Figure 1B - D Left ventricular LXRα expression in Wt and LXRα - Tg mice aged 12 weeks . ( B ) Western blot of LXRα in liver and LV tissue . B - D Left ventricular LXRα expression in Wt and LXRα - Tg mice aged 12 weeks . ( B ) Western blot of LXRα in liver and LV tissue . Quantification of ( C ) mRNA expression ( normalized to 36b4 ; n = 6 - 8 / group , * P < 0 . 00001 versus Wt ) and ( D ) protein expression ( normalized to GAPDH ; n = 4 / group , * P = 0 . 0001 versus Wt ) . E LV weight to tibia length ratios ( LV / tibia ) ; n = 15 / group . * P = 0 . 02 . F Assessment of mean arterial pressure ( MAP ) with in situ catheterization shows no significant differences . G Representative Masson ' s trichrome and wheat germ agglutinin ( WGA ) - FITC staining in cross - sectional LV , scale bars = 100 μm and 10 μm , respectively . H Relative mRNA levels of known LXRα - associated and target genes assessed with RT - PCR ; n = 8 / group . * P = 0 . 0003 , # P = 0 . 0004 , § P = 0 . 043 , † P = 0 . 0009 versus Wt .

O B-PANEL_START O O O O O O 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 B-PANEL_START O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue.B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue. Quantification of (C) mRNA expression (normalized to 36b4; n = 6-8/group, *P < 0.00001 versus Wt) and (D) protein expression (normalized to GAPDH; n = 4/group, *P = 0.0001 versus Wt).E LV weight to tibia length ratios (LV/tibia); n = 15/group. *P = 0.02.F Assessment of mean arterial pressure (MAP) with in situ catheterization shows no significant differences.G Representative Masson's trichrome and wheat germ agglutinin (WGA)-FITC staining in cross-sectional LV, scale bars = 100 μm and 10 μm, respectively.H Relative mRNA levels of known LXRα-associated and target genes assessed with RT-PCR; n = 8/group. *P = 0.0003, #P = 0.0004, §P = 0.043, †P = 0.0009 versus Wt.


Figure 2LV / tibia ratios in sham - and TAC - operated Wt and LXRα - Tgmice ; n = 21 Wt sham , n = 24 LXRα - Tg sham , n = 24 Wt TAC , n = 26 LXRα - TgTAC . * P < 0 . 00001 versus respective sham , # P = 0 . 00004 . Quantification of cardiomyocyte cross - sectional area from WGA - FITC - stained histological sections ; n = 5 Wt sham , n = 4 LXRα - Tg sham , n = 5 Wt TAC , n = 3 LXRα - TgTAC . * * P = 0 . 00001 versus Wt sham , * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 01 . Representative LV sections stained with WGA - FITC and Masson ' s trichrome to assess cardiomyocyte hypertrophy and fibrosis , respectively ; scale bars = 1 mm , 100 μm , 10 μm , from top to bottom . Whole area percentage of fibrosis in Masson ' s trichrome - stained sections ; n = 8 per group , except n = 7 in Wt TAC group . * P = 0 . 02 versus Wt sham , # P = 0 . 02 . Measurement of mRNA levels with RT - PCR to assess induction of fetal gene program , as well as hypertrophy ( Rcan1 ) and fibrosis ( Col1a1 , Ctgf ) ; n = 8 per group , except n = 7 in Wt TAC group . Myh6 / Myh7 : * P = 0 . 0001 versus Wt sham , # P = 0 . 00001 ; Anp : * P = 0 . 0005 versus LXRα - Tg sham , # P = 0 . 02 ; Bnp : * P = 0 . 0006 versus Wt sham , * * P = 0 . 009 versus LXRα - Tg sham , # P = 0 . 03 ; Acta1 : § P < 0 . 00001 versus Wt sham , * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 001 ; Rcan1 : * * P = 0 . 00004 versus Wt sham , * P = 0 . 05 versus LXRα - Tg sham , # P = 0 . 04 ; Col1a1 : * P = 0 . 00003 versus Wt sham ; Ctgf : § P < 0 . 00001 versus Wt sham , # P = 0 . 00001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2LV/tibia ratios in sham- and TAC-operated Wt and LXRα-Tgmice; n = 21 Wt sham, n = 24 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. *P < 0.00001 versus respective sham, #P = 0.00004.Quantification of cardiomyocyte cross-sectional area from WGA-FITC-stained histological sections; n = 5 Wt sham, n = 4 LXRα-Tg sham, n = 5 Wt TAC, n = 3 LXRα-TgTAC. **P = 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.01.Representative LV sections stained with WGA-FITC and Masson's trichrome to assess cardiomyocyte hypertrophy and fibrosis, respectively; scale bars = 1 mm, 100 μm, 10 μm, from top to bottom.Whole area percentage of fibrosis in Masson's trichrome-stained sections; n = 8 per group, except n = 7 in Wt TAC group. *P = 0.02 versus Wt sham, #P = 0.02.Measurement of mRNA levels with RT-PCR to assess induction of fetal gene program, as well as hypertrophy (Rcan1) and fibrosis (Col1a1, Ctgf); n = 8 per group, except n = 7 in Wt TAC group. Myh6/Myh7: *P = 0.0001 versus Wt sham, #P = 0.00001; Anp: *P = 0.0005 versus LXRα-Tg sham, #P = 0.02; Bnp: *P = 0.0006 versus Wt sham, **P = 0.009 versus LXRα-Tg sham, #P = 0.03; Acta1: §P < 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.001; Rcan1: **P = 0.00004 versus Wt sham, *P = 0.05 versus LXRα-Tg sham, #P = 0.04; Col1a1: *P = 0.00003 versus Wt sham; Ctgf: §P < 0.00001 versus Wt sham, #P = 0.00001.


Figure 3A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC ; n = 19 Wt sham , n = 22 LXRα - Tg sham , n = 24 Wt TAC , n = 26 LXRα - TgTAC . * * P < 0 . 00001 versus Wt sham , * P = 0 . 0005 versus LXRα - Tg sham , # P = 0 . 009 . B - D Hemodynamic measurements obtained in situ in sham - and TAC - operated mice ; n = 6 - 8 / group . ( B ) Mean arterial pressure ( MAP ) showed no significant differences . ( C ) LV end - systolic pressure ( LVESP ) . * * P < 0 . 00001 versus Wt sham , * P = 0 . 00004 versus LXRα - Tg sham , * P = 0 . 003 . ( D ) LV end - diastolic pressure ( LVEDP ) . * P = 0 . 02 versus Wt sham . B - D Hemodynamic measurements obtained in situ in sham - and TAC - operated mice ; n = 6 - 8 / group . ( B ) Mean arterial pressure ( MAP ) showed no significant differences . ( C ) LV end - systolic pressure ( LVESP ) . * * P < 0 . 00001 versus Wt sham , * P = 0 . 00004 versus LXRα - Tg sham , * P = 0 . 003 . ( D ) LV end - diastolic pressure ( LVEDP ) . * P = 0 . 02 versus Wt sham .

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

Figure 3A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC; n = 19 Wt sham, n = 22 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. **P < 0.00001 versus Wt sham, *P = 0.0005 versus LXRα-Tg sham, #P = 0.009.B-D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6-8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham.B-D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6-8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham.


Figure 4Representative 18F - FDG images with microPET in the coronal and axial planes in Wt and LXRα - Tg mice 5 weeks post - TAC . Myocardial FDG uptake measured as standard uptake value ( SUV ) ; n = 4 - 6 / group . * P = 0 . 04 versus Wt sham , * * P = 0 . 02 versus LXRα - Tg sham , # P = 0 . 03 , # # P = 0 . 01 . Quantification of myocardial glycogen content shows no significant differences ; n = 5 per sham group , n = 7 per TAC group . GLUT protein expression in LV tissue normalized to GAPDH ; n = 6 / group . Glut1 : * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 03 ; Glut4 : * P = 0 . 03 versus LXRα - Tg sham , # P = 0 . 01 .

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

Figure 4Representative 18F-FDG images with microPET in the coronal and axial planes in Wt and LXRα-Tg mice 5 weeks post-TAC.Myocardial FDG uptake measured as standard uptake value (SUV); n = 4-6/group. *P = 0.04 versus Wt sham, **P = 0.02 versus LXRα-Tg sham, #P = 0.03, ##P = 0.01.Quantification of myocardial glycogen content shows no significant differences; n = 5 per sham group, n = 7 per TAC group.GLUT protein expression in LV tissue normalized to GAPDH; n = 6/group. Glut1: *P = 0.01 versus LXRα-Tg sham, #P = 0.03; Glut4: *P = 0.03 versus LXRα-Tg sham, #P = 0.01.


Figure 5A LV / tibia ratios in sham - and TAC - operated WT and LXRα − / − mice ; n = 10 WT sham , n = 8 LXRα − / − sham , n = 12 WT TAC , n = 10 LXRα − / − TAC . * P = 0 . 006 versus WT sham , * * P = 0 . 00008 versus LXRα − / − sham . B , C Cardiac functional assessment at 5 weeks post - TAC . ( B ) Percent fractional shortening determined with echocardiography ; n = 10 WT sham , n = 7 LXRα − / − sham , n = 12 WT TAC , n = 10 LXRα − / − TAC . * P < 0 . 00001 versus respective sham , § P = 0 . 06 . ( C ) LV end - diastolic pressure ( LVEDP ) recorded in situ ; n = 10 WT sham , n = 7 LXRα − / − sham , n = 10 WT TAC , n = 9 LXRα − / − TAC . * P = 0 . 001 versus WT sham , * * P = 0 . 00005 versus LXRα − / − sham . D - G Relative mRNA gene expression as determined by RT - PCR , normalized to 36b4 ; n = 8 / group . Anp : no significant differences ; Bnp : * P = 0 . 01 versus WT sham , * * P = 0 . 03 versus LXRα − / − sham ; Myh6 / Myh7 : * P = 0 . 0002 versus WT sham , * * P = 0 . 0003 versus LXRα − / − sham ; Acta1 : * P = 0 . 001 versus respective sham . H Western blot detection of GLUT protein expression in LV tissue normalized to GAPDH , expressed as fold change , shows no significant differences among groups ; n = 6 - 8 / group . I Representative 18F - FDG - microPET scans . J Myocardial FDG uptake quantified as standard uptake value ( SUV ) ; n = 6 / group . * P = 0 . 0001 versus WT sham , # P = 0 . 047 .

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

Figure 5A LV/tibia ratios in sham- and TAC-operated WT and LXRα−/− mice; n = 10 WT sham, n = 8 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P = 0.006 versus WT sham, **P = 0.00008 versus LXRα−/−sham.B, C Cardiac functional assessment at 5 weeks post-TAC. (B) Percent fractional shortening determined with echocardiography; n = 10 WT sham, n = 7 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P < 0.00001 versus respective sham, §P = 0.06. (C) LV end-diastolic pressure (LVEDP) recorded in situ; n = 10 WT sham, n = 7 LXRα−/− sham, n = 10 WT TAC, n = 9 LXRα−/−TAC. *P = 0.001 versus WT sham, **P = 0.00005 versus LXRα−/− sham.D-G Relative mRNA gene expression as determined by RT-PCR, normalized to 36b4; n = 8/group. Anp: no significant differences; Bnp: *P = 0.01 versus WT sham, **P = 0.03 versus LXRα−/− sham; Myh6/Myh7: *P = 0.0002 versus WT sham, **P = 0.0003 versus LXRα−/− sham; Acta1: *P = 0.001 versus respective sham.H Western blot detection of GLUT protein expression in LV tissue normalized to GAPDH, expressed as fold change, shows no significant differences among groups; n = 6-8/group.I Representative 18F-FDG-microPET scans.J Myocardial FDG uptake quantified as standard uptake value (SUV); n = 6/group. *P = 0.0001 versus WT sham, #P = 0.047.


Figure 6A Assessment of 2 - deoxyglucose ( 2 - DG ) uptake from 4 independent experiments . * P = 0 . 03 versus Ad - cont . B , C Glut mRNA expression determined by RTD Western blot indicating Ad - LXRα - and PE - induced increases in global protein O - GlcNAcylation , which was abrogated following inhibition of HBP with DON . LXRα protein expression is shown , and GAPDH served as a loading control . E , F Modulation of Anp and Bnp mRNA levels by Ad - LXRα - induced O - GlcNAc signaling . Gene expression as determined by RT - PCR normalized to 36b4 , n = 5 per condition in the absence of PE , n = 4 per condition in the presence of PE . * P = 0 . 02 versus Ad - cont , * * P = 0 . 008 versus Ad - cont , # P = 0 . 03 , # # P = 0 . 02 . G Measurement of cell size , n = 5 per condition . * P = 0 . 008 versus Ad - cont , # P = 0 . 02 , # # P = 0 . 03 . H Representative images for the determination of cell size . Cells were stained with an antibody specific for LXRα ( green , indicated by arrow ) , DAPI for nuclei ( blue ) , and rhodamine - phalloidin for F - actin ( red ) ; scale bar = 50 μm .

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

Figure 6A Assessment of 2-deoxyglucose (2-DG) uptake from 4 independent experiments. *P = 0.03 versus Ad-cont.B, C Glut mRNA expression determined by RTD Western blot indicating Ad-LXRα- and PE-induced increases in global protein O-GlcNAcylation, which was abrogated following inhibition of HBP with DON. LXRα protein expression is shown, and GAPDH served as a loading control.E, F Modulation of Anp and Bnp mRNA levels by Ad-LXRα-induced O-GlcNAc signaling. Gene expression as determined by RT-PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.02.G Measurement of cell size, n = 5 per condition. *P = 0.008 versus Ad-cont, #P = 0.02, ##P = 0.03.H Representative images for the determination of cell size. Cells were stained with an antibody specific for LXRα (green, indicated by arrow), DAPI for nuclei (blue), and rhodamine-phalloidin for F-actin (red); scale bar = 50 μm.


Figure 7A Protein synthesis determined via leucine incorporation , n = 2 - 4 independent experiments . * P = 0 . 03 versus Ad - cont , § P = 0 . 06 versus Ad - cont . B Global O - GlcNAc protein expression with Western blot , GAPDH served as a control . C - F mRNA expression , normalized to 36b4 , n = 6 per condition , except n = 5 for si - LXRα control . Anp : * P = 0 . 009 versus Ad - cont , # P = 0 . 004 , # # P = 0 . 009 ; Bnp : * P = 0 . 004 versus Ad - cont , * * P = 0 . 002 versus Ad - cont ; Acta1 : * P = 0 . 002 versus Ad - cont , § P = 0 . 06 versus Ad - cont , # P = 0 . 02 ; Rcan1 : * P = 0 . 03 versus Ad - cont , * * P = 0 . 004 versus Ad - cont , # P = 0 . 004 .

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

Figure 7A Protein synthesis determined via leucine incorporation, n = 2-4 independent experiments. *P = 0.03 versus Ad-cont, §P = 0.06 versus Ad-cont.B Global O-GlcNAc protein expression with Western blot, GAPDH served as a control.C-F mRNA expression, normalized to 36b4, n = 6 per condition, except n = 5 for si-LXRα control. Anp: *P = 0.009 versus Ad-cont, #P = 0.004, ##P = 0.009; Bnp: *P = 0.004 versus Ad-cont, **P = 0.002 versus Ad-cont; Acta1: *P = 0.002 versus Ad-cont, §P = 0.06 versus Ad-cont, #P = 0.02; Rcan1: *P = 0.03 versus Ad-cont, **P = 0.004 versus Ad-cont, #P = 0.004.


Figure 8A , B Western blot analyses of global protein O - GlcNAc levels in left ventricles of mice with either ( A ) cardiac - specific LXRα overexpression or ( B ) LXRα deficiency and subjected to 5 weeks TAC . C , D Nuclear protein extracts were precipitated with agarose WGA in the absence or presence of GlcNAc , a competitor , and analyzed by Western blot with antibodies against Mef2c , GATA4 , or Nkx - 2 . 5 , known transcription factors of natriuretic peptides ; bands represent 3 pooled hearts per Wt and LXRα - Tg lanes , and ( D ) quantification is for n = 2 , expressed as fold change .

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

Figure 8A, B Western blot analyses of global protein O-GlcNAc levels in left ventricles of mice with either (A) cardiac-specific LXRα overexpression or (B) LXRα deficiency and subjected to 5 weeks TAC.C, D Nuclear protein extracts were precipitated with agarose WGA in the absence or presence of GlcNAc, a competitor, and analyzed by Western blot with antibodies against Mef2c, GATA4, or Nkx-2.5, known transcription factors of natriuretic peptides; bands represent 3 pooled hearts per Wt and LXRα-Tg lanes, and (D) quantification is for n = 2, expressed as fold change.


Figure 1B . Coronal section of a P3 spheroid embedded with paraffin and stained with DAPI ( blue ) , LDHA ( green ) , and LDHB ( red ) . Magnification boxes show different areas as depicted in the main image . Quantification of LDHA and LDHB staining was performed on the spheroid areas as indicated in the graphs . Scale bar : 100 µm . Data are represented as mean ± s . d . ( 5 - 8 fields per area of interest , one representative spheroid of 3 independent invading spheroids )

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Figure 1B. Coronal section of a P3 spheroid embedded with paraffin and stained with DAPI (blue), LDHA (green), and LDHB (red). Magnification boxes show different areas as depicted in the main image. Quantification of LDHA and LDHB staining was performed on the spheroid areas as indicated in the graphs. Scale bar: 100 µm. Data are represented as mean ± s.d. (5-8 fields per area of interest, one representative spheroid of 3 independent invading spheroids)


O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of LDHA and LDHB from P3 cells upon exposure to 21% or 0.1% O2 for 6, 24, 48, and 72 h. The graphs represent densitometry quantification of immunoblots normalized to tubulin (n = 4 independent experiments). Data are represented as mean ± s.d. and analyzed using One-Way ANOVA following Dunnett's multiple comparisons tests. LDHA: O2 21% vs O2 0.1% 6h, P>0.99; O2 21% vs O2 0.1% 24h, P=0.15; O2 21% vs O2 0.1% 48h, P=0.0009; O2 21% vs O2 0.1% 72h, P<0.0001. LDHB: O2 21% vs O2 0.1% 6h, P>0.99; O2 21% vs O2 0.1% 24h, P>0.99; O2 21% vs O2 0.1% 48h, P=0.97; O2 21% vs O2 0.1% 72h, P=0.88.


Figure 3A . Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR - Cas9 lentiviral vectors with scramble sequence ( sgControl ) or against LDHA ( sgLDHA ) , LDHB ( sgLDHB ) or both ( sgLDHA / B ) . Knockout ( KO ) cells were exposed to 21 % or 1 % O2 for 48 h . D . Left : P3 spheroid growth recorded over one week at 1 % O2 ( n = 3 independent experiments , one experiment including 16 spheroids per condition ) . Data are represented as mean and analyzed using Kruskal - Wallis test followed by Dunn ' s multiple comparisons test . Spheroid growth : sgCont vs sgLDHA , P = 0 . 0003 ; sgCont vs sgLDHB , P = 0 . 78 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Dead area : sgCont vs sgLDHA , P = 0 . 19 ; sgCont vs sgLDHB , P < 0 . 0001 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Right : Viability of spheroids at one week , incubated with calcein ( green ) or ethidium homodimer - 1 ( red ) . Scale bar : 200 µm . E . P3 spheroid invasion in collagen I gel , incubated 24 h at 21 % or 0 . 1 % O2 . Invasion rate is expressed as fold change from controls ( n = 4 independent experiments , one experiment including 6 - 8 spheroids per condition ) . Data are represented as mean and analyzed using Two - way ANOVA following by Tukey ' s multiple comparisons test : sgCont 21 % vs sgLDHA 21 % , P = 0 . 003 ; sgCont 0 . 1 % vs sgLDHA 0 . 1 % , P < 0 . 0001 ; sgCont 21 % vs sgLDHB 21 % , P < 0 . 0001 ; sgCont 0 . 1 % vs sgLDHB 0 . 1 % , P < 0 . 0001 ; sgCont 21 % vs sgLDHA / B 21 % , P < 0 . 0001 ; sgCont 0 . 1 % vs sgLDHA / B 0 . 1 % , P < 0 . 0001 . Images of representative of control or LDHA / B KO spheroids . Scale bar : 100 µm . F - G . A first cohort of mice was sacrificed when one mouse reached a limit point , brains was extract , snap frozen in liquid nitrogen , sliced and stained . For the second cohort , each mouse was sacrificed when it reached a limit point allowing the draw of survival curves . Left : Immunofluorescence staining of Nestin ( top and middle ) and CD31 ( bottom ) of control and LDHA / B KO P3 tumor section . Scale bars : 2 mm ( top ) , 250 µm ( middle ) and 100 µm ( bottom ) . Right : Graphs represent tumor core and invasion area of control and LDHA / B KO P3 tumors in mm2 ( n = 5 mice per group , average of 5 - 6 sections per tumor ) . Data are represented as mean . For F , data are analyzed using One - way ANOVA following by Dunnett ' s multiple comparisons test . Core : sgCont vs sgLDHA , P = 0 . 48 ; sgCont vs sgLDHB , P = 0 . 99 . Invasion : sgCont vs sgLDHA , P = 0 . 40 ; sgCont vs sgLDHB , P = 0 . 81 . For G , data are analyzed using unpaired t test . Core : sgCont vs sgLDHA / B , P = 0 . 005 . Invasion : sgCont vs sgLDHA / B , P = 0 . 01 .

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

Figure 3A. Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR-Cas9 lentiviral vectors with scramble sequence (sgControl) or against LDHA (sgLDHA), LDHB (sgLDHB) or both (sgLDHA/B). Knockout (KO) cells were exposed to 21% or 1% O2 for 48 h.D. Left: P3 spheroid growth recorded over one week at 1% O2 (n = 3 independent experiments, one experiment including 16 spheroids per condition). Data are represented as mean and analyzed using Kruskal-Wallis test followed by Dunn's multiple comparisons test. Spheroid growth: sgCont vs sgLDHA, P=0.0003; sgCont vs sgLDHB, P=0.78; sgCont vs sgLDHA/B, P<0.0001. Dead area: sgCont vs sgLDHA, P=0.19; sgCont vs sgLDHB, P<0.0001; sgCont vs sgLDHA/B, P<0.0001. Right: Viability of spheroids at one week, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 200 µm.E. P3 spheroid invasion in collagen I gel, incubated 24 h at 21% or 0.1% O2. Invasion rate is expressed as fold change from controls (n = 4 independent experiments, one experiment including 6-8 spheroids per condition). Data are represented as mean and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont 21% vs sgLDHA 21%, P=0.003; sgCont 0.1% vs sgLDHA 0.1%, P<0.0001; sgCont 21% vs sgLDHB 21%, P<0.0001; sgCont 0.1% vs sgLDHB 0.1%, P<0.0001; sgCont 21% vs sgLDHA/B 21%, P<0.0001; sgCont 0.1% vs sgLDHA/B 0.1%, P<0.0001. Images of representative of control or LDHA/B KO spheroids. Scale bar: 100 µm.F-G. A first cohort of mice was sacrificed when one mouse reached a limit point, brains was extract, snap frozen in liquid nitrogen, sliced and stained. For the second cohort, each mouse was sacrificed when it reached a limit point allowing the draw of survival curves. Left: Immunofluorescence staining of Nestin (top and middle) and CD31 (bottom) of control and LDHA/B KO P3 tumor section. Scale bars: 2 mm (top), 250 µm (middle) and 100 µm (bottom). Right: Graphs represent tumor core and invasion area of control and LDHA/B KO P3 tumors in mm2 (n = 5 mice per group, average of 5-6 sections per tumor). Data are represented as mean. For F, data are analyzed using One-way ANOVA following by Dunnett's multiple comparisons test. Core: sgCont vs sgLDHA, P=0.48; sgCont vs sgLDHB, P=0.99. Invasion: sgCont vs sgLDHA, P=0.40; sgCont vs sgLDHB, P=0.81. For G, data are analyzed using unpaired t test. Core: sgCont vs sgLDHA/B, P=0.005. Invasion: sgCont vs sgLDHA/B, P=0.01.


Figure 5A . Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21 % or 0 . 1 % O2 . Roman numbers indicate the respiratory complex number . The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21 % and 0 . 1 % O2 ( n = 3 independent experiments ) . B . Epifluorescence ( top ) and quantitative image analysis ( bottom ) of immune stained mitochondria ( TOM20 , green ) from LDH KO P3 cells ( Phalloidin , red ; DAPI , blue ) upon exposure to 0 . 1 % O2 ( n = 2 independent experiments , 28 - 32 cells per group ) . Data are represented as mean and analyzed using One - way ANOVA following by Tukey ' s multiple comparisons test . Mitochondrial mass : sgCont vs sgLDHA / B , P = 0 . 005 ; sgLDHA / sgLDHB vs sgLDHA / B , p = 0 . 006 . Network aspect ratio : sgCont vs sgLDHA / B , P = 0 . 03 ; sgLDHA vs sgLDHA / B , P = 0 . 02 ; sgLDHB vs sgLDHA / B , p = 0 . 04 . Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area . Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network . Scale bar : 10 µm . E . Kaplan - Meier survival curves of xenotransplanted mice with LDHA / B KO ( red ) or control ( blue ) P3 cells , treated with phenformin or irradiated with 10 Gy ( n = 6 - 8 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) test : sgCont Vehicle vs sgCont Phenformin , P = 0 . 019 ; sgCont Vehicle vs sgCont Irradiation , P = 0 . 0002 ; sgCont Irradiation vs sgLDHA / B Irradiation , P = 0 . 024 .

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

Figure 5A. Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21% or 0.1% O2. Roman numbers indicate the respiratory complex number. The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21% and 0.1% O2 (n = 3 independent experiments).B. Epifluorescence (top) and quantitative image analysis (bottom) of immune stained mitochondria (TOM20, green) from LDH KO P3 cells (Phalloidin, red; DAPI, blue) upon exposure to 0.1% O2 (n = 2 independent experiments, 28-32 cells per group). Data are represented as mean and analyzed using One-way ANOVA following by Tukey's multiple comparisons test. Mitochondrial mass: sgCont vs sgLDHA/B, P=0.005; sgLDHA/sgLDHB vs sgLDHA/B, p=0.006. Network aspect ratio: sgCont vs sgLDHA/B, P=0.03; sgLDHA vs sgLDHA/B, P=0.02; sgLDHB vs sgLDHA/B, p=0.04. Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area. Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network. Scale bar: 10 µm.E. Kaplan-Meier survival curves of xenotransplanted mice with LDHA/B KO (red) or control (blue) P3 cells, treated with phenformin or irradiated with 10 Gy (n = 6-8 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: sgCont Vehicle vs sgCont Phenformin, P=0.019; sgCont Vehicle vs sgCont Irradiation, P=0.0002; sgCont Irradiation vs sgLDHA/B Irradiation, P=0.024.


Figure 6D . Left : P3 spheroid growth recorded over 72 h during incubation with stiripentol at 0 . 1 % O2 ( n = 3 independent experiments , one experiment including 8 spheroids per condition ) . Data are represented as mean ± s . d . and growth at 72 h are analyzed using unpaired t test : P < 0 . 0001 . Right : Viability of spheroids at 72 h , incubated with calcein ( green ) or ethidium homodimer - 1 ( red ) . Scale bar : 100 µm . E . Left : P3 spheroid invasion in collagen I gel , incubated 24 h with 500 µM stiripentol at 0 . 1 % O2 . Invasion rate is expressed as fold change from control ( n = 3 independent experiments , one experiment including 8 spheroids per condition ) . Data are represented as mean and analyzed using unpaired t test : P < 0 . 0001 . Right : Representative images of invasive spheroids for each condition . Scale bar : 100 µm . F . Kaplan - Meier survival curves of xenotransplanted mice with P3 cells . Mice were treated either with vehicle ( blue ) or stiripentol ( red ) ( n = 15 - 16 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) test : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D. Left: P3 spheroid growth recorded over 72 h during incubation with stiripentol at 0.1% O2 (n = 3 independent experiments, one experiment including 8 spheroids per condition). Data are represented as mean ± s.d. and growth at 72 h are analyzed using unpaired t test: P<0.0001. Right: Viability of spheroids at 72 h, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 100 µm. E. Left: P3 spheroid invasion in collagen I gel, incubated 24 h with 500 µM stiripentol at 0.1% O2. Invasion rate is expressed as fold change from control (n = 3 independent experiments, one experiment including 8 spheroids per condition). Data are represented as mean and analyzed using unpaired t test: P<0.0001. Right: Representative images of invasive spheroids for each condition. Scale bar: 100 µm.F. Kaplan-Meier survival curves of xenotransplanted mice with P3 cells. Mice were treated either with vehicle (blue) or stiripentol (red) (n = 15-16 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: P=0.002


figf1a ) The number of somatic mutations discovered from each paired NPC case subjected to WES and key clinical parameters ( Supplementary Table 2a ) clustered according to the clinical stage . Indel , insertion or deletion . ( b ) Trinucleotide contexts of somatic mutations occurring at cytosine nucleotides in NPC . The font size of the nucleotides at the 3 ′ and 5 ′ positions is proportional to their frequencies ( Supplementary Fig . 3b ) . ( c ) Rates of arm - level and focal SCNVs ( Online Methods ) across different types of malignancies . Data regarding SCNVs of the following types were summarized from Zack et al . : RCC , renal cell carcinoma ; GBM , glioblastoma multiforme ; UCEC , uterine cervix ; COAD , colorectal adenocarcinoma ; BRCA , breast ; HNSC , head and neck squamous cell ; OV , ovary ; LUAD , lung adenocarcinoma ; BLCA , bladder ; LUSC , lung squamous cell .

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

figf1a) The number of somatic mutations discovered from each paired NPC case subjected to WES and key clinical parameters (Supplementary Table 2a) clustered according to the clinical stage. Indel, insertion or deletion. (b) Trinucleotide contexts of somatic mutations occurring at cytosine nucleotides in NPC. The font size of the nucleotides at the 3′ and 5′ positions is proportional to their frequencies (Supplementary Fig. 3b).(c) Rates of arm-level and focal SCNVs (Online Methods) across different types of malignancies. Data regarding SCNVs of the following types were summarized from Zack et al.: RCC, renal cell carcinoma; GBM, glioblastoma multiforme; UCEC, uterine cervix; COAD, colorectal adenocarcinoma; BRCA, breast; HNSC, head and neck squamous cell; OV, ovary; LUAD, lung adenocarcinoma; BLCA, bladder; LUSC, lung squamous cell.


figf2Data matrix showing frequent SCNVs , significantly mutated genes ( bold ; Benjamini - Hochberg FDR 0 . 2 calculated with CHASM ) and their associated pathway or family mutations discovered in cases with NPC analyzed by WES , targeted deep sequencing ( TS ) or SNP array ( SNP - a ) . The frequencies of the alterations are plotted on the right . The colors and shapes denoting different types of somatic events are also applied in Figure 5a . Columns , examined cases ; rows , genes ; hot spot , identical mutations have been registered in COSMIC ( see URLs ) ; LOH , loss of heterozygosity . The asterisk indicates that this tumor was also subjected to SNP array profiling . PIK3CA amplification in C666 cells has been previously described .

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

figf2Data matrix showing frequent SCNVs, significantly mutated genes (bold; Benjamini-Hochberg FDR 0.2 calculated with CHASM) and their associated pathway or family mutations discovered in cases with NPC analyzed by WES, targeted deep sequencing (TS) or SNP array (SNP-a). The frequencies of the alterations are plotted on the right. The colors and shapes denoting different types of somatic events are also applied in Figure 5a. Columns, examined cases; rows, genes; hot spot, identical mutations have been registered in COSMIC (see URLs); LOH, loss of heterozygosity. The asterisk indicates that this tumor was also subjected to SNP array profiling. PIK3CA amplification in C666 cells has been previously described.


figf3 ( b , c ) Significant correlations between dysregulation of chromatin modification and total number of somatic mutations ( b ) and overall survival probability ( c ) . Mann - Whitney test ( b ) and log - rank test ( c ) were used to calculate statistical significance . Horizontal bars , mean value ; HR , hazard ratio .

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figf3(b,c) Significant correlations between dysregulation of chromatin modification and total number of somatic mutations (b) and overall survival probability (c). Mann-Whitney test (b) and log-rank test (c) were used to calculate statistical significance. Horizontal bars, mean value; HR, hazard ratio.


figf4 ( a - d ) NPC cells stably expressing either negative control shRNA ( Scramble ) or pooled shRNAs targeting ARID1A ( shARID1A ) were subjected to anchorage - independent colony formation assay ( a ) , migration assay ( b ) , xenograft growth assay ( horizontal lines , mean values ) and western blot ( WB ) assay with the indicated antibodies ( d ) . ( e - g ) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation assay ( e ) ( e - g ) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation assay ( e ) , migration assay ( f ) e - g ) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation assay ( e ) , migration assay ( f ) and WB assay with the indicated antibodies ( g ) . ( h , i ) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage - independent colony formation assay ( h ) h , i ) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage - independent colony formation assay ( h ) and WB assay with indicated antibodies ( i ) . β - actin was examined as a loading control . The data shown in a , b , e , f and h represent the mean ± s . d . ; n = 3 for a , b , e and f ; n = 4 for h . * P 0 . 05 , * * P 0 . 01 . Student ' s t - test was used to calculate statistical significance in a - c , e , f and h ; the blots shown in d , g and i are representative of three total blots run .

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

figf4(a-d) NPC cells stably expressing either negative control shRNA (Scramble) or pooled shRNAs targeting ARID1A (shARID1A) were subjected to anchorage-independent colony formation assay (a), migration assay (b), xenograft growth assay (horizontal lines, mean values) and western blot (WB) assay with the indicated antibodies (d).(e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e)(e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f)e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f) and WB assay with the indicated antibodies (g).(h,i) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage-independent colony formation assay (h)h,i) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage-independent colony formation assay (h) and WB assay with indicated antibodies (i). β-actin was examined as a loading control. The data shown in a, b, e, f and h represent the mean ± s.d.; n = 3 for a, b, e and f; n = 4 for h. *P 0.05, **P 0.01. Student's t-test was used to calculate statistical significance in a-c, e, f and h; the blots shown in d, g and i are representative of three total blots run.


figf5 ( a ) Data matrix showing alterations of genes involved in the ERBB - PI3K signaling pathway and their related targeting molecules . Considering space limitations , the numbers of targeting agents available for each protein are shown in parentheses . Drugs currently in phase 2 clinical trials are marked in blue , those in phase 3 are in purple , and FDA - approved drugs are in brown ( see URLs ) . ( b ) Kaplan - Meier survival curves of NPC subjects either with or without genetic lesions in the ERBB - PI3K signaling pathway . Log - rank test was used to calculate statistical significance .

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

figf5(a) Data matrix showing alterations of genes involved in the ERBB-PI3K signaling pathway and their related targeting molecules. Considering space limitations, the numbers of targeting agents available for each protein are shown in parentheses. Drugs currently in phase 2 clinical trials are marked in blue, those in phase 3 are in purple, and FDA-approved drugs are in brown (see URLs).(b) Kaplan-Meier survival curves of NPC subjects either with or without genetic lesions in the ERBB-PI3K signaling pathway. Log-rank test was used to calculate statistical significance.


Figure 1Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs . Images of mESCs transduced with Rptor and Rictor shRNAs in 2iL . Scale bar , 400 µm . Relative proliferation of mESCs as in ( E ) ( mean values ± SD , P value was calculated using two - tailed unpaired Student ' s t test , * P < 0 . 05 , also hereafter in similar experiments ) . n = 3 biological replicates . Western blotting for RPTOR and the indicated pluripotency proteins in mESCs as in ( H ) ( left ) , and quantification of pluripotency proteins ( right ) ( mean values ± SD ) . n = 3 biological replicates . Images of mESCs transduced with Rptor siRNA on different days in 2iL . siNC , transduced with a negative control ; day 0 was the day of siRNA transfection , also hereafter in similar experiments . Scale bar , 400 µm .

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Figure 1Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs.Images of mESCs transduced with Rptor and Rictor shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, *P < 0.05, also hereafter in similar experiments). n = 3 biological replicates.  Western blotting for RPTOR and the indicated pluripotency proteins in mESCs as in (H) (left), and quantification of pluripotency proteins (right) (mean values ± SD). n = 3 biological replicates.Images of mESCs transduced with Rptor siRNA on different days in 2iL. siNC, transduced with a negative control; day 0 was the day of siRNA transfection, also hereafter in similar experiments. Scale bar, 400 µm.


Figure 2RNA - seq heatmaps of the indicated pluripotency gene sets in control and INK128 ( 200 nM ) treated mESCs in 2iL . RNA - seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs in 2iL .

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Figure 2RNA-seq heatmaps of the indicated pluripotency gene sets in control and INK128 (200 nM) treated mESCs in 2iL.   RNA-seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs in 2iL.


Figure 3Images of mESCs transduced with Eif4a1 , Eif4e and Eif4g1 shRNAs in 2iL . Scale bar , 400 µm . Relative proliferation of mESCs as in ( B ) ( mean values ± SD ) . n = 3 biological replicates . Western blotting for the indicated shRNAs and pluripotency proteins as in ( B ) ( left ) and quantification ( right ) ( mean values ± SD ) . n = 3 biological replicates .

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Figure 3Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (B) (mean values ± SD). n = 3 biological replicates.  Western blotting for the indicated shRNAs and pluripotency proteins as in (B) (left) and quantification (right) (mean values ± SD). n =3 biological replicates.


Figure 4Relative proliferation of mESCs as in ( B ) ( mean values ± SD , P value was calculated using two - tailed unpaired Student ' s t test , * * P < 0 . 01 , also hereafter in similar experiments ) . n = 3 biological replicates . Images of mESCs transduced with Larp1 shRNAs in 2iL , in control or INK128 ( 200 nM ) treated for three days . Scale bar , 400 µm . Relative proliferation of mESCs as in ( E ) ( mean values ± SD ) . n = 3 biological replicates .

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Figure 4Relative proliferation of mESCs as in (B) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, **P < 0.01, also hereafter in similar experiments). n = 3 biological replicates.Images of mESCs transduced with Larp1 shRNAs in 2iL, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.


Figure 5Relative enrichment heatmaps of mitochondrial ribosome subunit and ETC complex component mRNAs as in ( A ) . RT - qPCR for pluripotency and mitochondrial ribosome subunit genes as in ( E ) ( mean values ± SD ) . n = 3 biological replicates .

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Figure 5Relative enrichment heatmaps of mitochondrial ribosome subunit and ETC complex component mRNAs as in (A).RT-qPCR for pluripotency and mitochondrial ribosome subunit genes as in (E) (mean values ± SD). n = 3 biological replicates.


Figure 6OCR analysis of INK128 ( 200 nM ) treated mESCs in 2iL . qPCR for mtDNA copy number in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL ( mean values ± SD ) . n = 3 biological replicates . Western blotting for the indicated pluripotency proteins in mESCs as in ( F ) ( left ) , and quantification ( right ) ( mean values ± SD ) . n = 3 biological replicates .

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Figure 6OCR analysis of INK128 (200 nM) treated mESCs in 2iL.qPCR for mtDNA copy number in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.Western blotting for the indicated pluripotency proteins in mESCs as in (F) (left), and quantification (right) (mean values ± SD). n = 3 biological replicates.


Figure 7Images of mESCs transduced with Rptor or Rictor shRNAs in SL . Scale bar , 400 µm . Relative proliferation of mESCs as in ( A ) ( mean values ± SD ) . n = 3 biological replicates . Images of mESCs transduced with Eif4a1 , Eif4e and Eif4g1 shRNAs in SL . Scale bar , 400 µm . Western blotting for EIF4E , EIF4G1 and the indicated pluripotency proteins in cells with the indicated KD in the indicated culture conditions .

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Figure 7Images of mESCs transduced with Rptor or Rictor shRNAs in SL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (A) (mean values ± SD). n = 3 biological replicates.  Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in SL. Scale bar, 400 µm.Western blotting for EIF4E, EIF4G1 and the indicated pluripotency proteins in cells with the indicated KD in the indicated culture conditions.


Figure 1A . Single grey - scale and coloured merged images of immunocytochemical staining against PSD - 95 , synaptophysin - 1 ( SYP1 ) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse . Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 ( n = 12 neurons from 3 different cultures ) . B . Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse . Presynaptic compartments are indicated in pink while postsynaptic in blue . White arrows show the 15 - nm gold particles linked to NPC1 ( d - dendrite , sv - synaptic vesicles , m - mitochondria ) . C , D . Western blots against NPC1 and actin - β ( ACTB ) in total brain ( C ) and synaptosomes ( D ) from wt and NPC1nmf164 mice . Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control ( wt ) values ( n = 4 mice , 3 month - old , unpaired Student t test , * * psynaptosomes = 0 . 0037 ) . E . Western blots against NPC1 and actin - β ( ACTB ) in total brain extracts from wt and Npc1 - null mice ( NPC1KO ) containing the same amount of protein ( n = 2 mice , 6 week - old ) . F . Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice ( n = 4 mice , 3 month - old ) . G . Western blots against NPC1 and actin - β ( ACTB ) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide ( CHX ) . Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control ( wt non cLTP induced ) values ( n = 3 mice , 3 month - old , 2 - way ANOVA , * * * p < 0 . 001 ) . H . To the left Western blots against NPC1 and actin - β ( ACTB ) in synaptosomal extracts from wt and NPC1nmf164 mice used as input for the immunoprecipitation assays shown in the right . Immunoprecipitates were pulled down with an anti - ubiquitin antibody and without it for negative control and analysed by western blot against NPC1 . MG132 was used as proteasome inhibitor . Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs ( left ) ( n = 3 , one - way ANOVA , * pWT + MG - 132 = 0 . 0496 , pNPC1nmf1 + MG - 132 = 0 . 0358 ) and of NPC1 associated to ubiquitin ( right ) ( n = 3 mice , 3 month - old , one - way ANOVA , * pNPC1nmf164 = 0 . 0237 , pNPC1nmf164 + MG - 132 = 0 . 0386 .

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Figure 1A. Single grey-scale and coloured merged images of immunocytochemical staining against PSD-95, synaptophysin-1 (SYP1) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse. Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 (n=12 neurons from 3 different cultures).B. Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse. Presynaptic compartments are indicated in pink while postsynaptic in blue. White arrows show the 15-nm gold particles linked to NPC1 (d-dendrite, sv-synaptic vesicles, m-mitochondria).C, D. Western blots against NPC1 and actin-β (ACTB) in total brain (C) and synaptosomes (D) from wt and NPC1nmf164 mice. Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt) values (n=4 mice, 3 month-old, unpaired Student t test, **psynaptosomes= 0.0037).E. Western blots against NPC1 and actin-β (ACTB) in total brain extracts from wt and Npc1-null mice (NPC1KO) containing the same amount of protein (n=2 mice, 6 week-old).F. Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice (n=4 mice, 3 month-old).G. Western blots against NPC1 and actin-β (ACTB) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non cLTP induced) values (n=3 mice, 3 month-old, 2-way ANOVA, ***p < 0.001).H. To the left Western blots against NPC1 and actin-β (ACTB) in synaptosomal extracts from wt and NPC1nmf164 mice used as input for the immunoprecipitation assays shown in the right. Immunoprecipitates were pulled down with an anti-ubiquitin antibody and without it for negative control and analysed by western blot against NPC1. MG132 was used as proteasome inhibitor. Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs (left) (n=3, one-way ANOVA, *pWT+MG-132=0.0496, pNPC1nmf1+MG-132=0.0358) and of NPC1 associated to ubiquitin (right) (n=3 mice, 3 month-old, one-way ANOVA, *pNPC1nmf164= 0.0237, pNPC1nmf164+MG-132= 0.0386.


Figure 2A . Representative electron micrographs of synapses in the hippocampal CA1 region in wt and NPC1nmf164 mice ( black arrows indicate post - synaptic densities , d - dendrite , sv - synaptic vesicles , m - mitochondria , MLB - multilamellar bodies ) . B . Mean ± SEM synapse density ( n = 3 mice , 3 month - old , unpaired Student t test , * p = 0 . 0377 ) . C . Mean ± SEM synaptic vesicle density ( 3 , 088 vesicles from 101 wt and 121 NPC1nmf164 synapses from 3 different mice ( 3 month - old ) per group , unpaired Student t test , * * * p < 0 . 0001 ) . D . Synaptic vesicle diameter ( 528 wt and 507 NPC1nmf164 vesicles from 3 different mice ( 3 month - old ) per group ) . E . Mean ± SEM post - synaptic density length ( 101 wt and 121 NPC1nmf164 post - synaptic densities from 3 different mice ( 3 month - old ) per group , unpaired Student t test , * * * p < 0 . 0001 ) . F . Mean ± SEM post - synaptic density thickness ( n = 3 mice , 3 month - old , unpaired Student t test , * * * p < 0 . 0001 ) . G . Mean ± SEM cholesterol levels in synaptosomes ( n = 4 mice , 3 month - old , unpaired Student t test , * p = 0 . 0476 ) from wt and NPC1nmf164 mice expressed as percentage of wt mice .

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Figure 2A. Representative electron micrographs of synapses in the hippocampal CA1 region in wt and NPC1nmf164 mice (black arrows indicate post-synaptic densities, d-dendrite, sv-synaptic vesicles, m-mitochondria, MLB-multilamellar bodies).B. Mean ± SEM synapse density (n=3 mice, 3 month-old, unpaired Student t test, *p = 0.0377).C. Mean ± SEM synaptic vesicle density (3,088 vesicles from 101 wt and 121 NPC1nmf164 synapses from 3 different mice (3 month-old) per group, unpaired Student t test, ***p < 0.0001).D. Synaptic vesicle diameter (528 wt and 507 NPC1nmf164 vesicles from 3 different mice (3 month-old) per group).E. Mean ± SEM post-synaptic density length (101 wt and 121 NPC1nmf164 post-synaptic densities from 3 different mice (3 month-old) per group, unpaired Student t test, ***p < 0.0001).F. Mean ± SEM post-synaptic density thickness (n=3 mice, 3 month-old, unpaired Student t test, ***p < 0.0001).G. Mean ± SEM cholesterol levels in synaptosomes (n=4 mice, 3 month-old, unpaired Student t test, *p = 0.0476) from wt and NPC1nmf164 mice expressed as percentage of wt mice.


Figure 3The following properties of Schaffer collateral - CA1 synaptic responses were recorded from wt and NPC1nmf164 mice . A . Basal synaptic transmission expressed as mean ± SEM EPSP slope ( n = 9 slices from 5 WT mice and n = 8 slices from 5 NPC1nmf164 mice , all mice 10 week - old , unpaired Student t test , * p150μA = 0 . 0368 , p200μA = 0 . 0138 ) ; B . Mean ± SEM fibre volley amplitude ( n = 10 slices from 5 WT mice and n = 7 slices from 5 NPC1nmf164 mice , all mice 10 week - old ) ; C . Mean ± SEM paired pulse facilitation ( n = 11 slices from 5 WT mice and n = 10 slices from 5 NPC1nmf164 mice , all mice 10 week - old , unpaired Student t test , * * p = 0 . 0011 ) ; D . LTP expressed as mean ± SEM percentage of EPSP slope over baseline ( n = 7 slices form 5 WT mice for WT and n = 10 slices from 5 NPC1nmf164 mice , all mice 10 week - old , unpaired Student t test , * * * p < 0 . 0001 in the last 10 min recording ) The following properties of Schaffer collateral - CA1 synaptic responses were recorded from wt and NPC1nmf164 mice . E . LTD expressed as mean ± SEM percentage of EPSP slope over baseline ( n = 7 slices from 5 WT mice and n = 12 slices from 5 NPC1nmf164 mice , all mice 10 week - old ) . The behavioural tests were recorded from wt and NPC1nmf164 mice . F . Mean ± SEM exploration time of objects in novel and familiar location in the Object Placement Recognition test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * p = 0 . 0167 ) . The behavioural tests were recorded from wt and NPC1nmf164 mice . G . Mean ± SEM percentage of entry to the novel arm of the Y maze test ( n = 10 mice , 10 week - old ; unpaired Student t test , * * p = 0 . 0015 ) . H . Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test ( n = 10 mice , 10 week - old ; unpaired Student t test , * * * p = 0 . 0002 ) . I . Mean ± SEM distance covered during a 5 min period in the Open Field test ( n = 10 mice , 10 week - old ) .

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Figure 3The following properties of Schaffer collateral-CA1 synaptic responses were recorded from wt and NPC1nmf164 mice. A. Basal synaptic transmission expressed as mean ± SEM EPSP slope (n= 9 slices from 5 WT mice and n=8 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, * p150μA = 0.0368, p200μA = 0.0138); B. Mean ± SEM fibre volley amplitude (n=10 slices from 5 WT mice and n= 7 slices from 5 NPC1nmf164 mice, all mice 10 week-old); C. Mean ± SEM paired pulse facilitation (n=11 slices from 5 WT mice and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, **p = 0.0011); D. LTP expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices form 5 WT mice for WT and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, ***p < 0.0001 in the last 10 min recording) The following properties of Schaffer collateral-CA1 synaptic responses were recorded from wt and NPC1nmf164 mice. E. LTD expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice and n=12 slices from 5 NPC1nmf164 mice, all mice 10 week-old).The behavioural tests were recorded from wt and NPC1nmf164 mice. F. Mean ± SEM exploration time of objects in novel and familiar location in the Object Placement Recognition test (n=10 mice, 10 week-old, 2-way ANOVA, *p = 0.0167).The behavioural tests were recorded from wt and NPC1nmf164 mice. G. Mean ± SEM percentage of entry to the novel arm of the Y maze test (n=10 mice, 10 week-old; unpaired Student t test, **p = 0.0015). H. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old; unpaired Student t test, ***p = 0.0002). I. Mean ± SEM distance covered during a 5 min period in the Open Field test (n=10 mice, 10 week-old).


Figure 4A . Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction ( n = 6 , unpaired Student t test , * pwt = 0 . 0402B . Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh - scr or sh - NPC1 RNAs . Black bars indicate that the sh - NPC1 lane was not consecutive to the others but belongs to the same Western blot . Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt , non - transfected controls . C . Representative images of the cholesterol binding probe mCherry - D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh - scr or sh - NPC1 RNAs . Z - stack images acquired using a Nikon A1R + confocal microscope were used to generate 3D images of the neurons . Western blots against NPC1 actin - β ( ACTB ) in total extracts ( T ) and biotin - streptavidin immunoprecipitates ( S ) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not . Graphs show mean ± SEM of the levels of biotinylated - surface NPC1 as a percentage of the total amount of each protein ( n = 3 mice , 3 month - old , 2 - way ANOVA , * pNPC1 = 0 . 0251Western blots against CYP46A1 and actin - β ( ACTB ) in total extracts ( T ) and biotin - streptavidin immunoprecipitates ( S ) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not . Graphs show mean ± SEM of the levels of biotinylated - surface CYP46A1 as a percentage of the total amount of each protein ( n = 3 mice , 3 month - old , 2 - way ANOVA , * pCYP46A1 = 0 . 0371 ) . F . Representative images of GluA1 immunocytochemical staining before and after cLTP in non - permeabilized ( surface ) and permeabilized ( total ) cultured hippocampal neurons expressing sh - scr or sh - NPC1 RNAs . Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions , as a percentage of the sh - scr baseline ( n = 20 images per condition from 2 different experiments , 2 - way ANOVA , * * * psh - scr < 0 . 001 ) .

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Figure 4A. Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction (n=6, unpaired Student t test, *pwt = 0.0402B. Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh-scr or sh-NPC1 RNAs. Black bars indicate that the sh-NPC1 lane was not consecutive to the others but belongs to the same Western blot. Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt, non-transfected controls.C. Representative images of the cholesterol binding probe mCherry-D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh-scr or sh-NPC1 RNAs. Z-stack images acquired using a Nikon A1R+ confocal microscope were used to generate 3D images of the neurons.Western blots against NPC1 actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface NPC1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA, *pNPC1 = 0.0251Western blots against CYP46A1 and actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface CYP46A1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA , *pCYP46A1 = 0.0371).F. Representative images of GluA1 immunocytochemical staining before and after cLTP in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs. Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions, as a percentage of the sh-scr baseline (n=20 images per condition from 2 different experiments, 2-way ANOVA, ***psh-scr < 0.001).


Figure 5A . Western blots against CYP46A1 and ACTB in extracts of total brain and synaptosomes from wt and NPC1nmf164 mice as a percentage of wt values . Graphs show 3 biological replicates mean ± SEM of CYP46A1 level normalized to ACTB in arbitrary units . B . Quantitative PCR of Cyp46 of total brain and synaptosomes from wt and NPC1nmf164 mice . 3 biological replicates mean ± SEM is shown . C . Mean ± SEM cholesterol levels in EFV - treated or non - treated synaptosomes from wt ( n = 5 mice , 14 - week - old , unpaired Student t test , * p = 0 . 0233 ) and NPC1nmf164 ( n = 4 mice , 14 week - old , unpaired Student t test , * p = 0 . 0188 ) mice . EFV values are expressed as percentage of their corresponding non - treated controls in wt and NPC1nmf164 conditions . D . Basal synaptic transmission in EFV - treated and non - treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope ( n = 9 slices from 5 WT mice , n = 11 slices EFV - treated from 5 WT mice , n = 8 slices from 5 NPC1nmf164 mice and n = 13 slices EFV - treated from 5 NPC1nmf164 mice , all mice 14 - week - old , 2 - way ANOVA , * p = 0 . 0117 ) . E . LTP in EFV - treated and non - treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline ( n = 7 slices from 5 WT mice , n = 9 slices EFV - treated from 5 WT mice , n = 10 slices from 5 NPC1nmf164 mice and n = 11 slices EFV - tretaed from 5 NPC1nmf164 mice , all mice 14 - week - old , 2 - way ANOVA , * * * p < 0 . 0001 ) . F . Mean ± SEM paired pulse facilitation in EFV - treated and non - treated hippocampal slices from wt and NPC1nmf164 mice ( n = 11 slices from 5 WT mice , n = 9 slices EFV - treated from 5 WT mice , n = 10 slices from 5 NPC1nmf164 mice and n = 11 slices EFV - tretaed from 5 NPC1nmf164 mice , all mice 14 - week - old , 2 - way ANOVA , * * pNPC1nmf164 = 0 . 0027 , * pNPC1nmf164 + EFV = 0 . 0229 ) .

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Figure 5A. Western blots against CYP46A1 and ACTB in extracts of total brain and synaptosomes from wt and NPC1nmf164 mice as a percentage of wt values. Graphs show 3 biological replicates mean ± SEM of CYP46A1 level normalized to ACTB in arbitrary units.B. Quantitative PCR of Cyp46 of total brain and synaptosomes from wt and NPC1nmf164 mice. 3 biological replicates mean ± SEM is shown.C. Mean ± SEM cholesterol levels in EFV-treated or non-treated synaptosomes from wt (n=5 mice, 14-week-old, unpaired Student t test, *p = 0.0233) and NPC1nmf164 (n=4 mice, 14 week-old, unpaired Student t test, *p = 0.0188) mice. EFV values are expressed as percentage of their corresponding non-treated controls in wt and NPC1nmf164 conditions.D. Basal synaptic transmission in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope (n=9 slices from 5 WT mice, n=11 slices EFV-treated from 5 WT mice, n=8 slices from 5 NPC1nmf164 mice and n=13 slices EFV-treated from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, *p = 0.0117).E. LTP in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, ***p < 0.0001).F. Mean ± SEM paired pulse facilitation in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice (n=11 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, **pNPC1nmf164=0.0027, *pNPC1nmf164+EFV=0.0229).


Figure 6Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non - permeabilized ( surface ) and permeabilized ( total ) cultured hippocampal neurons expressing sh - scr or sh - NPC1 RNAs and treated or not with EFV . Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP , as a percentage of the sh - scr baseline ( n = 20 images per condition from 2 separate experiments , 2 - way ANOVA , * * * psh - scr < 0 . 001 , * * psh - scr + EFV < 0 . 01 , * * * psh - NPC1 + EFV < 0 . 001 ) .

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Figure 6Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs and treated or not with EFV. Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP, as a percentage of the sh-scr baseline (n=20 images per condition from 2 separate experiments, 2-way ANOVA, ***psh-scr < 0.001, **psh-scr+EFV < 0.01, ***psh-NPC1+EFV < 0.001).


Figure 7The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV A . Mean ± SEM 24 ( S ) - hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt ( n = 6 mice , 14 week - old and 16 week - old , 2 - way ANOVA , * * p6 weeks = 0 . 0023 ) and NPC1nmf164 ( n = 5 mice , 14 week - old and 16 week - old , 2 - way ANOVA , * * * p6 weeks < 0 . 0001 ; * * * p8 weeks < 0 . 0001 ) mice . The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV B . Mean ± SEM body weight in grams in wt and NPC1nmf164 mice treated or not with EFV ( n = 4 mice , unpaired Student t - test , * * * pNPC1nmf164 < 0 . 0001 , * * pNPC1nmf164 + EFV = 0 . 0048 ) The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV C . Mean ± SEM discrimination index as a normalized ratio of time spent with novel and familiar objects in the Object Placement Recognition test ( n = 6 mice , 10 week - old , 2 - way ANOVA , * * pWT / NPC1nmf164 = 0 . 0065 , * * * pNPC1nmf164 / NPC1nmf164 + EFV = 0 . 0008 ) . The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV D . Mean ± SEM percentage of entries in the novel arm of the Y maze test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * * * p < 0 . 0001 ) . E . Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * * pNPC1 < 0 . 0025 , * pNPC1 + EFV < 0 . 0181 ) . F . Mean ± SEM percentage of freezing time in the Cued Fear Conditioning test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * pNPC1 < 0 . 0453 , * * pNPC1 + EFV < 0 . 0054 ) . The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV G . Mean ± SEM cholesterol level in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV expressed as percentage of the wt non - treated samples ( n = 4 mice , 14 week - old , 2 - way ANOVA , * pNPC1 < 0 . 0476 , * pNPC1 + EFV < 0 . 0118 ) . The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV H . Representative fluorescence images of the CA1 hippocampal region from wt and NPC1nmf164 mice treated or not with EFV and stained with filipin and an antibody against LAMP1 . White arrows indicate lysosome enlargement in the hippocampus of non - treated NPC1nmf164 mice . Graph to the right shows mean ± SEM fluorescence intensity associated to filipin per area in arbitrary units ( n = 4 mice , 14 - week old , unpaired Student t - test , * * pNPC1nmf164 = 0 . 0011 , * pNPC1nmf164 + EFV = 0 . 0273 ) ) . I . Survival graph for wt and NPC1nmf164 mice treated or not with EFV ( n = 5 mice , 2 - way ANOVA , * * * p < 0 . 0001 ) .

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

Figure 7The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV A. Mean ± SEM 24(S)-hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt (n=6 mice, 14 week-old and 16 week-old, 2-way ANOVA, **p6 weeks = 0.0023) and NPC1nmf164 (n=5 mice, 14 week-old and 16 week-old, 2-way ANOVA, ***p6 weeks < 0.0001; ***p8 weeks < 0.0001) mice.The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV B. Mean ± SEM body weight in grams in wt and NPC1nmf164 mice treated or not with EFV (n=4 mice, unpaired Student t-test, ***pNPC1nmf164 < 0.0001, **pNPC1nmf164+EFV = 0.0048)The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV C. Mean ± SEM discrimination index as a normalized ratio of time spent with novel and familiar objects in the Object Placement Recognition test (n=6 mice, 10 week-old, 2-way ANOVA, **pWT/NPC1nmf164 = 0.0065, ***pNPC1nmf164/NPC1nmf164+EFV = 0.0008).The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV D. Mean ± SEM percentage of entries in the novel arm of the Y maze test (n=10 mice, 10 week-old, 2-way ANOVA, ***p < 0.0001). E. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, **pNPC1 < 0.0025, *pNPC1+EFV < 0.0181). F. Mean ± SEM percentage of freezing time in the Cued Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, *pNPC1 < 0.0453, **pNPC1+EFV < 0.0054). The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV G. Mean ± SEM cholesterol level in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV expressed as percentage of the wt non-treated samples (n=4 mice, 14 week-old, 2-way ANOVA, *pNPC1 < 0.0476, *pNPC1+EFV < 0.0118).The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV H. Representative fluorescence images of the CA1 hippocampal region from wt and NPC1nmf164 mice treated or not with EFV and stained with filipin and an antibody against LAMP1. White arrows indicate lysosome enlargement in the hippocampus of non-treated NPC1nmf164 mice. Graph to the right shows mean ± SEM fluorescence intensity associated to filipin per area in arbitrary units (n=4 mice, 14-week old, unpaired Student t-test, **pNPC1nmf164 = 0.0011, *pNPC1nmf164+EFV = 0.0273)). I. Survival graph for wt and NPC1nmf164 mice treated or not with EFV (n=5 mice, 2-way ANOVA, ***p < 0.0001).


Figure 1 ( B ) Co - immunoprecipitation of HA - tagged Btz - WT or Btz - HD with Myc - tagged GFP or eIF4AIII from transfected S2R + cells using Myc - Trap Magnetic Agarose beads . The upper Western blot is blotted with anti - Myc and the lower with anti - HA . Input samples are shown on the left and immunoprecipitates on the right . Btz - WT co - immunoprecipitates with eIF4AIII , but Btz - HD does not , although the protein is equally stable . The asterisk represents a likely cleavage product of Btz . ( C - F ) show stage 10 egg chambers stained with anti - Vasa ( C - F ) or anti - GFP ( E ' , F ' ) . ( C ) wild type ; ( D ) germline clones homozygous for the null allele btz2 ; ( E ) btz2 germline clones rescued with Btz - WT ; ( F ) btz2 germline clones rescued with Btz - HD . Posterior is to the right . Scale bars , 80 μm . Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction - defective transgene . Wild - type Btz - GFP , but not the mutant form , is also slightly enriched at the posterior pole .

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Figure 1(B) Co-immunoprecipitation of HA-tagged Btz-WT or Btz-HD with Myc-tagged GFP or eIF4AIII from transfected S2R+ cells using Myc-Trap Magnetic Agarose beads. The upper Western blot is blotted with anti-Myc and the lower with anti-HA. Input samples are shown on the left and immunoprecipitates on the right. Btz-WT co-immunoprecipitates with eIF4AIII, but Btz-HD does not, although the protein is equally stable. The asterisk represents a likely cleavage product of Btz.(C-F) show stage 10 egg chambers stained with anti-Vasa (C-F) or anti-GFP (E', F'). (C) wild type; (D) germline clones homozygous for the null allele btz2; (E) btz2 germline clones rescued with Btz-WT; (F) btz2 germline clones rescued with Btz-HD. Posterior is to the right. Scale bars, 80 μm. Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction-defective transgene. Wild-type Btz-GFP, but not the mutant form, is also slightly enriched at the posterior pole.


Figure 2 ( L - N ) electron micrographs showing cross - sections of larval muscle 6 in segment A3 , with the dorsal surface to the right . Scale bars , 10 μm . ( L ) btz2 / + ; ( M ) Df ( 3R ) BSC497 / + ; ( N ) btz2 / Df ( 3R ) BSC497 . Yellow arrows indicate examples of mitochondria interspersed between muscle fibers , and red arrows indicate mitochondria at the dorsal surface . Scale bars , 2 μm . ( O ) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated . * * * , p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant by Mann - Whitney test . n = 9 ( btz / + ) , n = 11 ( Df / + , btz / Df ) , n = 6 ( btz / Df ; G14 > btz , C57 - GAL4 / + ) , n = 4 ( btz / Df ; Btz - WT , C57 > eIF4AIII RNAi ) , n = 5 ( btz / Df ; Btz - HD ) , n = 10 ( C57 > btz RNAi ) , or n = 3 ( C57 > mago RNAi ) . Error bars show mean ± SEM .

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Figure 2(L-N) electron micrographs showing cross-sections of larval muscle 6 in segment A3, with the dorsal surface to the right. Scale bars, 10 μm. (L) btz2/+; (M) Df(3R)BSC497/+; (N) btz2/ Df(3R)BSC497. Yellow arrows indicate examples of mitochondria interspersed between muscle fibers, and red arrows indicate mitochondria at the dorsal surface. Scale bars, 2 μm.(O) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated. ***, p<0.001; **p<0.01; *p<0.05; ns, not significant by Mann-Whitney test. n=9 (btz/+), n=11 (Df/+, btz/Df), n=6 (btz/Df; G14>btz, C57-GAL4/+), n=4 (btz/Df; Btz-WT, C57>eIF4AIII RNAi), n=5 (btz/Df; Btz-HD), n=10 (C57>btz RNAi), or n=3 (C57>mago RNAi). Error bars show mean ± SEM.


Figure 3 ( A - J ) confocal images of the NMJ on larval muscles 6 and 7 in segment A3 , stained with anti - HRP ( magenta ) to label the nerve and anti - Dlg ( green ) as a postsynaptic marker of synaptic boutons . ( A ) w1118 control ; ( B ) btz2 / + ; ( C ) btz2 / Df ( 3R ) BSC497 ; ( D ) UAS - btz / + ; da - GAL4 , btz2 / Df ( 3R ) BSC497 ; ( E ) BG380 - GAL4 / + ; UAS - btz / + ; btz2 / Df ( 3R ) BSC497 ; ( F ) 24B - GAL4 / UAS - btz ; btz2 / Df ( 3R ) BSC497 ; ( G ) BG380 - GAL4 / + ; 24B - GAL4 / UAS - btz ; btz2 / Df ( 3R ) BSC497 ; ( H ) BG380 - GAL4 / + ; UAS - btz RNAi / + ; ( I ) mhc - GAL4 / UAS - btz RNAi ; ( J ) repo - GAL4 / UAS - btz RNAi . Scale bar , 30 μm . Insets in ( A , C ) show enlargements of the boxed regions , illustrating the closer spacing of boutons in btz mutants . ( K , L ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; ns , not significant by unpaired t - test with Welch ' s correction . n = 23 ( w ; da - GAL4 , btz / Df ) , n = 6 ( btz / + ) , n = 10 ( Df / + , UAS - BtzGFP / + ; btz / Df ) , n = 8 ( btz / Df ) , n = 22 ( da > btz - GFP ; btz / Df ) , n = 13 ( BG380 - GAL4 ; btz / Df ; BG380 + 24B > Btz - GFP ; btz / Df ; da - GAL4 / + ) , n = 5 ( BG380 > Btz - GFP ; btz / Df ) , n = 11 ( 24B - GAL4 ; btz / Df ; da > btz RNAi ) , n = 12 ( 24B > Btz - GFP ; btz / Df ; btz RNAi / + ) , n = 9 ( BG380 + 24B - GAL4 ; btz / Df ) , n = 15 ( BG380 - GAL4 / + ) , n = 14 ( BG380 > btz RNAi ; repo > btz RNAi ) , n = 16 ( mhc - GAL4 / + ) , n = 28 ( mhc > btz RNAi ) , or n = 21 ( repo - GAL4 / + ) . Error bars show mean ± SEM . NMJ size is reduced in btz mutants and rescued by expressing UAS - btz with da - GAL4 or with the combination of the BG380 - GAL4 and 24B - GAL4 drivers , but not with any single driver . Expressing UAS - btz RNAi with da - GAL4 or in motor neurons with BG380 - GAL4 , muscles with mhc - GAL4 or glia with repo - GAL4 also reduces NMJ size .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) to label the nerve and anti-Dlg (green) as a postsynaptic marker of synaptic boutons. (A) w1118 control; (B) btz2/+; (C) btz2/Df(3R)BSC497; (D) UAS-btz/+; da-GAL4, btz2/Df(3R)BSC497; (E) BG380-GAL4/+; UAS-btz/+; btz2/Df(3R)BSC497; (F) 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (G) BG380-GAL4/+; 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (H) BG380-GAL4/+; UAS-btz RNAi/+; (I) mhc-GAL4/UAS-btz RNAi; (J) repo-GAL4/UAS-btz RNAi. Scale bar, 30 μm. Insets in (A, C) show enlargements of the boxed regions, illustrating the closer spacing of boutons in btz mutants.(K, L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not significant by unpaired t-test with Welch's correction. n=23 (w; da-GAL4, btz/Df), n=6 (btz/+), n=10 (Df/+, UAS-BtzGFP/+; btz/Df), n=8 (btz/Df), n=22 (da>btz-GFP; btz/Df), n=13 (BG380-GAL4; btz/Df; BG380+24B>Btz-GFP; btz/Df; da-GAL4/+), n=5 (BG380>Btz-GFP; btz/Df), n=11 (24B-GAL4; btz/Df; da>btz RNAi), n=12 (24B>Btz-GFP; btz/Df; btz RNAi/+), n=9 (BG380+24B-GAL4; btz/Df), n=15 (BG380-GAL4/+), n=14 (BG380>btz RNAi; repo>btz RNAi), n=16 (mhc-GAL4/+), n=28 (mhc>btz RNAi), or n=21 (repo-GAL4/+). Error bars show mean ± SEM. NMJ size is reduced in btz mutants and rescued by expressing UAS-btz with da-GAL4 or with the combination of the BG380-GAL4 and 24B-GAL4 drivers, but not with any single driver. Expressing UAS-btz RNAi with da-GAL4 or in motor neurons with BG380-GAL4, muscles with mhc-GAL4 or glia with repo-GAL4 also reduces NMJ size.


Figure 4 ( I ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 by unpaired t - test or Mann - Whitney test . n = 15 ( BG380 - GAL4 ) , n = 12 ( BG380 > btz RNAi ; BG380 > mago RNAi ; BG380 > eIF4AIII RNAi ) , n = 16 ( btz / + ) , n = 13 ( btz / Df ) , or n = 10 ( Btz - WT ; btz / Df ; Btz - HD ; btz / Df ) . Error bars show mean ± SEM . NMJ size is reduced by knocking down btz in motor neurons , but slightly increased by knocking down mago or eIF4AIII . The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction - defective btz transgenes .

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

Figure 4(I) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001 by unpaired t-test or Mann-Whitney test. n=15 (BG380-GAL4), n=12 (BG380>btz RNAi; BG380>mago RNAi; BG380>eIF4AIII RNAi), n=16 (btz/+), n=13 (btz/Df), or n=10 (Btz-WT; btz/Df; Btz-HD; btz/Df). Error bars show mean ± SEM. NMJ size is reduced by knocking down btz in motor neurons, but slightly increased by knocking down mago or eIF4AIII. The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction-defective btz transgenes.


Figure 5 ( L ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; ns , not significant by unpaired t - test or Mann - Whitney test . n = 19 ( UAS - daw RNAi / + ) , n = 9 ( BG380 > daw RNAi ; btz / Df ; tub > daw ) , n = 23 ( mhc > daw RNAi ) , n = 10 ( repo > daw RNAi ; tub > daw ) , n = 12 ( btz / Df ; UAS - daw ) , or n = 14 ( btz / Df ; tub > btz ) . Error bars show mean ± SEM . Knocking down daw in motor neurons , muscles or glia reduces NMJ size . Expressing UAS - daw with tub - GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS - btz , but has no significant effect on NMJ size in a wild - type background .

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 5(L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ns, not significant by unpaired t-test or Mann-Whitney test. n=19 (UAS-daw RNAi/+), n=9 (BG380>daw RNAi; btz/Df; tub>daw), n=23 (mhc>daw RNAi), n=10 (repo>daw RNAi; tub>daw), n=12 (btz/Df; UAS-daw), or n=14 (btz/Df; tub>btz). Error bars show mean ± SEM. Knocking down daw in motor neurons, muscles or glia reduces NMJ size. Expressing UAS-daw with tub-GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS-btz, but has no significant effect on NMJ size in a wild-type background.