Figure 9Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( A , B ) TDP - 43 knockdown ( DIV6 + 5 ) significantly reduces dendrite branching compared to control : at 25 , 37 . 5 and 87 . 5 µm radius p < 0 . 05 , at 50 µm p < 0 . 01 and from 62 . 5 to 75 µm p < 0 . 001 . Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( C , D ) VPS4B transfection ( DIV6 + 4 ) significantly reduces dendrite complexity : at 25 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 and from 87 . 5 to 100 µm p < 0 . 01 . Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( E , F ) Two days after shRNA transfection neurons were treated with 1 nM NRG1 - beta 1 ( NRG1 ) or vehicle and analyzed at DIV6 + 5 . shCtrl vs . shTDP : at 25 and 87 . 5 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 . shCtrl vs . shCtrl + NRG1 : from 37 . 5 to 62 . 5 µm p < 0 . 05 . shTDP vs . shTDP + NRG1 : no significant difference . Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( G , H ) Transfection of the indicated combinations of shCtrl , shTDP , ErbB4 or vector control ( DIV6 + 5 ) . shCtrl + Ctrl vs . shTDP + Ctrl : from 25 to 75 µm radius p < 0 . 001 , at 87 and 112 . 5 µm p < 0 . 01 and at 100 µm p < 0 . 05 . shTDP + Ctrl vs . shTDP + ErbB4 : from 25 to 100 µm radius p < 0 . 001 , at 112 . 5 µm p < 0 . 01 . shCtrl + Ctrl vs . shTDP + ErbB4 : no significant differences .

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

Figure 9Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (A, B) TDP-43 knockdown (DIV6+5) significantly reduces dendrite branching compared to control: at 25, 37.5 and 87.5 µm radius p < 0.05, at 50 µm p < 0.01 and from 62.5 to 75 µm p < 0.001.Primary hippocampal neurons (DIV6+4) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (C, D) VPS4B transfection (DIV6+4) significantly reduces dendrite complexity: at 25 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001 and from 87.5 to 100 µm p < 0.01.Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (E, F) Two days after shRNA transfection neurons were treated with 1 nM NRG1-beta 1 (NRG1) or vehicle and analyzed at DIV6+5. shCtrl vs. shTDP: at 25 and 87.5 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001. shCtrl vs. shCtrl + NRG1: from 37.5 to 62.5 µm p < 0.05. shTDP vs. shTDP+NRG1: no significant difference.Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (G, H) Transfection of the indicated combinations of shCtrl, shTDP, ErbB4 or vector control (DIV6+5). shCtrl + Ctrl vs. shTDP + Ctrl: from 25 to 75 µm radius p < 0.001, at 87 and 112.5 µm p < 0.01 and at 100 µm p < 0.05. shTDP + Ctrl vs. shTDP + ErbB4: from 25 to 100 µm radius p < 0.001, at 112.5 µm p < 0.01. shCtrl + Ctrl vs. shTDP + ErbB4: no significant differences.


Figure 1 ( A , B ) In situ cryo - electron tomogram displaying the proximal region of a mature mother centriole ( A ) and procentriole ( B ) . Mature centriole , MC ; procentriole , PC ; mitochondria , mito ; vacuole , vac ; white dashed line , lamella edge . Scale bars , 100 nm . ( C ) Side view z - projection of cartwheels containing the central hub and several CIDs from a mature centriole . Central hub , CH ; cartwheel inner densities , CID . Scale bar , 20 nm . ( D ) Cross section of the cartwheel - containing regions from a mature centriole . Microtubule triplet , MTT ; spokes , SP . Scale bar , 200 nm . ( E ) Side view z - projection of cartwheels containing the central hub and several CIDs from a procentriole . Scale bar , 20 nm . ( F ) Cross section of the cartwheel - containing regions from a procentriole . Scale bar , 200 nm . ( G ) Nine - fold symmetrized cross sections of the cartwheel - containing region from a mature centriole ( left side ) and a procentriole ( right side ) . Dashed white circle , central hub . Scale bars , 100 nm . ( H ) Longitudinal periodicity measurements of the central hub and CIDs . Central hub , blue ; CID , red . Mean values are displayed above the data range . Blue data points are measured distance between individual units of the central hub , red data points are measured distances between individual units of the cartwheel inner density . Mature centriole , central hub , n = 30 , mean = 4 . 1 + / - 0 . 67 ( S . D ) ; mature centriole , cartwheel inner density , n = 18 , mean = 8 . 7 + / - 1 . 6 ( S . D ) ; procentriole , central hub , n = 10 , mean = 4 . 0 + / - 0 . 66 ( S . D ) ; procentriole , cartwheel inner density , n = 5 , mean = 8 . 6 + / - 1 . 2 ( S . D ) . ( I , J ) Nine - fold symmetrized central hub z - projections , starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5 . 4 nm steps in a mature centriole ( I ) and a procentriole ( J ) . Red arrow , CID ; blue arrow ; central hub . Scale bar : 20 nm .

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

Figure 1(A, B) In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm.(C) Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner densities, CID. Scale bar, 20 nm.(D) Cross section of the cartwheel-containing regions from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm.(E) Side view z-projection of cartwheels containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm.(F) Cross section of the cartwheel-containing regions from a procentriole. Scale bar, 200 nm.(G) Nine-fold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm.(H) Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 +/- 0.67 (S.D); mature centriole, cartwheel inner density, n = 18, mean = 8.7 +/- 1.6 (S.D); procentriole, central hub, n = 10, mean = 4.0 +/- 0.66 (S.D); procentriole, cartwheel inner density, n = 5, mean = 8.6 +/- 1.2 (S.D).(I, J) Nine-fold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm.


Figure 2 ( A , B , C ) Cryo - electron tomograms of the proximal regions of a P . tetraurelia centriole ( A ) , a N . gruberi centriole ( B ) , and a H . sapiens procentriole ( C ) . White arrows denote a broken cartwheel ; procentriole , PC ; mature centriole , MC ; Scale bar , 100 nm . Note that most N . gruberi and H . sapiens centrioles were heavily compressed during the cryo - EM preparation , as previously described ( Guichard et al . , 2010 ) . The displayed N . gruberi centriole illustrates the damage caused by compression . The periodicities of N . gruberi and H . sapiens cartwheels were measured only on regions that were not damaged ( D ) Cross sections from cartwheel - containing region of P . tetraurelia centrioles . Scale bar , 50 nm . ( E ) Zoomed side view of cartwheel from P . tetraurelia , displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow . Scale bar , 25 nm . ( F ) Cross section from cartwheel - containing region of N . gruberi , ( G ) Zoomed side view of cartwheel from N . gruberi displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow . ( H ) Cross section from cartwheel - containing region of H . sapiens centriole . ( I ) Zoomed side view of cartwheel from H . sapiens , displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow . ( J ) Longitudinal periodicity of the central hub and CIDs in P . tetraurelia , N . gruberi , and H . sapiens . Mean values are displayed above data range . Black lines indicate the mean and the standard deviation . Blue data points are measured distance between individual units of the central hub , red data points are measured distances between individual units of the cartwheel inner density . P . tetraurelia , central hub , n = 10 , mean = 4 . 3 + / - 0 . 38 ( S . D ) ; N . gruberi , central hub , n = 10 , mean = 4 . 4 + / - 0 . 53 ( S . D ) ; H . sapiens , central hub , n = 10 , mean = 4 . 2 + / - 0 . 68 ( S . D ) ; P . tetraurelia , cartwheel inner density , n = 8 , mean = 8 . 4 + / - 1 . 3 ( S . D ) ; N . gruberi , cartwheel inner density , n = 8 , mean = 8 . 3 + / - 1 . 8 ( S . D ) ; H . sapiens , cartwheel inner density , n = 8 , mean = 8 . 1 + / - 2 . 5 ( S . D ) . ( K Proximal protrusion length of the cartwheel beyond the microtubule triplets in C . reinhardtii , P . tetraurelia , N . gruberi , and H . sapiens . Internal cartwheel inside the microtubule barrel , dark blue ( INT ) ; external cartwheel beyond the microtubule wall , light blue ( EXT ) . Mean values are displayed above each bar plot with the black lines indicating the standard deviation ( K ) . Mature C . reinhardtii , n = 4 , external cartwheel length = 37 . 6 + / - 3 . 4 , internal cartwheel length 74 . 2 + / - 16 . 6 ; C . reinhardtii procentriole , n = 2 , external cartwheel mean length = 52 . 8 + / - 22 . 0 , internal cartwheel length 26 . 5 + / - 29 . 7 ; P . tetraurelia , n = 23 , external cartwheel mean length = 11 . 5 + / - 9 . 2 , internal cartwheel length 66 . 6 + / - 15 . 7 ; N . gruberi , n = 19 , external cartwheel mean length = 41 . 0 + / - 22 . 5 , internal cartwheel length 259 . 0 + / - 87 . 2 ; human procentriole , n = 7 , external cartwheel length 23 . 1 + / - 10 . 3 , internal cartwheel length 154 . 7 + / - 47 . 7 . Error reported is standard deviation . Scale bar , 50 nm . The start of each microtubule wall is delineated by a dashed white line ( L ) . Mature C . reinhardtii , n = 4 , external cartwheel length = 37 . 6 + / - 3 . 4 , internal cartwheel length 74 . 2 + / - 16 . 6 ; C . reinhardtii procentriole , n = 2 , external cartwheel mean length = 52 . 8 + / - 22 . 0 , internal cartwheel length 26 . 5 + / - 29 . 7 ; P . tetraurelia , n = 23 , external cartwheel mean length = 11 . 5 + / - 9 . 2 , internal cartwheel length 66 . 6 + / - 15 . 7 ; N . gruberi , n = 19 , external cartwheel mean length = 41 . 0 + / - 22 . 5 , internal cartwheel length 259 . 0 + / - 87 . 2 ; human procentriole , n = 7 , external cartwheel length 23 . 1 + / - 10 . 3 , internal cartwheel length 154 . 7 + / - 47 . 7 . Error reported is standard deviation . Scale bar , 50 nm .

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

Figure 2(A, B, C) Cryo-electron tomograms of the proximal regions of a P. tetraurelia centriole (A), a N. gruberi centriole (B), and a H. sapiens procentriole (C). White arrows denote a broken cartwheel; procentriole, PC; mature centriole, MC; Scale bar, 100 nm. Note that most N. gruberi and H. sapiens centrioles were heavily compressed during the cryo-EM preparation, as previously described (Guichard et al., 2010). The displayed N. gruberi centriole illustrates the damage caused by compression. The periodicities of N. gruberi and H. sapiens cartwheels were measured only on regions that were not damaged(D) Cross sections from cartwheel-containing region of P. tetraurelia centrioles. Scale bar, 50 nm. (E) Zoomed side view of cartwheel from P. tetraurelia, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Scale bar, 25 nm.(F) Cross section from cartwheel-containing region of N. gruberi, (G) Zoomed side view of cartwheel from N. gruberi displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.(H) Cross section from cartwheel-containing region of H. sapiens centriole. (I) Zoomed side view of cartwheel from H. sapiens, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.(J) Longitudinal periodicity of the central hub and CIDs in P. tetraurelia, N. gruberi, and H. sapiens. Mean values are displayed above data range. Black lines indicate the mean and the standard deviation. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. P. tetraurelia, central hub, n = 10, mean = 4.3 +/- 0.38 (S.D); N. gruberi, central hub, n = 10, mean = 4.4 +/- 0.53 (S.D); H. sapiens, central hub, n = 10, mean = 4.2 +/- 0.68 (S.D); P. tetraurelia, cartwheel inner density, n = 8, mean = 8.4 +/- 1.3 (S.D); N. gruberi, cartwheel inner density, n = 8, mean = 8.3 +/- 1.8 (S.D); H. sapiens, cartwheel inner density, n = 8, mean = 8.1 +/- 2.5 (S.D).(K Proximal protrusion length of the cartwheel beyond the microtubule triplets in C. reinhardtii, P. tetraurelia, N. gruberi, and H. sapiens. Internal cartwheel inside the microtubule barrel, dark blue (INT); external cartwheel beyond the microtubule wall, light blue (EXT). Mean values are displayed above each bar plot with the black lines indicating the standard deviation (K). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 +/- 3.4, internal cartwheel length 74.2 +/- 16.6; C. reinhardtii procentriole, n = 2, external cartwheel mean length = 52.8 +/- 22.0, internal cartwheel length 26.5 +/- 29.7; P. tetraurelia, n = 23, external cartwheel mean length = 11.5 +/- 9.2, internal cartwheel length 66.6 +/- 15.7; N. gruberi, n = 19, external cartwheel mean length = 41.0 +/- 22.5, internal cartwheel length 259.0 +/- 87.2; human procentriole, n = 7, external cartwheel length 23.1 +/- 10.3, internal cartwheel length 154.7 +/- 47.7. Error reported is standard deviation. Scale bar, 50 nm.The start of each microtubule wall is delineated by a dashed white line (L). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 +/- 3.4, internal cartwheel length 74.2 +/- 16.6; C. reinhardtii procentriole, n = 2, external cartwheel mean length = 52.8 +/- 22.0, internal cartwheel length 26.5 +/- 29.7; P. tetraurelia, n = 23, external cartwheel mean length = 11.5 +/- 9.2, internal cartwheel length 66.6 +/- 15.7; N. gruberi, n = 19, external cartwheel mean length = 41.0 +/- 22.5, internal cartwheel length 259.0 +/- 87.2; human procentriole, n = 7, external cartwheel length 23.1 +/- 10.3, internal cartwheel length 154.7 +/- 47.7. Error reported is standard deviation. Scale bar, 50 nm.


Figure 3 ( A ) Top view of cartwheel reconstruction from P . tetraurelia . Scale bar , 50 nm . Dark orange arrow marks the D2 - rod . Purple arrow marks part of the pinhead . Dotted yellow box with arrows denotes the central hub - focused reslice shown in panel B ; dashed white box with arrows denotes the spoke - focused reslice shown in panel D . ( B ) Reslice of central hub - containing region with spokes in P . tetraurelia . Scale bar , 50 nm . Dashed light yellow line denotes the zoomed view shown in panels C , black line with arrows indicates the ~ 25 nm repeat distance between D1 densities formed by merged spokes . Dark orange arrow marks the D2 - rod . ( C ) Zoomed view displaying periodic repeats of the central hub ( CH ) and several cartwheel inner densities ( CIDs ) in P . tetraurelia . CIDs , red arrowheads and red plot profile ; CH , blue arrowheads and blue plot profile . Overlay between CID and CH peaks is plotted on the right in red and blue . Mean distance between CID peaks , red 8 . 6 + / - . 8 ( n = 5 ) . Distances between CH peaks split into two distinct populations : smaller ( within a ring - pair ) , green 3 . 1 + / - 1 . 3 ( n = 6 ) ; larger ( between ring - pairs ) , blue 5 . 5 + / - . 7 ( n = 5 ) ; average periodicity , black 4 . 2 + / - 1 . 3 ( n = 11 ) . ( D ) Serial z - projections of ~ 4 nm thickness from one cartwheel repeat unit of P . tetraurelia . Left - most z - projections display the central hub , right - most projection shows the microtubule wall . Blue circles delineate one ring - pair . White arrows mark individual emanating spokes . White arrowheads mark fused spokes . White dashed line follows the tilt of the spokes . Black arrow indicates the final merged spoke ( D1 density ) . Dark orange arrow marks the D2 - rod . Scale bar , 50 nm . ( E ) Top view of cartwheel reconstruction from C . reinhardtii . Scale bar , 50 nm . Dark orange arrow marks the D2 - rod . Purple arrow marks part of the pinhead . Dotted yellow box with arrows denotes the central hub - focused reslice shown in panel F ; dashed white box with arrows denotes the spoke - focused reslice shown in panel H . ( F ) Reslice of central hub - containing region with spokes in C . reinhardtii . Scale bar , 50 nm . Dashed light yellow line denotes the zoomed view shown in panel G , black line with arrows indicates the ~ 25 nm repeat distance between D1 densities formed by merged spokes . Dark orange arrow marks the D2 - rod . ( G ) Zoomed view displaying periodic repeats of the central hub ( CH ) and several cartwheel inner densities ( CIDs ) in C . reinhardtii . CIDs , red arrowheads and red plot profile ; CH , blue arrowheads and blue plot profile . Overlay between CID and CH peaks is plotted on the right in red and blue . Mean distance between CID peaks , red 8 . 8 + / - 1 . 0 ( n = 5 ) . Distances between CH peaks split into two distinct populations : smaller ( within a ring - pair ) , green 3 . 5 + / - 1 . 3 ( n = 6 ) ; larger ( between ring - pairs ) , blue 5 . 1 + / - . 4 ( n = 5 ) ; average periodicity , black 4 . 2 + / - 1 . 3 ( n = 11 ) . ( H ) Serial z - projections of ~ 4 nm thickness from one cartwheel repeat unit of C . reinhardtii . Left - most z - projections display the central hub , right - most projection shows the microtubule wall . Blue circles delineate one ring - pair . White arrows mark individual emanating spokes . White arrowheads mark fused spokes . White dashed line follows the tilt of the spokes . Black arrow indicates the final merged spoke ( D1 density ) . Dark orange arrow marks the D2 - rod . Scale bar , 50 nm .

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

Figure 3(A) Top view of cartwheel reconstruction from P. tetraurelia. Scale bar, 50 nm. Dark orange arrow marks the D2-rod. Purple arrow marks part of the pinhead. Dotted yellow box with arrows denotes the central hub-focused reslice shown in panel B; dashed white box with arrows denotes the spoke-focused reslice shown in panel D.(B) Reslice of central hub-containing region with spokes in P. tetraurelia. Scale bar, 50 nm. Dashed light yellow line denotes the zoomed view shown in panels C, black line with arrows indicates the ~25 nm repeat distance between D1 densities formed by merged spokes. Dark orange arrow marks the D2-rod.(C) Zoomed view displaying periodic repeats of the central hub (CH) and several cartwheel inner densities (CIDs) in P. tetraurelia. CIDs, red arrowheads and red plot profile; CH, blue arrowheads and blue plot profile. Overlay between CID and CH peaks is plotted on the right in red and blue. Mean distance between CID peaks, red 8.6 +/- .8 (n= 5). Distances between CH peaks split into two distinct populations: smaller (within a ring-pair), green 3.1 +/- 1.3 (n= 6); larger (between ring-pairs), blue 5.5 +/- .7 (n= 5); average periodicity, black 4.2 +/- 1.3 (n= 11).(D) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of P. tetraurelia. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.(E) Top view of cartwheel reconstruction from C. reinhardtii. Scale bar, 50 nm. Dark orange arrow marks the D2-rod. Purple arrow marks part of the pinhead. Dotted yellow box with arrows denotes the central hub-focused reslice shown in panel F; dashed white box with arrows denotes the spoke-focused reslice shown in panel H.(F) Reslice of central hub-containing region with spokes in C. reinhardtii. Scale bar, 50 nm. Dashed light yellow line denotes the zoomed view shown in panel G, black line with arrows indicates the ~25 nm repeat distance between D1 densities formed by merged spokes. Dark orange arrow marks the D2-rod.(G) Zoomed view displaying periodic repeats of the central hub (CH) and several cartwheel inner densities (CIDs) in C. reinhardtii. CIDs, red arrowheads and red plot profile; CH, blue arrowheads and blue plot profile. Overlay between CID and CH peaks is plotted on the right in red and blue. Mean distance between CID peaks, red 8.8 +/- 1.0 (n= 5). Distances between CH peaks split into two distinct populations: smaller (within a ring-pair), green 3.5 +/- 1.3 (n= 6); larger (between ring-pairs), blue 5.1 +/- .4 (n= 5); average periodicity, black 4.2 +/- 1.3 (n= 11).(H) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of C. reinhardtii. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.


Figure 4 ( A ) Cryo - electron tomogram of P . tetraurelia centriole . Blue arrow denotes cartwheel , orange arrow denotes inner scaffold . White dashed box delimits the inset represented in ( D , E ) . Red lines and arrows indicate the position and the direction of the cross - sections made in ( B ) . Scale bar , 100 nm . ( B ) Nine - fold symmetrizations of serial cross - sections taken along the proximal to distal axis in P . tetraurelia . Each section is a z - projection of 20 . 7 nm . White dashed circles delineate the structures highlighted in C . Scale bar , 60 nm . ( C ) Zoomed images of proximal centriole substructures from nine - fold symmetrizations of P . tetraurelia along the proximal - distal axis . Each panel corresponds to the above image from panel B . Purple arrow , pinhead ; light green arrow , triplet base ; turquoise arrow , A - C linker ; orange arrow , inner scaffold . ( D ) Side view showing the transition from pinhead to inner scaffold in P . tetraurelia . ( G ) Cryo - electron tomogram of C . reinhardtii centriole . Blue arrow denotes cartwheel , orange arrow denotes inner scaffold . White dashed box delimits the inset represented in ( J , K ) . Red lines and arrows indicate the position and the direction of the cross - sections made in ( H ) . Scale bar , 100 nm . ( H ) Nine - fold symmetrizations of serial cross - sections taken along the proximal to distal axis in C . reinhardtii . Each section is a z - projection of 20 . 7 nm . White dashed circles delineate the structures highlighted in I . Scale bar , 60 nm . ( I ) Zoomed images of proximal centriole substructures from nine - fold symmetrizations of C . reinhardtii along the proximal - distal axis . Each panel corresponds to the above image from panel H . Purple arrow , pinhead ; light green arrow , triplet base ; turquoise arrow , A - C linker ; orange arrow , inner scaffold . ( J , K ) Side views showing the transition from pinhead to inner scaffold in C . reinhardtii from two consecutive sections from a tomogram . ( M , N ) Positioning of the different structures along the proximal length from representative P . tetraurelia ( M ) and C . reinhardtii ( N ) centrioles . Distance between the ends of the pinhead and cartwheel regions is denoted by zone 1 ( for quantification , see Figure EV4E ) . Distance between end of the pinhead region and start of the inner scaffold region is denoted by zone 2 ( O ) Cartwheel and A - C linker length in C . reinhardtii ( n = 5 centrioles ) , P . tetraurelia ( n = 16 centrioles ) , N . gruberi ( n = 11 centrioles ) , and H . sapiens ( n = 5 centrioles ) . Means and standard deviations of the mean are displayed above the range . A - C linker , turquoise ; cartwheel , blue ; microtubule triplets , grey .

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

Figure 4(A) Cryo-electron tomogram of P. tetraurelia centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (D, E). Red lines and arrows indicate the position and the direction of the cross-sections made in (B). Scale bar, 100 nm.(B) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in P. tetraurelia. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in C. Scale bar, 60 nm.(C) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of P. tetraurelia along the proximal-distal axis. Each panel corresponds to the above image from panel B. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.(D) Side view showing the transition from pinhead to inner scaffold in P. tetraurelia.(G) Cryo-electron tomogram of C. reinhardtii centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (J, K). Red lines and arrows indicate the position and the direction of the cross-sections made in (H). Scale bar, 100 nm.(H) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in C. reinhardtii. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in I. Scale bar, 60 nm.(I) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of C. reinhardtii along the proximal-distal axis. Each panel corresponds to the above image from panel H. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.(J, K) Side views showing the transition from pinhead to inner scaffold in C. reinhardtii from two consecutive sections from a tomogram.(M, N) Positioning of the different structures along the proximal length from representative P. tetraurelia (M) and C. reinhardtii (N) centrioles. Distance between the ends of the pinhead and cartwheel regions is denoted by zone 1 (for quantification, see Figure EV4E). Distance between end of the pinhead region and start of the inner scaffold region is denoted by zone 2(O) Cartwheel and A-C linker length in C. reinhardtii (n= 5 centrioles), P. tetraurelia (n= 16 centrioles), N. gruberi (n= 11 centrioles), and H. sapiens (n= 5 centrioles). Means and standard deviations of the mean are displayed above the range. A-C linker, turquoise; cartwheel, blue; microtubule triplets, grey.


Figure 1Representative immunofluorescence ( IF ) staining of Ki67 and TDP - 43 in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Nuclei were stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; upper panel in blue ) or neural marker NeuN ( lower panel in green ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Quantification of cells or neurons with Ki67 immunoreactivity and TDP - 43 mislocalization from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0007 by t - test . Representative data of reverse - transcription PCR ( left panel ) or Western blot ( right panel ) for cell cycle - related genes and semi - quantification of the expression levels in the frontal cortices and hippocampus from the 6 - mon - old WT and FTLD - TDP Tg mice . N = 5 mice per group , data are presented as mean ± SEM ( % ) , statistical analysis by multiple t - test with FDR correction , Q = 1 % . * P < 0 . 05 , * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 and * * * * P ≤ 0 .

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

Figure 1Representative immunofluorescence (IF) staining of Ki67 and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.Quantification of cells or neurons with Ki67 immunoreactivity and TDP-43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0007 by t-test.Representative data of reverse-transcription PCR (left panel) or Western blot (right panel) for cell cycle-related genes and semi-quantification of the expression levels in the frontal cortices and hippocampus from the 6-mon-old WT and FTLD-TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t-test with FDR correction, Q = 1%. *P < 0.05, **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001


Figure 2Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Scale bar : 50 μm . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * p = 0 . 0114 by t - test . Representative IF staining of γH2AX and TDP - 43 in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Nuclei were stained with DAPI ( upper panel in blue ) or NeuN ( middle panel in green ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Lower panel : quantification of cells or neurons with γH2AX immunoreactivity and TDP - 43 proteinopathies from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0008 by t - test . Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD - TDP Tg mice . Nuclei were stained with DAPI ( upper panel ) or NeuN ( middle panel ) . Scale bar : 50 μm . Subregions , scale bar : 15 μm . Lower panel : quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0004 by 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *p = 0.0114 by t-test.Representative IF staining of γH2AX and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP-43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0008 by t-test.Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0004 by t-test.


Figure 3Top , representative western blot data of HDAC1 and TDP - 43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 - mon - old of FTLD - TDP or WT mice . Bottom , semi - quantification of HDAC1 and TDP - 43 expression levels . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * * * * p < 0 . 0001 by multiple t - tests . Top , representative western blot data of HDAC1 and TDP - 43 in the cytosolic and nuclear extracts . Bottom , quantification of HDAC1 and TDP - 43 levels . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * * * p = 0 . 0005 ( TDP - 43 ) or 0 . 0004 ( HDAC1 ) ; * * * * p < 0 . 0001 by multiple t - tests . IF staining of HDAC1 and SMI - 32 in the regions of frontal cortices from WT and FTLD - TDP Tg mice . Scale bar : 150 μm . Blocked area in left bottom picture is showed in a magnified view in the right bottom picture . Scale bar : 50 μm . Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6 - mon - old of FTLD - TDP and WT mice . TSA : nuclear extracts that were treated with Trichostatin A ( TSA , an HDAC inhibitor ) as a negative control for HDAC1 - transferred fluorescent activity during the HDAC1 activity assay . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0415 by t - test . Left , representative western blot data of nuclear acetylated - histone H3 ( Lys 9 / 14 ) and total - histone H3 in 6 - mon - old of WT and FTLD - TDP Tg mice . Right , quantification of nuclear acetylated - histone H3 / total - histone H3 ratio . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0147 by 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Top, representative western blot data of HDAC1 and TDP-43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6-mon-old of FTLD-TDP or WT mice. Bottom, semi-quantification of HDAC1 and TDP-43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** p < 0.0001 by multiple t-tests.Top, representative western blot data of HDAC1 and TDP-43 in the cytosolic and nuclear extracts. Bottom, quantification of HDAC1 and TDP-43 levels. N = 5 mice per group, data are presented as mean ± SEM (%), *** p = 0.0005 (TDP-43) or 0.0004 (HDAC1); **** p < 0.0001 by multiple t-tests.IF staining of HDAC1 and SMI-32 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Scale bar: 150 μm. Blocked area in left bottom picture is showed in a magnified view in the right bottom picture. Scale bar: 50 μm.Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6-mon-old of FTLD-TDP and WT mice. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. N = 5 mice per group, data are presented as mean ± SEM (%), *p = 0.0415 by t-test.Left, representative western blot data of nuclear acetylated-histone H3 (Lys 9/14) and total-histone H3 in 6-mon-old of WT and FTLD-TDP Tg mice. Right, quantification of nuclear acetylated-histone H3/total-histone H3 ratio. N = 5 mice per group, data are presented as mean ± SEM (%), * p = 0.0147 by t-test.


Figure 4Left graph : IF staining of TDP - 43 and HDAC1 during progression of TDP - 43 proteinopathies in the frontal cortices from the FTLD - TDP Tg and WT mice . Nuclei were stained with DAPI ( in blue ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Right histogram : Quantification of co - localized TDP - 43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1 - , 6 - , and 12 - month - old FTLD - TDP Tg mice . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM . * Nucleus : Tg 1m vs . Tg 6m or 12m ; # Cytosol : Tg 1m vs . Tg 6m or 12m ; @ Tg 6m vs Tg 12m . * * * * / # # # # / @ @ @ @ p < 0 . 0001 by multiple comparison . Left graph : IF staining of γH2AX and HDAC1 in the frontal cortices from the 12 - month - old FTLD - TDP Tg and WT mice . Nuclei were stained with DAPI ( in blue ) . Arrow head : γH2AX + nucleus ; arrow : mislocalized HDAC1 . Scale bar : 50 μm . Right histogram : Quantification of γH2AX + and HDAC1 mislocalized cells in the WT or 1 - , 6 - , and 12 - month - old FTLD - TDP Tg mice . n = 4 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM . * * p = 0 . 0079 , * * * * p < 0 . 0001 by multiple comparison .

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

Figure 4Left graph: IF staining of TDP-43 and HDAC1 during progression of TDP-43 proteinopathies in the frontal cortices from the FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co-localized TDP-43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1m vs. Tg 6m or 12m; #Cytosol: Tg 1m vs. Tg 6m or 12m; @ Tg 6m vs Tg 12m. ****/####/@@@@ p < 0.0001 by multiple comparison.Left graph: IF staining of γH2AX and HDAC1 in the frontal cortices from the 12-month-old FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Arrow head: γH2AX+ nucleus; arrow: mislocalized HDAC1. Scale bar: 50 μm. Right histogram: Quantification of γH2AX+ and HDAC1 mislocalized cells in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 4 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. ** p = 0.0079, **** p < 0.0001 by multiple comparison.


Figure 5Left panel : flag - tagged full - length HDAC1 was overexpressed with myc - tagged TDP - 43 in HEK - 293T cells ; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP - 43 and flag . Right panel : myc - tagged TDP - 43 was overexpressed with flag - tagged full - length HDAC1 in HEK - 293T cells ; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP - 43 . Upper left : flag - tagged full - length HDAC1 ( b . Ⅰ ) or various truncation mutations ( b . Ⅱ - Ⅳ ) were overexpressed with myc - tagged TDP - 43 ; the catalytic domain is indicated in red . Lower panel : the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP - 43 . Upper panel : Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP - 43 in WT and FTLD - TDP Tg mice . Lower histogram : Quantification of immunoprecipitation results of HDAC1 and TDP - 43 in WT and Tg mice . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0149 , * * * p = 0 . 0003 by t - test . Western - blot of HDAC1 and TDP - 43 in urea - soluble fractions . N = 5 mice per group .

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

Figure 5Left panel: flag-tagged full-length HDAC1 was overexpressed with myc-tagged TDP-43 in HEK-293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP-43 and flag. Right panel: myc-tagged TDP-43 was overexpressed with flag-tagged full-length HDAC1 in HEK-293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP-43.Upper left: flag-tagged full-length HDAC1 (b.Ⅰ) or various truncation mutations (b.Ⅱ-Ⅳ) were overexpressed with myc-tagged TDP-43; the catalytic domain is indicated in red. Lower panel: the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP-43.Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP-43 in WT and FTLD-TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP-43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * p =0.0149, *** p =0.0003 by t-test.Western-blot of HDAC1 and TDP-43 in urea-soluble fractions. N = 5 mice per group.


Figure 6Escape latency of mice in Morris water maze task . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical analysis by two - way ANOVA with Bonferroni ' s multiple comparisons . # P < 0 . 05 , * * * P ≤ 0 . 001 and * * * * / # # # # P ≤ 0 . 0001 Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison . The representative searching path of mice in the probe test . Scores of mice with respect to the number of times they crossed the hidden platform , time spent searching in the target quadrant , and the velocity of swimming in the probe test at 24 h after escape training . * * p = 0 . 009 , * * * p = 0 . 0002 by multiple comparison . Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison . Scores of the discrimination index in the novel object recognition test . Scores of mice in the rotarod test . Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

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

Figure 6Escape latency of mice in Morris water maze task. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by two-way ANOVA with Bonferroni's multiple comparisons. #P < 0.05, ***P ≤ 0.001 and ****/####P ≤ 0.0001 Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.The representative searching path of mice in the probe test.Scores of mice with respect to the number of times they crossed the hidden platform, time spent searching in the target quadrant, and the velocity of swimming in the probe test at 24 h after escape training. ** p = 0.009, *** p = 0.0002 by multiple comparison. Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.Scores of the discrimination index in the novel object recognition test.   Scores of mice in the rotarod test.   Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.


Figure 7Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment . The nuclear extracts of the frontal cortices and hippocampus from 8 - mon - old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1 , and were then conducted to HDAC1 activity assay . N = 6 mice per group . TSA : nuclear extracts that were treated with Trichostatin A ( TSA , an HDAC inhibitor ) as a negative control for HDAC1 - transferred fluorescent activity during the HDAC1 activity assay . * p = 0 . 0203 ( WT vs Tg - Veh ) or 0 . 0408 ( Tg - Veh vs Tg + 5104434 ) by multiple comparison . Data information : All data are presented as mean ± SEM ( % Representative data of western blot for nuclear acetylated - histone 3 ( Lys 9 / 14 ) , total histone H3 , and quantification of protein levels . N = 6 mice per group . * p = 0 . 0105 or * * * p = 0 . 0008 by multiple comparison . Data information : All data are presented as mean ± SEM ( % ) Representative data of western blot for nuclear HDAC1 levels and semi - quantification of the expression levels . N = 6 mice per group . * * p = 0 . 0022 ( Tg ) / 0 . 0012 ( Tg + 5104434 ) by multiple comparison . Data information : All data are presented as mean ± SEM ( % ) Left panel : representative data of western blot for cell cycle - and DNA damage - related proteins . Right histogram : semi - quantification of protein levels . N = 6 per group . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical analysis by multiple t - test with FDR correction , Q = 1 % . * * P ≤ 0 . 01 ; # # # P ≤ 0 . 001 and * * * * / # # # # P ≤ 0 . 0001 . Data information : All data are presented as mean ± SEM ( % ) IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8 - mon - old of WT + Vehicle , Tg + Vehicle , Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * p = 0 . 0421 or * * * * p < 0 . 0001 by multiple comparison . Data information : number of cells in each view is presented . Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice , 8 - mon - old WT + Vehicle , Tg + Vehicle and Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * p = 0 . 0414 or * * * p = 0 . 0006 by multiple comparison . Data information : All data are presented as mean ± SEM ( % ) IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice , 12 - month - old WT + Vehicle , Tg + Vehicle and Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * * p = 0 . 002 and * * * * p < 0 . 0001 by multiple comparison . Data information : where number of cells in each view is presented .

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

Figure 7Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment. The nuclear extracts of the frontal cortices and hippocampus from 8-mon-old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1, and were then conducted to HDAC1 activity assay. N = 6 mice per group. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. * p = 0.0203 (WT vs Tg-Veh) or 0.0408 (Tg-Veh vs Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%Representative data of western blot for nuclear acetylated-histone 3 (Lys 9/14), total histone H3, and quantification of protein levels. N = 6 mice per group. * p = 0.0105 or *** p = 0.0008 by multiple comparison. Data information: All data are presented as mean ± SEM (%)Representative data of western blot for nuclear HDAC1 levels and semi-quantification of the expression levels. N = 6 mice per group. ** p = 0.0022 (Tg)/ 0.0012 (Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%)Left panel: representative data of western blot for cell cycle- and DNA damage-related proteins. Right histogram: semi-quantification of protein levels. N = 6 per group. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by multiple t-test with FDR correction, Q = 1%. **P ≤ 0.01; ###P ≤ 0.001 and ****/####P ≤ 0.0001. Data information: All data are presented as mean ± SEM (%)IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8-mon-old of WT+Vehicle, Tg+Vehicle, Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. * p = 0.0421 or **** p < 0.0001 by multiple comparison. Data information: number of cells in each view is presented.Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 8-mon-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm.* p = 0.0414 or *** p = 0.0006 by multiple comparison. Data information: All data are presented as mean ± SEM (%)IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 12-month-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. ** p = 0.002 and **** p < 0.0001 by multiple comparison. Data information: where number of cells in each view is presented.


Figure 8Representative IF staining of TDP - 43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD - TDP . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Quantification of cells with co - mislocalized HDAC1 and TDP - 43 from each view of microscope . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test . Linear regression analysis of cells with HDAC1 mislocalization and TDP - 43 proteinopathies . Total cell counts : 3000 per samples . P = 0 . 0001 by Pearson correlation analysis . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test . Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD - TDP from each view of microscope . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Quantification of cells with γH2AX and Ki67 immunoreactivity . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test . Linear regression analysis of cells with DNA damage and aberrant cell cycle activity . Cell counts : 40 - 113 ( normal ) and 418 - 887 ( FTLD ) per samples . p < 0 . 0001 by Pearson correlation analysis . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test . Dot - blot of HDAC1 and TDP - 43 in urea - soluble fractions from the frontal cortex of normal individuals and patients with FTLD - TDP . Quantification of HDAC1 and TDP - 43 expression levels in urea - soluble fractions . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Representative IF staining of TDP-43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD-TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.Quantification of cells with co-mislocalized HDAC1 and TDP-43 from each view of microscope. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.Linear regression analysis of cells with HDAC1 mislocalization and TDP-43 proteinopathies. Total cell counts: 3000 per samples. P = 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD-TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Cell counts: 40 -113 (normal) and 418-887 (FTLD) per samples. p < 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.Dot-blot of HDAC1 and TDP-43 in urea-soluble fractions from the frontal cortex of normal individuals and patients with FTLD-TDP.Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Figure 2 ( A ) Volcano plot of phosphorylation changes in iKras PDAC cells in response to KrasG12D induction . Phospho - to total proteomic changes were quantified from three independent biological replicates of Dox treated vs . untreated cells , via TMT ( Dataset EV8 ) . 872 phosphorylations were found to be significantly increased , while 82 phosphorylations were significantly decreased ( FDR < 0 . 05 ) . Significantly changing phosphorylations on RBPs whose RNA - binding activity was significantly increased ( red ) or decreased ( blue ) as per Fig 1C are highlighted on the plot . ( E ) KrasG12D induction enhances phosphorylation of CK2α ( T360 / S362 ) , in an Erk1 / 2 - dependent manner . IKras PDAC cells were grown in the absence of Dox for 48 hrs , before its addition to the indicated cells , with or without Trametinib ( 10 nM ) , for a further 24 hrs . Cells were then lysed and analyzed by immunoblotting ( IB ) with the indicated antibodies . The red triangle marks the main phospho band which overlaps with the total CK2α . ( G ) Analysis of the RNA - binding activity of WT , Phospho - defective ( S4A ) , and phospho - mimicking ( S4D ) mutants of Ncl by OOPS . IKras PDAC cells were transiently transfected with myc - tagged WT , S4A , and S4D Ncl constructs . Cells were subsequently grown in the absence of Dox for 48 hrs , followed by Dox addition for 24 hrs to induce KrasG12D expression . Cells were then subjected to OOPS to isolate the interface ( RNA - bound proteins ) , or whole cell lysis ( total lysate ) , followed by immunoblotting with anti - Ncl antibody . For comparison , Gapdh , which also binds RNA but does not show a change in its RNA - binding activity ( Dataset EV1 ) , was also blotted for . A fraction of each interface or total lysate sample was also subjected to RNA extraction , which was resolved and quantified by capillary electrophoresis ( CE ) as loading control .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot of phosphorylation changes in iKras PDAC cells in response to KrasG12D induction. Phospho- to total proteomic changes were quantified from three independent biological replicates of Dox treated vs. untreated cells, via TMT (Dataset EV8). 872 phosphorylations were found to be significantly increased, while 82 phosphorylations were significantly decreased (FDR < 0.05). Significantly changing phosphorylations on RBPs whose RNA-binding activity was significantly increased (red) or decreased (blue) as per Fig 1C are highlighted on the plot.(E) KrasG12D induction enhances phosphorylation of CK2α (T360/S362), in an Erk1/2-dependent manner. IKras PDAC cells were grown in the absence of Dox for 48 hrs, before its addition to the indicated cells, with or without Trametinib (10 nM), for a further 24 hrs. Cells were then lysed and analyzed by immunoblotting (IB) with the indicated antibodies. The red triangle marks the main phospho band which overlaps with the total CK2α.(G) Analysis of the RNA-binding activity of WT, Phospho-defective (S4A), and phospho-mimicking (S4D) mutants of Ncl by OOPS. IKras PDAC cells were transiently transfected with myc-tagged WT, S4A, and S4D Ncl constructs. Cells were subsequently grown in the absence of Dox for 48 hrs, followed by Dox addition for 24 hrs to induce KrasG12D expression. Cells were then subjected to OOPS to isolate the interface (RNA-bound proteins), or whole cell lysis (total lysate), followed by immunoblotting with anti-Ncl antibody. For comparison, Gapdh, which also binds RNA but does not show a change in its RNA-binding activity (Dataset EV1), was also blotted for. A fraction of each interface or total lysate sample was also subjected to RNA extraction, which was resolved and quantified by capillary electrophoresis (CE) as loading control.


Figure 3 ( B ) Immunofluorescence analysis of WT , S4A , and S4D Myc - Ncl subcellular localization . IKras PDAC cells ectopically expressing either WT , S4A , or S4D Myc - Ncl were fixed and immunostained with anti - Myc - tag antibody , along with anti - Fibrillarin ( Fbl ) antibody as a Nucleolar marker , and Hoechst , followed by confocal microscopy analysis . ( C ) Distribution of WT , S4A , and S4D Myc - Ncl crosslink sites across the annotated 47S pre - rRNA genomic region . Peak heights represent mean crosslink intensities from three independent iCLIP replicate experiments . ( D ) Quantification of the percentage of crosslinks in each region of the 47S pre - rRNA for WT , S4A , and S4D Myc - Ncl . Percentage of crosslinks relative to 47S total were quantified from three independent biological replicate iCLIP experiments . ( E ) Quantification of the proportional density of crosslinks in each region of the 47S pre - rRNA for WT , S4A , and S4D Myc - Ncl . Proportional density was calculated from three independent iCLIP experiments by normalizing the number of crosslinks in each region to the sequence length of that region .

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

Figure 3(B) Immunofluorescence analysis of WT, S4A, and S4D Myc-Ncl subcellular localization. IKras PDAC cells ectopically expressing either WT, S4A, or S4D Myc-Ncl were fixed and immunostained with anti-Myc-tag antibody, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker, and Hoechst, followed by confocal microscopy analysis.(C) Distribution of WT, S4A, and S4D Myc-Ncl crosslink sites across the annotated 47S pre-rRNA genomic region. Peak heights represent mean crosslink intensities from three independent iCLIP replicate experiments.(D) Quantification of the percentage of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Percentage of crosslinks relative to 47S total were quantified from three independent biological replicate iCLIP experiments. (E) Quantification of the proportional density of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Proportional density was calculated from three independent iCLIP experiments by normalizing the number of crosslinks in each region to the sequence length of that region.


Figure 4 ( A ) Nascent RNA imaging in control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , grown for 48hrs in presence or absence of Dox , prior to pulse labeling with FUrd . Cells were then fixed and immunostained with anti - FUrd antibody ( green ) to visualize nascent RNA , along with anti - Fibrillarin ( Fbl ) antibody as a Nucleolar marker ( red ) , and Hoechst ( blue ) as the Nuclear stain , followed by confocal microscopy analysis . ( B ) Quantification of Nucleolar FUrd levels in images from ( A ) . FUrd fluorescence densities in single nucleoli were quantified from 182 - 220 individual cells per condition , combined from 2 independent biological replicate experiments ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . ( C ) RT - qPCR analysis of 5 ' ETS - containing pre - rRNA transcript levels in control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , and grown for 48hrs in presence or absence of Dox , before RT - qPCR analysis with a specific probe against the mouse 5 ' ETS region . A probe against mouse Actb mRNA was used as loading control for normalization . A total of 4 biological replicate experiments were quantified ( * * * : P < 0 . 001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . ( E ) Assessment of the overall protein synthesis rates in control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D , by puromycinylation . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , and grown for 48hrs in presence or absence of Dox , before pulse labelling with Puromycin ( 10 μg / ml ) for 15 min to label nascent proteins . Cells were subsequently lysed and analyzed by immunoblotting with the indicated antibodies . ( F ) Quantification of puromycin incorporation from ( E ) , as an indicator of the overall protein synthesis rate . Total Erk1 / 2 levels were used as loading control . A total of 4 independent biological replicate experiments were quantified . Error bars depict SD . ( * * : P < 0 . 01 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . ( ( G ) Nascent RNA imaging of WT , S4A , and S4D Myc - Ncl expressing iKras PDAC cells , in presence or absence of KrasG12D . Vectors encoding Myc - tagged WT , S4A , and S4D Ncl were transiently transfected into iKras PDAC cells , before reseeding and growing the cells for 48hrs in presence or absence of Dox . Cells were then subjected to pulse labeling with FUrd , fixation , and immunostaining with anti - FUrd antibody ( green ) , anti - Myc - tag antibody ( red ) , and Hoechst ( blue ) , followed by confocal microscopy analysis . Scale bar = 10 µm . ( H ) Quantification of FUrd levels in Myc - positive nucleoli from ( E ) . FUrd fluorescence densities in single nucleoli were quantified from 160 - 281 individual cells per condition , combined from three independent biological replicate experiments ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . ( ( I ) RT - qPCR analysis of 5 ' ETS - containing pre - rRNA transcript levels in WT , S4A , and S4D Myc - Ncl expressing iKras PDAC cells , in presence or absence of KrasG12D . Vectors encoding Myc - tagged WT , S4A , and S4D Ncl were transiently transfected into iKras PDAC cells , before reseeding and growing the cells for 48hrs in presence or absence of Dox , followed by RT - qPCR analysis with a specific probe against the mouse 5 ' ETS region . A probe against mouse Actb mRNA was used as loading control for normalization . A total of 5 to 8 biological replicate experiments per condition were quantified ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) .

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

Figure 4(A) Nascent RNA imaging in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, grown for 48hrs in presence or absence of Dox, prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody (green) to visualize nascent RNA, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 182-220 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).(C) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 4 biological replicate experiments were quantified (***: P < 0.001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).(E) Assessment of the overall protein synthesis rates in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D, by puromycinylation. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before pulse labelling with Puromycin (10 μg/ml) for 15 min to label nascent proteins. Cells were subsequently lysed and analyzed by immunoblotting with the indicated antibodies. (F) Quantification of puromycin incorporation from (E), as an indicator of the overall protein synthesis rate. Total Erk1/2 levels were used as loading control. A total of 4 independent biological replicate experiments were quantified. Error bars depict SD. (**: P < 0.01; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). ((G) Nascent RNA imaging of WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox. Cells were then subjected to pulse labeling with FUrd, fixation, and immunostaining with anti-FUrd antibody (green), anti-Myc-tag antibody (red), and Hoechst (blue), followed by confocal microscopy analysis. Scale bar = 10 µm. (H) Quantification of FUrd levels in Myc-positive nucleoli from (E). FUrd fluorescence densities in single nucleoli were quantified from 160-281 individual cells per condition, combined from three independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). ((I) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox, followed by RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 5 to 8 biological replicate experiments per condition were quantified (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).


Figure 5 ( A ) Colony formation of control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , followed by clonogenic assay for 7 days in presence or absence of Dox . Colonies were visualized by Crystal Violet staining . ( B ) Quantification of Crystal Violet staining levels from ( A ) . A total of 6 biological replicate experiments were quantified . Error bars depict SD . ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from two way ANOVA with Šídák ' s multiple comparisons test ) . ( ( C ) 3D proliferation of control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , before being reseeded onto 3D Collagen - I gels , with or without Dox , and allowed to grow for 48 hrs . Cells were subsequently imaged live by phase contrast microscopy . Scale bar = 200 µm . ( D ) Analysis of the relative percentage of viable cells in 3D collagen - I cultures from ( C ) . Cells were subjected to luminescence - based viability assay by CellTiter - Glo to quantify the percentage of viable cells . A total of 3 biological replicate experiments were quantified . Error bars depict SD . ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from two way ANOVA with Šídák ' s multiple comparisons test ) . (

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 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

Figure 5(A) Colony formation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, followed by clonogenic assay for 7 days in presence or absence of Dox. Colonies were visualized by Crystal Violet staining. (B) Quantification of Crystal Violet staining levels from (A). A total of 6 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). ((C) 3D proliferation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, before being reseeded onto 3D Collagen-I gels, with or without Dox, and allowed to grow for 48 hrs. Cells were subsequently imaged live by phase contrast microscopy. Scale bar = 200 µm. (D) Analysis of the relative percentage of viable cells in 3D collagen-I cultures from (C). Cells were subjected to luminescence-based viability assay by CellTiter-Glo to quantify the percentage of viable cells. A total of 3 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). (


Figure 6 ( A ) Dose response analysis of CX - 5461 impact on nascent rRNA expression . IKras PDAC cells were grown in the absence of Dox for 48hrs . Cells were subsequently treated with or without Dox for 24 hrs to induce KrasG12D expression , followed by pre - treatment with the indicated concentrations of CX - 5461 for 30 mins prior to pulse labeling with FUrd . Cells were then fixed and immunostained with anti - FUrd antibody to visualize nascent RNA ( green ) , anti - Ncl antibody to reveal the Nucleolus ( red ) , and Hoechst ( blue ) as the Nuclear stain , followed by confocal microscopy analysis . ( B ) Quantification of Nucleolar FUrd levels in images from ( A ) . FUrd fluorescence densities in single nucleoli were quantified from 251 - 479 individual cells per condition , combined from 2 independent biological replicate experiments ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . ( G ) MRI imaging of orthotopic iKras tumors in untreated or CX - 5461 treated mice . IKras PDAC cells were engrafted into the pancreas of Dox - fed nude mice and allowed to form tumors for 4 days . Animals were then divided into two groups , with the first group treated by daily oral administration of CX - 5461 ( 50mg / kg ) for a further 10 days , whilst the second group was left untreated for the same period . T2 scans were taken on the indicated days , post - engraftment . Green areas mark the tumors . ( K ) H & E analysis of the extracted pancreas tissues from ( I ) . Representative pancreatic tissue images from the untreated and CX - 5461 treated mice , showing typical PDAC histology with malignant ductal structures surrounded by stroma in the untreated , but a largely normal pancreas histology with a small malignant ( M ) component ( marked by the dashed line ) in the treated mice .

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

Figure 6(A) Dose response analysis of CX-5461 impact on nascent rRNA expression. IKras PDAC cells were grown in the absence of Dox for 48hrs. Cells were subsequently treated with or without Dox for 24 hrs to induce KrasG12D expression, followed by pre-treatment with the indicated concentrations of CX-5461 for 30 mins prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody to visualize nascent RNA (green), anti-Ncl antibody to reveal the Nucleolus (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 251-479 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).(G) MRI imaging of orthotopic iKras tumors in untreated or CX-5461 treated mice. IKras PDAC cells were engrafted into the pancreas of Dox-fed nude mice and allowed to form tumors for 4 days. Animals were then divided into two groups, with the first group treated by daily oral administration of CX-5461 (50mg/kg) for a further 10 days, whilst the second group was left untreated for the same period. T2 scans were taken on the indicated days, post-engraftment. Green areas mark the tumors.(K) H&E analysis of the extracted pancreas tissues from (I). Representative pancreatic tissue images from the untreated and CX-5461 treated mice, showing typical PDAC histology with malignant ductal structures surrounded by stroma in the untreated, but a largely normal pancreas histology with a small malignant (M) component (marked by the dashed line) in the treated mice.


Figure 1B . The truncated receptor encoded by RNA1 is endogenously expressed . HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C ( RNA1 ) . SDS - lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 ( Figure EV1 ) . Mousehypothalamus was exposed 10 times longer . In brain , a band around 75kDa of unknown origin is detected by the antiserum ( indicated by a star ) . D . RT - PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition . Below is a quantification of three independent experiments . A statistical evaluation showed significant differences between oligo # 5 ( p = 0 . 00008 ) and oligo # 5 - 3 ( p = 0 . 0019 ) and control .

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 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

Figure 1B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).D. RT-PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition. Below is a quantification of three independent experiments. A statistical evaluation showed significant differences between oligo#5 (p=0.00008) and oligo#5-3 (p= 0.0019) and control.


Figure 2A . Titration of oligo # 5 . An increasing amount of oligo # 5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT - PCR . - RT : negative control without reverse transcriptase . B . Quantification of three independent experiments . The half - maximal effect was seen at 13 . 1 nM concentration . C . Influence of oligo # 5 shortening on alternative splicing of the serotonin 2C receptor . Oligo # 5 : 5 ' AGU AUU GAG CAU AGC CGC 3 ' ( 18mer ) ; Oligo # 5 - 3 : 5 ' GAG CAU AGC CGC 3 ' ( 12mer ) ; Oligo # 5 - 10 : 5 ' G CAU AGC CGC 3 ' ( 10mer ) were used in transfection assays analyzed by RT - PCR . D . Statistical analysis of the effect of oligonucleotide shortening . The effect of all oligos testes is significant , oligo # 5 : p = 0 . 0015 ; oligo # 5 - 3 : p = 0 . 031 ; oligo # 5 - 10 : p = 0 . 048 , n = 6 . E . Influence of cy3 label on the 5 ' and 3 ' end of oligo # 5 . Using a phosphate linker , Cy3 was added to the 5 ' or 3 ' phosphate , respectively and the oligonucleotides assayed by RT - PCR . Oligo # 5 with cy3 at the 5 ' end showed 86 % inclusion , with Cy3 at the 3 ' end 17 % exon inclusion . F . Statistical analysis of the effect of oligonucleotide modification . The effect is significant , p = 0 . 009 , n = 4 .

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 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 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

Figure 2A. Titration of oligo#5. An increasing amount of oligo#5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT-PCR. -RT: negative control without reverse transcriptase.B. Quantification of three independent experiments. The half-maximal effect was seen at 13.1 nM concentration.C. Influence of oligo#5 shortening on alternative splicing of the serotonin 2C receptor. Oligo#5: 5' AGU AUU GAG CAU AGC CGC 3' (18mer); Oligo#5-3: 5' GAG CAU AGC CGC 3' (12mer); Oligo#5-10: 5' G CAU AGC CGC 3' (10mer) were used in transfection assays analyzed by RT-PCR.D. Statistical analysis of the effect of oligonucleotide shortening. The effect of all oligos testes is significant, oligo#5: p= 0.0015; oligo#5-3: p=0.031; oligo#5-10: p= 0.048, n=6.E. Influence of cy3 label on the 5' and 3' end of oligo#5. Using a phosphate linker, Cy3 was added to the 5' or 3' phosphate, respectively and the oligonucleotides assayed by RT-PCR. Oligo#5 with cy3 at the 5' end showed 86% inclusion, with Cy3 at the 3' end 17% exon inclusion.F. Statistical analysis of the effect of oligonucleotide modification. The effect is significant, p=0.009, n=4.


Figure 3D . In vitro splicing assay : Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract ( NE ) under splicing conditions for two hours at 30 ºC . The structures of the in vitro splicing products are shown on the right . LI and LII are two lariats formed . Numbers indicate the expected product lengths . E . Quantification of selected bands from four independent experiments . The percentage of each form in the total products is shown . The differences of splice products ( Va + b - MINX ) is significant , p = 0 . 0055 ; n = 3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3D. In vitro splicing assay: Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract (NE) under splicing conditions for two hours at 30 ºC. The structures of the in vitro splicing products are shown on the right. LI and LII are two lariats formed. Numbers indicate the expected product lengths.E. Quantification of selected bands from four independent experiments. The percentage of each form in the total products is shown. The differences of splice products (Va+b-MINX) is significant, p=0.0055; n=3.