B ) mGFP - TRBP but not GFP - TRBP blocks the BDNF - induced decrease in miR - 16 - 5p activity in dual - fluorescence sensor assay . Hippocampal neurons were transfected with 200ng miR16 - or control - sensor ( see Fig 5A ) plus 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - TRBP . Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti - GFP antibody . BDNF led to a decrease in miR - 16 - 5p activity in GFP - transfected neurons ( p = 0 . 03 , n = 4 , t - test type 3 ) , which was significantly different from mGFP - TRBP - expressing neurons ( p = 0 . 04 , n = 4 , t - test type 2 , error bars are s . d . ) .

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B) mGFP-TRBP but not GFP-TRBP blocks the BDNF-induced decrease in miR-16-5p activity in dual-fluorescence sensor assay. Hippocampal neurons were transfected with 200ng miR16- or control-sensor (see Fig 5A) plus 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-TRBP. Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti-GFP antibody. BDNF led to a decrease in miR-16-5p activity in GFP-transfected neurons (p=0.03, n=4, t-test type 3), which was significantly different from mGFP-TRBP-expressing neurons (p=0.04, n=4, t-test type 2, error bars are s.d.).


C & D ) BDNF - induced dendritogenesis is blocked by mGFP - TRBP overexpression . C ) Hippocampal neurons ( 4DIV ) were transfected for three days with 200ng dsRed and 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - BDNF . Neurons were treated with BDNF or vehicle control 24 hours before fixation . Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites ( scale bars are 10μm ) . D ) The total number of intersections was calculated in Sholl analysis , and values were normalized to control treatment in each of three independent experiments . BDNF led to a significant increase in dendritic complexity ( p = 0 . 04 ; t - test type 3 ) , which was significantly different in cells transfected with mGFP - TRBP , but not GFP - TRBP ( n = 3 , p = 0 . 03 ; t - test type 2 , error bars ; s . d . ) .

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C&D) BDNF-induced dendritogenesis is blocked by mGFP-TRBP overexpression. C) Hippocampal neurons (4DIV) were transfected for three days with 200ng dsRed and 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-BDNF. Neurons were treated with BDNF or vehicle control 24 hours before fixation. Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites (scale bars are 10μm). D) The total number of intersections was calculated in Sholl analysis, and values were normalized to control treatment in each of three independent experiments. BDNF led to a significant increase in dendritic complexity (p=0.04; t-test type 3), which was significantly different in cells transfected with mGFP-TRBP, but not GFP-TRBP (n=3, p=0.03; t-test type 2, error bars; s.d.).


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 .

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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.


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 ) .

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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 .

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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 .

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 .

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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 .

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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.


LV / 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 .

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LV/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 .

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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 .

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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 .

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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 .

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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.


A 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 .

A 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 .

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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.


Representative 18F - FDG images with microPET in the coronal and axial planes in Wt and LXRα - Tg mice 5 weeks post - TAC .

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Representative 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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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 .

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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 .

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 .

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J Myocardial FDG uptake quantified as standard uptake value (SUV); n = 6/group. *P = 0.0001 versus WT sham, #P = 0.047.


A Assessment of 2 - deoxyglucose ( 2 - DG ) uptake from 4 independent experiments . * P = 0 . 03 versus Ad - cont .

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A Assessment of 2-deoxyglucose (2-DG) uptake from 4 independent experiments. *P = 0.03 versus Ad-cont.


B , C Glut mRNA expression determined by

B, C Glut mRNA expression determined by RT


D 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 .

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D 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 .

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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 .

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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 .

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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.


A Protein synthesis determined via leucine incorporation , n = 2 - 4 independent experiments . * P = 0 . 03 versus Ad - cont , § P = 0 . 06 versus Ad - cont .

A 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 .

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 .

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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.


A , 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 .

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A, 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 .

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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.


C ) Results of a competitive uptake assay showing inhibition of 0 . 2 µM L - [ 3H ] Trp or L - [ 3H ] Leu uptake in MhsT - WT - expressing MQ614 cells , measured for 30 seconds in the absence ( - ) or presence of 12 µM of the natural amino acids ( the one - letter code is used ) or 4 - fluoro - phenylalanine ( 4FP ) . Data are the mean ± S . E . M . of three independent experiments each performed as technical triplicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.


A ) Uptake of 0 . 1 μM L - [ 3H ] Leu was measured for 10 - second periods in the presence or absence of the indicated natural amino acids and 4 - fluorophenylalanine ( 4FP , all compounds tested at 100 μM final concentration ) in MQ614 expressing MhsT - WT or - M236F .

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A) Uptake of 0.1 μM L-[3H]Leu was measured for 10-second periods in the presence or absence of the indicated natural amino acids and 4-fluorophenylalanine (4FP, all compounds tested at 100 μM final concentration) in MQ614 expressing MhsT-WT or -M236F.


B , C ) Time course of 0 . 1 µM L - [ 3H ] Leu ( B ) or 0 . 1 µM L - [ 3H ] Trp ( C ) by MQ614 expressing MhsT - WT ( ) or - M236F ( ) , or MQ614 transformed with the control plasmid , pQE60 ( ) .

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B, C) Time course of 0.1 µM L-[3H]Leu (B) or 0.1 µM L-[3H]Trp (C) by MQ614 expressing MhsT-WT () or -M236F (), or MQ614 transformed with the control plasmid, pQE60 ().


D , E ) Kinetics of L - [ 3H ] Leu ( D ) or L - [ 3H ] Trp ( E ) uptake .

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D, E) Kinetics of L-[3H]Leu (D) or L-[3H]Trp (E) uptake.


( A ) EGFR degradation was followed in MSD , MPS ‐ IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization . The cells were then immediately lysed and subjected to anti ‐ EGFR blotting . The amount of remaining EGFR was quantified by densitometry analysis ( Image J ) of the blot and expressed in the chart as % of the EGFR amount present at time T0 ( 100 % ) . The values in the chart represent the mean ± s . e . m . values of three independent experiments .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) EGFR degradation was followed in MSD, MPS‐IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti‐EGFR blotting. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T0 (100%). The values in the chart represent the mean±s.e.m. values of three independent experiments.


( B ) MSD , MPS ‐ IIIA and WT MEFs cells loaded with dextran ( alexafluor ‐ 594 conjugated ) were labelled with anti ‐ LAMP1 antibody and the percentage of dextran co ‐ localizing with LAMP1 was evaluated . The chart displays merge values ( means ± s . e . m . ) that represent the percentage of dextran co ‐ localizing with LAMP1 measured in 15 different cells of triplicate experiments .

O O 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O

(B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments.


( C ) The rate of lysosome fusion with autophagosomes was monitored in MSD , MPS ‐ IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al , 2007 ) . The rate of autophagosome maturation reflected the percentage of the LC3 ' unfused ' ( green / red fluorescence ratio ) at each time ( 1 and 3 h ) after bafilomycin removal ( T0 ) . The percentage of LC3 ' unfused ' was displayed versus the value at T0 ( assumed to be 100 % ) . Values are represented as means ± s . e . m . of triplicate experiments . * P0 . 05 , Student ' s t ‐ test : ( A ) : WT versus MSD and WT versus MPS ‐ IIIA ; ( B , C ) : WT versus MSD and WT versus MPS ‐ IIIA at each time point . Scale bar : 10 μm ( B , C ) .

O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 'unfused' (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 'unfused' was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).


( A ) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein ( see Materials and methods section for details ) .

(A) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein (see Materials and methods section for details).


( B ) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS ‐ IIIA MEFs ( arrowheads and enlarged images ) .

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(B) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS‐IIIA MEFs (arrowheads and enlarged images).


( C , D ) Total lipids extracted from the indicated endolysosomal membrane samples ( 30 μg of proteins ) were either ( C ) subjected to a phosphate assay to quantify the bulk of phospholipids or ( D ) separated by TLC . Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining . CHOL , cholesterol ; LBPA lysobisphosphatidic acid ; PC , phosphatidylcoline ; PE phosphatidylethanolamine ; PI phosphatidyinositol ; SM sphingomyelin . ( A , C ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus MSD and WT versus MPS ‐ IIIA . Scale bar , 10 μm ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).


( A ) Endolysosomal membranes from MSD , MPS ‐ IIIA and WT MEFs were treated with 1 % Triton X ‐ 114 and loaded on a sucrose gradient . Immunoblots with Flotillin ‐ 1 identified DRMs in fractions 2 , 3 and 4 ( arrows ) . The fractions at the bottom of the gradient ( 12 and 13 ) correspond to high ‐ density detergent soluble fractions , whereas the remaining ones were defined as intermediate fractions ( intermediate ‐ I : 5 , 6 , 7 8 ; intermediate ‐ II : 9 , 10 and 11 ) . The percentage of Flotillin ‐ 1 in DRMs was calculated from the densitometric quantification of immunoblots .

O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

(A) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots.


( B ) Immuno ‐ EM of GM1 lipid was carried out in WT , MSD and MPS ‐ IIIA MEFs by staining cells with anti ‐ cholera toxin B antibodies ( see Materials and methods section ) . The number of GM1 ‐ positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT .

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(B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT.


( C ) Endolysosomal membranes from MSD , MPS ‐ IIIA and WT MEFs were stained with C ‐ laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value ( see Materials and methods section for details ) . Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft ( DRMs ) and soluble fractions .

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(C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions.


( D ) Equal aliquots from either DRMs or soluble fractions were pooled , the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions .

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(D) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions.


( E ) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient . Values represent the mean ± s . e . m . values of three experiments ( A - D ) . * P0 . 05 , Student ' s t ‐ test : ( A - C ) : WT versus MSD and WT versus MPS ‐ IIIA ; ( D ) : WT versus MSD and WT versus MPS ‐ IIIA for each fraction . Scale bar : 0 . 3 μm ( B ) .

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(E) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient. Values represent the mean±s.e.m. values of three experiments (A-D). *P0.05, Student's t‐test: (A-C): WT versus MSD and WT versus MPS‐IIIA; (D): WT versus MSD and WT versus MPS‐IIIA for each fraction. Scale bar: 0.3 μm (B).


( A ) VAMP7 , Vti1b and syntaxin 7 distributions in endo ‐ lysosomal membranes from MSD , MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions . To simplify the analysis the DRM , the intermediate ‐ I , the intermediate ‐ II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting . SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment . In MSD and MPS ‐ IIIA MEFs , all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes . Cholesterol modulation results in a change of SNARE distribution .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) VAMP7, Vti1b and syntaxin 7 distributions in endo‐lysosomal membranes from MSD, MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions. To simplify the analysis the DRM, the intermediate‐I, the intermediate‐II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting. SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment. In MSD and MPS‐IIIA MEFs, all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes. Cholesterol modulation results in a change of SNARE distribution.


( B ) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots ( ImageJ densitometry analysis ) and displayed as relative amount versus WT .

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(B) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots (ImageJ densitometry analysis) and displayed as relative amount versus WT.


( C , E ) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in ( C ) endolysosomal membranes and ( E ) total cell lysates from WT ( untreated and cholesterol treated ) and MSD MEFs ( untreated and MβCD treated ) . In the graphs , the protein levels were displayed as relative amount versus WT .

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(C, E) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in (C) endolysosomal membranes and (E) total cell lysates from WT (untreated and cholesterol treated) and MSD MEFs (untreated and MβCD treated). In the graphs, the protein levels were displayed as relative amount versus WT.


( D ) WT and MSD MEFs transfected with GFP - VAMP7 were stained with anti ‐ GFP for immuno ‐ EM . The enlarged image shows internalization of GFP - VAMP7 particles ( arrow ) .

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(D) WT and MSD MEFs transfected with GFP-VAMP7 were stained with anti‐GFP for immuno‐EM. The enlarged image shows internalization of GFP-VAMP7 particles (arrow).


( F ) Syntaxin 5 , Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs . Syntaxin 5 immunoblot shows two bands ( * , 35 kDa and * * , 42 kDa ) corresponding to the two isoforms of the protein .

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(F) Syntaxin 5, Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs. Syntaxin 5 immunoblot shows two bands (*, 35 kDa and **, 42 kDa) corresponding to the two isoforms of the protein.


( G ) Distribution profile of Rab7 in WT and MSD lysosomal membranes . Values represent the mean ± s . e . m . values of three independent experiments ( B , C , E ) . * P0 . 05 , Student ' s t ‐ test : ( B ) : WT versus MSD , WT versus MPS ‐ IIIA , WT versus WT + cholesterol , MSD versus MSD + MβCD , and MPS ‐ IIIA versus MβCD for each analysed SNARE ; ( C , E ) : WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD for each analysed protein . Scale bar : 0 . 3 μm ( D ) .

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(G) Distribution profile of Rab7 in WT and MSD lysosomal membranes. Values represent the mean±s.e.m. values of three independent experiments (B, C, E). *P0.05, Student's t‐test: (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD, and MPS‐IIIA versus MβCD for each analysed SNARE; (C, E): WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each analysed protein. Scale bar: 0.3 μm (D).


( A ) SDS ‐ resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total , detergent insoluble ( DRM ) and detergent soluble ( Sol . ) endo ‐ lysosomal membrane fractions derived from MSD and WT MEFs . The SDS ‐ resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD . Immunoblots revealed the presence of low molecular weight complexes ( * , 50 - 60 kDa ) and high molecular weight complexes ( * * , > 80 kDa ) . The percentage of Vti1b in SDS ‐ resistant complexes in total endolysosomal membranes ( bottom ‐ left chart ) and the amount of Vti1b ‐ containing SDS ‐ resistant complexes in DRM and soluble fractions ( bottom ‐ right chart ) were calculated by the densitometric quantification of the correspondent immunoblots ( ImageJ ) . Values represent the mean ± s . e . m . values of three independent measurements . * P0 . 05 , Student ' s t ‐ test : WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD ( bottom ‐ left chart ) ; WT versus MSD , WT versus WT + cholesterol and MSD versus MSD + MβCD for each fraction ( bottom ‐ right chart ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50-60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart).


( B ) Syntaxin 7 and VAMP7 were co ‐ immunoprecipitated with Vti1b using anti ‐ Vti1b antibodies in WT ( untreated or cholesterol treated ) and in MSD ( not treated or MβCD treated ) MEFs . The amount of Vti1b precipitated in each cell line is also shown .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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-MEASURED_VAR O O O O O O O O O

(B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown.


( C ) SDS ‐ resistant complexes are decorated by anti ‐ syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

(C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs.


( D ) Membrane ‐ associated α ‐ SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates ( total ) , intracellular membranes recovered after centrifugation from a post ‐ nuclear supernatant fraction ( membrane associated ) and cell lysates devoid of membranes ( cytosolic released ) derived from MSD ( untreated or MβCD treated ) and WT ( untreated or cholesterol treated ) MEFs .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.


( A ) MSD , MPS ‐ IIIA and WT MEFs were subjected to a triple labelling with anti ‐ VAMP7 , anti ‐ Vti1b and anti ‐ LAMP1 antibodies . The merges between VAMP7 and Vti1b ( double merges in yellow ) and between VAMP7 , Vti1b and LAMP1 ( triple merges in white ) are shown ( see also enlarged images showing the extent of co ‐ localization in different regions of the cells ) . The VAMP7 - Vti1b co ‐ localization was quantified in 15 different cells and displayed as % of Vti1b co ‐ localizing with VAMP7 ( means ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O

(A) MSD, MPS‐IIIA and WT MEFs were subjected to a triple labelling with anti‐VAMP7, anti‐Vti1b and anti‐LAMP1 antibodies. The merges between VAMP7 and Vti1b (double merges in yellow) and between VAMP7, Vti1b and LAMP1 (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The VAMP7-Vti1b co‐localization was quantified in 15 different cells and displayed as % of Vti1b co‐localizing with VAMP7 (means±s.e.m.).


( B ) Co ‐ localization of Vti1b with epsinR was quantified by double ‐ labelling experiments in MSD and MPS ‐ IIIA ( untreated and MβCD treated ) and in control WT ( untreated and cholesterol treated ) MEFs . The chart displays merge values ( means ± s . e . m . ) that represent the percentage of Vti1b co ‐ localizing with epsinR measured in 15 different cells .

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(B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells.


( C ) Vti1b trafficking was monitored by FRAP analysis in WT ( untreated and cholesterol treated ) and MSD ( untreated and MβCD treated ) MEFs transfected with GFP - Vti1b ( see Materials and methods section for details ) . FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach ( 100 % ) and are representative of 10 recordings from different cells .

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(C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.


( A ) Blood ammonia in 8 - 9 - week - old C57BL / 6J wild - type ( WT ) mice ( n = 8 ) and Becn1F121A mice with constitutive activation of autophagy ( n = 8 ) at baseline and 30 min after i . p . injection of NH4Cl ( 10 mmol / kg ) . * * p < 0 . 01 ( Unpaired t - test ) . ns : not statistically significant difference .

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(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.


( B ) Urinary orotic acid of 12 - week - old spf - ash mice treated with TB - 1 ( 15 mg / kg , i . p ) or vehicle at various times as indicated by the arrows ( n = 5 mice / group ) . * p < 0 . 05 ( Two - way ANOVA ) .

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(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).


( C ; D ) Western blotting and densitometric quantifications of autophagy markers ( LC3II : autophagosomes ; p62 and NBR1 : cargo receptors ) in livers of spf - ash mice harvested after ten days of treatment with TB - 1 or vehicle . β - actin was used as loading control . n = 5 mice / group . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) .

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(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).


( E ) Survival curves of spf - ash mice fed with a high protein diet ( HPD ) for ten days and treated with TB - 1 alone or combined with scavenger drug and L - Arginine , or treated with scavenger drug and L - Arginine , or left untreated . WT control were included ( n = 5 / group ) . * * * p < 0 . 001 ( Log - rank Mantel - Cox test ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

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(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( F ) Blood ammonia levels determined after 4 days under HPD ( n = 5 mice / group ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

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(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( A ) Survival curves of AslNeo / Neo mice and age - matched wild - type ( WT ) controls treated with TB - 1 ( 15 mg / kg , i . p . , every 48 hours starting at day 10 of age ) or vehicle . WT + Vehicle n = 8 ; WT + TB - 1 n = 8 ; AslNeo / Neo + Vehicle n = 20 ; AslNeo / Neo + TB - 1 n = 16 . * p < 0 . 05 ( Log - rank Mantel - Cox test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( B ) Isotopic enrichment of 15N - labeled urea in blood , 20 min after i . p . injection of 15NH4Cl tracer ( 4 mmol / kg ) in AslNeo / Neo mice treated with TB - 1 ( n = 15 ) or vehicle ( n = 9 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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(B) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( C ) Representative Western blotting bands of LC3 , p62 and NBR1 in livers of AslNeo / Neo mice treated with TB - 1 or vehicle . GAPDH and β - actin were used as loading controls . ( D ) Densitometric quantifications . AslNeo / Neo + Vehicle n = 5 ; AslNeo / Neo + TB - 1 n = 9 . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. (D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( E ) Argininosuccinate in dried blood spots ( DBS ) of WT and AslNeo / Neo mice injected with TB - 1 or vehicle ( n = 4 - 14 mice / group ) . * * * p < 0 . 001 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( F ) Isotopic enrichment of 15N - labeled argininosuccinate in livers of AslNeo / Neo mice treated with TB - 1 ( n = 11 ) or vehicle ( n = 5 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( A ) Hematoxylin and eosin ( H & E , upper panels ) and periodic acid Schiff ( PAS , lower panels ) staining of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bars : 500 µm .

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(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.


( B ) Computational analysis of PAS staining ( n ≥ 4 mice / group ) . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M .

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(B) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.


( C ) Quantification of hepatic glycogen in vehicle ( n = 6 ) - and TB - 1 - treated AslNeo / Neo mice ( n = 12 ) compared to WT ( n = 5 ) controls . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M .

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(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.


( D ) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo / Neo mice treated with either with TB - 1 or vehicle ( n = 4 mice / group ) . * p < 0 . 05 ( One - way ANOVA ) . GAPDH was used as loading control . Data information : All values are shown as averages ± S . E . M .

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(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.


( E ) Representative electron microscopy images of liver samples harvested from WT and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bar : 900 nm .

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(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.


( A ) Representative electron microscopy images of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . False color on the images indicates glycogen within the nuclei . Scale bar : 1 μm . ( B ) Quantification of nuclei containing glycogen ( approx . 180 nuclei were analyzed in total per condition , n = 3 mice / group ) .

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(A) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm. (B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group).


( A ) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h . Then , WB was used to measure p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein expression . " Nor " = normal medium . Actin served as the loading control . The red text " - Cys " indicates that cystine deficiency most significantly activates AMPK and its substrates .

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(A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates.


( F ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein in 293T cells transfected with shRNAs targeting LKB1 , CaMKK2 , TAK1 or a non - targeting control ( NTC ) that were treated with cystine - deficient medium for 8 h . Actin served as the loading control .

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(F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control.


( I ) 293T cells cultured with cystine - deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti - AMPKα , followed by WB analysis with anti - AMPKα and anti - CaMKK2 .

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(I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.


( E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK ACC , Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine - deficient medium for 8 h . Actin served as the loading control .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control.


( J ) WB analysis of p - AMPK and total AMPK protein expression in CARS - overexpressing 293T cells and RCC4 cells that were treated with 1 µg / ml STO - 609 or DMSO for 8 h . Actin served as the loading control .

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(J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.


( D , E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor , AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine - deficient medium for 8 h ( D ) or transfected with Flag - CARS for 48 h ( E ) . Actin served as the loading control .

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(D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control.


( F ) 293T cells were transfected with HA - AMPKγ2 alone or with Flag - CARS for 48 h and then cultured with cystine - deficient medium or complete medium for 8 h . Cell lysates were immunoprecipitated with anti - Flag , and a WB analysis was performed .

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(F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.


( B ) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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(B) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( C , D ) The crystal violet assay ( C ) and apoptosis rate assay ( D ) were performed in 293T cells transfected with shRNAs targeting NTC , CARS , CaMKK2 or AMPKγ2 and further treated with cystine - deficient medium for 24 h with or without 1 mM AICAR . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( F WB analysis of p - AMPK , total AMPK , CARS , AMPKγ2 , and CaMKK2 protein expression with 786 - O cells transfected with Flag - EV , Flag - CARS , Flag - CaMKK2 or Flag - AMPKγ2 and further treated with cystine - deficient medium for 8 h . Actin served as the loading control . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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(F WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) produced with lopinavir ( LPV , 0 - 100 nM ) detecting p24 antibody .

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(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.


Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated THP - 1 shSAMHD1 cells . Mean ± SD , n = 3 .

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Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3.


Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated U87 cells . Mean ± SD , n = 3 .

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Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3.


ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . G ) CXCL - 10 protein in cell supernatants from D - F ( ELISA ) .

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PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA).


( H ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 2 U RT / ml 30 nM LPV - treated HIV - 1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib . A control was stimulated with 1 ng / ml IFNβ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.


( I ) RT products from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 3 U RT / ml LPV - treated HIV - 1 GFP viruses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.


( J ) Immunoblot of HIV - 1 R9 BaL virus particles ( 2 × 1011 genomes ) produced with LPV ( 0 - 100 nM ) detecting p24 .

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(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.


( K , L ) ISG qRT - PCR ( K ) and infection data ( L ) from primary monocyte - derived macrophages ( MDM ) infected for 24 h with LPV - treated HIV - 1 R9 BaL viruses ( 0 . 2 U RT / ml ) . Data are collated from two donors ( represented by circles and squares ) , n = 3 . Horizontal line represents the median . For experiments in which the virus dose used was normalised by RT activity , the number of genome copies was also measured by qPCR of virus . This gave dose equivalents of within 2 - 3 fold of RT equivalents .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.


( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) with varying proportions of ΔCA - SP1 protease cleavage mutation detecting p24 .

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(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24.


( B ) Titration of HIV - 1 GFP ΔCA - SP1 viruses on U87 cells . Mean ± SD , n = 3 .

(B) Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n=3.


( C ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


D ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . E ) CXCL - 10 protein in supernatants from ( C , D ) ( ELISA ) .

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PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E) CXCL-10 protein in supernatants from (C, D) (ELISA).


( F ) RT products from THP - 1 cells transduced for 24 h with 6 × 109 genomes / ml ( approx . 0 . 5 U RT / ml ) HIV - 1 GFP ΔCA - SP1 viruses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) RT products from THP-1 cells transduced for 24 h with 6×109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses.


( G ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 016 - 0 . 2 U RT / ml ) . Data are shown as individual measurements , representative of 2 repeats .

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(G) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 - 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats.


( H ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 mutant , or stimulated with 4 μg / ml cGAMP as a control , in the presence of DMSO vehicle or 2 μM ruxolitinib .

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(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.


( I ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 3 × 108 genomes / ml or 1 . 5 × 109 genomes / ml , equivalent to 0 . 1 U RT / ml and 0 . 5 U RT / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).


( J ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib .

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(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.


primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . K ) CXCL - 10 protein in supernatants from ( J ) ( ELISA ) .

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primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K) CXCL-10 protein in supernatants from (J) (ELISA).


( A ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

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(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.


THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . B ) CXCL - 10 protein in supernatants from ( A ) ( ELISA ) .

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THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir. B) CXCL-10 protein in supernatants from (A) (ELISA).


ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.


( E ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ΔCA - SP1 ( WT ) , 75 % ΔCA - SP1 , 75 % ΔCA - SP1 carrying a mutation in reverse transcriptase ( 75 % ΔCA - SP1 RT D185E ) or 75 % ΔCA - SP1 carrying a mutation in integrase ( 75 % ΔCA - SP1 INT D116N ) ( 3 . 75 × 109 , 7 . 5 × 109 and 1 . 5 × 1010 genomes / ml ) .

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(E) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % ΔCA-SP1 (WT), 75 % ΔCA-SP1, 75 % ΔCA-SP1 carrying a mutation in reverse transcriptase (75 % ΔCA-SP1 RT D185E) or 75 % ΔCA-SP1 carrying a mutation in integrase (75 % ΔCA-SP1 INT D116N) (3.75×109, 7.5×109 and 1.5×1010 genomes/ml).


( F ) CXCL - 10 protein in supernatants from ( E ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

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(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.


( A ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING or MAVS , or a gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP 75 % ΔCA - SP1 ( 0 . 4 U RT / ml ) or stimulated by transfection with either HT - DNA ( 0 . 1 μg / ml ) or poly I : C ( 0 . 5 μg / ml ) .

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(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).


IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line stimulated for 24 h with poly I : C ( transfection , 0 . 5 μg / ml ) , HT - DNA ( transfection , 0 . 1 μg / ml ) , LPS ( 50 ng / ml ) or cGAMP ( transfection , 0 . 5 μg / ml )

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IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml)


IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml )

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IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml)


PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) ( C ) . D ) CXCL - 10 protein in supernatants from ( C ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

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PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C). D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.


( E ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

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(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).


( F ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

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(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).


PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . G ) CXCL - 10 protein in supernatants from ( E ) .

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PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). G) CXCL-10 protein in supernatants from (E).


PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( H ) CXCL - 10 protein in supernatants from ( F ) ( ELISA ) .

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PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). (H) CXCL-10 protein in supernatants from (F) (ELISA).


( I ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 control cells transduced for 48 h with WT , DRV - treated ( DRV , 12 . 5 nM ) or HIV - 1 GFP containing 90 % ΔCA - SP1 ( 1 × 1010 genomes / ml ) in the presence of DMSO vehicle , 2 μM ruxolitinib or 0 . 5 μg / ml H151 .

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(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.


Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with a fixed dose of HIV - 1 GFP ( 5 × 107 genomes / ml ) and increasing doses of HIV - LUC ΔCA - SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with a fixed dose of HIV-1 GFP (5×107 genomes/ml) and increasing doses of HIV-LUC ΔCA-SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with LPV - treated HIV - LUC viruses Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with LPV-treated HIV-LUC viruses Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


( A ) Infection data for THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).


THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . B ) IFIT - 1 reporter activity in supernatant from ( A ) .

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THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM). B) IFIT-1 reporter activity in supernatant from (A).


ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).


( E ) CXCL - 10 protein in supernatants of THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).