denotes p < 0 . 001 . Red dashed line indicates the control ' s expression level .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody. (A) Immunoblotting of input (left panels) and anti-GFP immunoprecipitates (right panels) to show GFP-reporter levels and poly-ubiquitination. (B) Quantification of HA-ubiquitin levels normalized to total GFP- NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05, and *** denotes p<0.001. Red dashed line indicates the control's expression level.


Figure 1ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP - PNP and aRF1 / aPelota . The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins , forming the post - splitting complex . rps : small subunit ribosomal proteins .

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

Figure 1ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP-PNP and aRF1/aPelota. The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins, forming the post-splitting complex. rps: small subunit ribosomal proteins.


Figure 2Yeast survival of ABCE1 variants ( S . solfataricus colored , S . cerevisiae in gray ) . Most residues connecting to 30S in the post - SC show a growth defect when exchanged for a small one ( alanine ) or a negative charge ( glutamate ) . + + no effect , + growth defect , - lethal .

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

Figure 2Yeast survival of ABCE1 variants (S. solfataricus colored, S. cerevisiae in gray). Most residues connecting to 30S in the post-SC show a growth defect when exchanged for a small one (alanine) or a negative charge (glutamate). ++ no effect, + growth defect, - lethal.


Figure 3Mutations in the α - helices of hinge 2 prevent 30S binding while the Y592A / Y593A ( C - terminus ) and L353Y ( A - loop in NBSII ) exchanges do not influence ribosome binding . 70S splitting efficiency normalized to wildtype . Hinge 2 mutations Y592A / Y593A , R565E and S580E display strongly impaired splitting activity . Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line . ATP turnover per ABCE1 is not affected in all tested mutants .

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

Figure 3Mutations in the α-helices of hinge 2 prevent 30S binding while the Y592A/Y593A (C-terminus) and L353Y (A-loop in NBSII) exchanges do not influence ribosome binding.70S splitting efficiency normalized to wildtype. Hinge 2 mutations Y592A/Y593A, R565E and S580E display strongly impaired splitting activity. Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line. ATP turnover per ABCE1 is not affected in all tested mutants.


Figure 1C . Total USV number in YFO and AFO . Data information : Data are presented in the box plot or pie chart format . The central band in each box represents the median ; boxes indicate the middle quartiles , and whiskers extend to the minimum and maximum values . * p < 0 . 05 , determined using Student ' s or Welch t - 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 O O O O O O O O

Figure 1C. Total USV number in YFO and AFO. Data information: Data are presented in the box plot or pie chart format. The central band in each box represents the median; boxes indicate the middle quartiles, and whiskers extend to the minimum and maximum values. *p<0.05, determined using Student's or Welch t-test.


Figure 2D . A heat map of the 16 hyper - and 96 hypo - methylated DMRs in sperm from aged mice ordered by their chromosomal positions . M indicates the average .

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Figure 2D. A heat map of the 16 hyper- and 96 hypo-methylated DMRs in sperm from aged mice ordered by their chromosomal positions. M indicates the average.


Figure 4A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by nominal GSEA p - value .

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 4A. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by nominal GSEA p-value.


Figure 5A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by volcano or nominal GSEA P - value . NES , normalized enrichment score .

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 5A. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by volcano or nominal GSEA P-value. NES, normalized enrichment score.


Figure 6C . Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Satb2 + / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Satb2 - / Ctip2 - : layer 6 . Scale bar : 100 μm . F . Cell number in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test . D . Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Tbr1 - / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Tbr1 + / Ctip2 - : layer 6 . Scale bar : 100 μm . G . Cell density in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 6C. Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Satb2+/Ctip2-: layer 2-4, Ctip2high: layer 5, Satb2-/Ctip2-: layer 6. Scale bar: 100 μm. F. Cell number in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.D. Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Tbr1-/Ctip2-: layer 2-4, Ctip2high: layer 5, Tbr1+/Ctip2-: layer 6. Scale bar: 100 μm. G. Cell density in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


Figure 7C . Representative image of 5 - mC immunofluorescence in the sperm of saline - and 5 - Aza - treated mice ( ST and AT , respectively ) . Scale bar : 100 μm . D . Quantification analysis of 5 - mC immunofluorescence in individual sperm cells from STO ( n = 1 , 673 sperm from 4 male mice ) and ATO ( n = 2 , 675 sperm from 4 male mice ) . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test . F . Overall USV number in STO and ATO . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7C. Representative image of 5-mC immunofluorescence in the sperm of saline- and 5-Aza-treated mice (ST and AT, respectively). Scale bar: 100 μm. D. Quantification analysis of 5-mC immunofluorescence in individual sperm cells from STO (n=1,673 sperm from 4 male mice) and ATO (n=2,675 sperm from 4 male mice).Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.F. Overall USV number in STO and ATO. Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.