qRT - PCR of CYP46A1 mRNA in LN229 GBM # P3 transfected with lentivirus expressing CYP46A1 ( lenti - CYP46A1 ) or control ( lenti - Ctrl ) . GAPDH was used as an internal control . Shown are means ( n = 3 ) . LN229 : * * * P < 0 . 0001 ; GBM # P3 : * * * P < 0 . 0001 . Statistical significance was determined by two - sided Student ' s t ‐ .

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qRT-PCR analysis of CYP46A1 mRNA levels in LN229 and GBM#P3 cells transfected with lentivirus expressing CYP46A1 (lenti-CYP46A1) or control sequence (lenti-Ctrl). GAPDH was used as an internal control. Shown are means and SEM (n = 3). LN229: ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by two-sided Student's t‐test.


Western blot to confirm CYP46A1 overexpression in GBM .

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Western blot analysis to confirm CYP46A1 overexpression in GBM cells.


Growth curves for GBM in vitro infected with lenti - Ctrl or lenti - CYP46A1 derived from trypan blue . Shown are means ( n = 3 ) . LN229 : * * P = 0 . 003 ; GBM # P3 : * * P = 0 . 002 . Statistical significance was determined by two - sided Student ' s t ‐ .

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Growth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t‐test.


Colony forming for GBM infected with lenti - Ctrl or lenti - CYP46A1 . Shown are means ( n = 3 ) . LN229 : * P = 0 . 013 ; GBM # P3 : * P = 0 . 012 . Statistical significance was determined by two - sided Student ' s t ‐ .

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Colony forming assay for GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Shown are means and SEM (n = 3). LN229: *P = 0.013; GBM#P3: *P = 0.012. Statistical significance was determined by two-sided Student's t‐test.


Representative H & E of orthotopic tumours derived from GBM infected with lenti - Ctrl or lenti - CYP46A1 . Scale bar = 2 mm .

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Representative H&E staining of orthotopic tumours derived from GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Scale bar = 2 mm.


Kaplan - Meier survival curve of tumour bearing mice injected with GBM infected with lenti - Ctrl or lenti - CYP46A1 ( n = 5 per group ) . A log - rank was used to assess statistical significance .

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Kaplan-Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti-Ctrl or lenti-CYP46A1 (n = 5 per group). A log-rank test was used to assess statistical significance.


for CYP46A1 , PCNA caspase - 3 in intracranial tumours . Scale bar = 100 μm .

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IHC for CYP46A1, PCNA and cleaved caspase-3 protein levels in the intracranial tumours. Scale bar = 100 μm.


24OHC in spent media from LN229 ( left ) GBM # P3 ( right ) transduced with lenti - Ctrl or lenti - CYP46A1 measured using targeted LC - MS /

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24OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MS


IC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay.


Flow cytometry to detect annexin V - FITC PI to determine of LN229 GBM # P3 undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h . Data are shown as mean ± ( n = 3 ) .

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Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3).


Colony forming ability of GBM lines treated with 24OHC ( 0 - 20 μM ) for 14 days .

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Colony forming ability of GBM cell lines treated with 24OHC (0 - 20 μM) for 14 days.


Western blot of apoptosis marker c - PARP , c - caspase 3 PCNA in lysates ( 20 µg ) from GBM treated with 24OHC ( 0 - 20 μM ) for 72 h .

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Western blot analysis of the apoptosis marker c-PARP, c-caspase 3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0 - 20 μM) for 72 h.


Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC ( 0 - 20 μM ) . Scale bar = 100 μm . Graphic representation of of tumoursphere formation . Data are shown as mean ± ( n = 3 ) . GBM # P3 : * P = 0 . 0192 , * * * P = 0 . 0006 , * * * P = 0 . 0002 ; GBM # BG7 : * * P = 0 . 0017 , * * * P < 0 . 0001 , * * * P < 0 . 0001 ; GBM # BG5 : * * P = 0 . 0027 , * * * P = 0 . 0001 , * * * P < 0 . 0001 .

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Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001.


Intracellular of cholesterol in GBM treated with 20 μM of 24OHC or DMSO for 72 h quantified using Invitrogen ™ Amplex ™ Red Cholesterol Kit normalized to total . Shown are means ( n = 3 ) . LN229 : * * P = 0 . 002 ; GBM # P3 : * * P = 0 . 011 . Statistical significance was determined by two - sided Student ' s t ‐ .

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Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen™ Amplex™ Red Cholesterol Assay Kit and normalized to total protein. Shown are means and SEM (n = 3). LN229: **P = 0.002; GBM#P3: **P = 0.011. Statistical significance was determined by two-sided Student's t‐test.


Representative of filipin in GBM treated with different concentrations of 24OHC ( 0 - 20 μM ) for 72 h . Scale bar = 30 μm .

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Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC (0 - 20 μM) for 72 h. Scale bar = 30 μm.


Growth curves for LN229 treated with DMSO , 0 . 5 μg / mL cholesterol or 10 μM 24OHC in presence or absence of 0 . 5 μg / mL cholesterol using trypan blue .

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Growth curves for LN229 cells treated with DMSO, 0.5 μg/mL cholesterol or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol using trypan blue staining.


Flow cytometry to detect annexin V - FITC PI to determine of GBM undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in presence or absence of 0 . 5 μg / mL cholesterol for 72 h . Data are shown as mean ± ( n = 3 ) . LN229 : * * P = 0 . 0021 , * * P = 0 . 0046 ; GBM # P3 : * * P = 0 . 0012 , * P = 0 . 0193 .

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Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0021, **P = 0.0046; GBM#P3: **P = 0.0012, *P = 0.0193.


Western blot of c - PARP in GBM treated with DMSO or 10 μM 24OHC in presence or absence of 0 . 5 μg / mL cholesterol for 72 h .

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Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h.


Tumoursphere formation assays for GBM # P3 GBM # BG7 treated with DMSO or 10 μM 24OHC in presence or absence of 0 . 5 μg / mL cholesterol . Scale bar = 100 μm . Data are shown as mean ± ( n = 3 ) . GBM # P3 : * * P = 0 . 0032 , * P = 0 . 031 ; GBM # BG7 : * * * P = 0 . 0008 , * P = 0 . 0242 .

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Tumoursphere formation assays for GBM#P3 and GBM#BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol. Scale bar = 100 μm. Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0032, *P = 0.031; GBM#BG7: ***P = 0.0008, *P = 0.0242.


CCK - 8 to determine relative survival in LN229 or GBM # P3 after treatment with vehicle control or different concentrations of MβCD for 72 h . Data are shown as mean ± ( n = 3 ) . LN229 : * * P = 0 . 0051 , * * * P = 0 . 0002 ; GBM # P3 : * * P = 0 . 0018 , * * * P < 0 . 0001 .

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CCK-8 assay to determine relative cell survival in LN229 or GBM#P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0051, ***P = 0.0002; GBM#P3: **P = 0.0018, ***P < 0.0001.


of differentially expressed transcripts in - seq data from GBM # P3 treated with DMSO or 20 μM 24OHC for 72 h . Gene expression data was mean z - transformed for display , colored red for high expression blue for low expression .

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Heatmap of differentially expressed transcripts in RNA-seq data from GBM#P3 cells treated with DMSO or 20 μM 24OHC for 72 h. Gene expression data was mean z-transformed for display, and colored red for high expression and blue for low expression.


GSEA plot showing normalized enrichment scores ( NES ) for cholesterol homeostasis , positive regulation of cholesterol efflux , SREBP targets proliferation signatures using - seq data from 24OHC DMSO treated .

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GSEA plot showing normalized enrichment scores (NES) for cholesterol homeostasis, positive regulation of cholesterol efflux, SREBP targets and stem cell proliferation signatures using RNA-seq data from 24OHC and DMSO treated cells.


Volcano plot highlighting up - regulated ( red ) down - regulated ( blue ) genes on - seq data from GBM # P3 after 24OHC treatment . LXR targets , SREBP targets stemness transcription factors are specifically included .

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Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included.


qRT - PCR of 24OHC - regulated genes in GBM # P3 after treatment with 20 μM 24OHC for 72 h . GAPDH was used as an internal control . Data are shown as mean ± ( n = 3 ) .

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qRT-PCR analysis of 24OHC-regulated genes in GBM#P3 cells after treatment with 20 μM 24OHC for 72 h. GAPDH was used as an internal control. Data are shown as the mean ± SEM (n = 3).


viability performed on GBM # P3 after 3 days of treatment with DMSO or 5 - 10 μM LXR - 623 in presence or absence of 10 μM 24OHC . Data are shown as mean ± ( n = 3 ) .

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Cell viability assay performed on GBM#P3 cells after 3 days of treatment with DMSO or 5 -10 μM LXR-623 in the presence or absence of 10 μM 24OHC. Data are shown as the mean ± SEM (n = 3).


Western blot of cholesterol metabolism - related in LN229 or GBM # P3 after treatment with 24OHC ( 0 - 20 μM ) .

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Western blot analysis of cholesterol metabolism-related proteins in LN229 or GBM#P3 after treatment with 24OHC (0 - 20 μM).


Growth curves for LN229 transfected with control vector or SREBP1 plasmid treated with 24OHC ( 0 - 40 μM ) as assessed by a trypan blue . Data are shown as mean ± ( n = 3 ) .

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Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC (0 - 40 μM) as assessed by a trypan blue test. Data are shown as the mean ± SEM (n = 3).


Colony forming ability of GBM lines treated with DMSO or 20 μM fatostatin for 14 days .

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Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days.


Representative of filipin in LN229 ( up ) or LN18 ( down ) treated with different of fatostatin ( 0 - 20 μM ) . Scale bar = 30 μm .

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Representative images of filipin staining in LN229 (up) or LN18 (down) cells treated with different concentration of fatostatin (0 - 20 μM). Scale bar = 30 μm.


Intracellular concentrations of cholesterol in GBM after treatment with 20 μM EFV or DMSO normalized to total . Data are shown as mean ± ( n = 3 ) . LN229 : * P = 0 . 013 ; GBM # P3 : * * P = 0 . 0023 .

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Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein. Data are shown as the mean ± SEM (n = 3). LN229: *P = 0.013; GBM#P3: **P = 0.0023.


Filipin of GBM after treatment with EFV ( 0 - 20 μM ) . Scale bar = 30 μm .

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Filipin staining of GBM cells after treatment with EFV (0 - 20 μM). Scale bar = 30 μm.


IC50 curves for EFV in GBM determined using OD readings ( 450nm ) from CCK - 8 .

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IC50 curves for EFV in GBM cells determined using OD readings (450nm) from the CCK-8 assay.


24OHC in spent media from LN229 ( left ) or GBM # P3 ( right ) treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC - MS / MS .

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24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC-MS/MS.


of colonies formed by GBM lines after treatment with different concentrations of EFV ( 0 - 20 μM ) . Data are shown as mean ± ( n = 3 ) . LN229 : * * P = 0 . 0056 , * * * P < 0 . 0001 ; LN18 : * * * P < 0 . 0001 .

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Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0 - 20 μM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001.


Flow cytometry to detect annexin V - FITC PI to determine of GBM undergoing apoptosis after treatment with EFV ( 0 - 20 μM ) for 72 h . Data are shown as mean ± ( n = 3 ) . LN229 : * * * P = 0 . 0006 , * * * P < 0 . 0001 ; GBM # P3 : * * * P < 0 . 0001 .

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Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV (0 - 20 μM) for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0006, ***P < 0.0001; GBM#P3: ***P < 0.0001.


Tumoursphere formation assays for GSCs treated with different concentrations of EFV ( 0 - 20 μM ) . Scale bar = 100 μm . Graphic representation of of tumoursphere formation ( right ) . Data are shown as mean ± ( n = 3 ) . GBM # P3 : * * P = 0 . 0011 , * * * P = 0 . 0003 , * * * P < 0 . 0001 ; GBM # BG7 : * * * P < 0 . 0001 ; GBM # BG5 : * * P = 0 . 0037 , * * P = 0 . 0015 , * * * P = 0 . 0002 .

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Tumoursphere formation assays for GSCs treated with different concentrations of EFV (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation (right). Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0011, ***P = 0.0003, *** P < 0.0001; GBM#BG7: ***P < 0.0001; GBM#BG5: **P = 0.0037, **P = 0.0015, ***P = 0.0002.


Western blot of cholesterol homeostasis related ( LDLR , ABCA1 , P - SREBP1 N - SREBP1 ) , c - PARP , PCNA SOX2 in GBM treated with EFV ( 0 - 20 μM ) for 72 h .

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Western blot analysis of cholesterol homeostasis related proteins (LDLR, ABCA1, P-SREBP1 and N-SREBP1), c-PARP, PCNA and SOX2 in GBM cells treated with EFV (0 - 20 μM) for 72 h.


survival curves of tumour - bearing mice implanted with LN229 or GBM # P3 after EFV or PBS treatment ( n = 5 per group ) . A log - rank was used to assess statistical significance .

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The survival curves of tumour-bearing mice implanted with LN229 or GBM#P3 cells after EFV or PBS treatment (n = 5 per group). A log-rank test was used to assess statistical significance.


Bioluminescent corresponding of tumours in mice implanted with GBM # P3 at days 7 ( n = 5 per group , P = 0 . 58 ) 21 ( n = 5 per group , P = 0 . 005 ) . Statistical significance was determined by two - sided Student ' s t ‐ . of tumour - bearing mice after 3 of EFV or PBS treatment . Data are shown as mean ± . Statistical significance was determined by two - sided Student ' s t ‐ .

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Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM#P3 cells at days 7 (n = 5 per group, P = 0.58) and 21 (n = 5 per group, P = 0.005). Statistical significance was determined by two-sided Student's t‐test. Body weight of tumour-bearing mice after 3 weeks of EFV or PBS treatment. Data are shown as the mean ± SEM. Statistical significance was determined by two-sided Student's t‐test.


( A to D ) 10 - to 11 - week - old mice were treated with vehicle control or AT845 at vector doses indicated followed for 12 ( N = 7 or 8 per cohort ) . ( A ) Vector copy number , ( B ) GAA , ( C ) GAA activity , ( D ) glycogen content in cardiac skeletal . ( C to D ) Statistical : two - way ANOVA , Dunnett ' s . Data are presented as box - - whisker plots with Tukey whiskers that show minimum , median , maximum . Asterisks ( * ) indicate significant differences compared with untreated Gaa - / - mice ( black ) or wild - type mice ( red ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 . See also Appendix Table S1 .

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(A to D) 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C to D) Statistical analysis: two-way ANOVA, Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice (black) or wild-type mice (red). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P <0.0001. See also Appendix Table S1.


Representative of H & E ( top ) PAS ( bottom ) of quadriceps in wild - type Gaa - / - mice untreated or treated with escalating doses of AT845 ( 3 × 1013 , 1 × 1014 , 3 × 1014 vg / kg ) . Scale bar , 80 µm . ( See also Appendix Table S1 )

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Representative images of H&E (top) and PAS (bottom) staining of the quadriceps in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). Scale bar, 80 µm. (See also Appendix Table S1)


A - D ( A , B ) curves ( C , D ) grip response - ability to grip onto an inverted wire screen for a 60 - second period - in wild - type Gaa - / - mice untreated or treated with escalating doses of AT845 ( 3 × 1013 , 1 × 1014 , 3 × 1014 vg / kg ) . groups had n = 9 at timepoints , except 6 week timepoint ( ) 12 week timepoint ( ) for Gaa - / - mice , which had n = 8 per group . mice dosed with 3 × 1013 , 1 × 1014 , 3 × 1014 vg / kg had a 22 % , 11 % , 0 % failure rate at week 12 , respectively ; in comparison , control WT mice had a 22 % failure rate control Gaa - / - mice had a 38 % failure . mice dosed with 3 × 1013 , 1 × 1014 , 3 × 1014 vg / kg had a 56 % , 33 % , 11 % failure rate at week 12 , respectively ; in comparison , control WT mice had a 11 % failure rate control Gaa - / - mice had a 100 % failure . Bars represent median . Data show a dose - dependent improvement in grip response following treatment with AT845 . Statistical : two - way ANOVA , Dunnett ' s . Asterisks ( * ) indicate significant differences compared with untreated Gaa - / - mice at each respective time point . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

O O O O O O O O B-EXP_ASSAY O O O 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-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O 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-D (A, B) Body weight curves and (C, D) grip response - ability to grip onto an inverted wire screen for a 60-second period - in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). All groups had n=9 at all timepoints, except the 6 week timepoint (females) and the 12 week timepoint (males and females) for the Gaa-/- mice, which had n=8 per group. Female mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 22%, 11%, and 0% failure rate at week 12, respectively; in comparison, the female control WT mice had a 22% failure rate and the control Gaa-/- mice had a 38% failure. Male mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 56%, 33%, and 11% failure rate at week 12, respectively; in comparison, the male control WT mice had a 11% failure rate and the control Gaa-/- mice had a 100% failure. Bars represent median. Data show a dose-dependent improvement in grip response following treatment with AT845. Statistical analysis: two-way ANOVA, Dunnett's test. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice at each respective time point. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


A , B Equal numbers of cynomolgus monkeys were treated with vehicle control ( n = 4 ) or escalating doses AT845 ( n = 6 ) followed for approximately 12 . ( A ) Vector copy number ( B ) GAA activity in heart , quadriceps , triceps , diaphragm , liver . Statistical : mixed - effects with Dunnett ' s . Data are presented as box - - whisker plots with Tukey whiskers that show minimum , median , maximum values . Asterisks ( * ) indicate significant differences compared with vehicle control NHPs . * , P < 0 . 05 ; * * , P < 0 . 01 .

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A, B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control (n=4) or escalating doses AT845 (n=6) and followed for approximately 12 weeks. (A) Vector copy number and (B) GAA activity in heart, quadriceps, triceps, diaphragm, and liver. Statistical analysis: mixed-effects analysis with Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values. Asterisks (*) indicate significant differences compared with vehicle control NHPs. *, P<0.05; **, P<0.01.


A - C cynomolgus monkeys were treated with vehicle control ( n = 2 ) , AT845 ( n = 3 ) at 6 × 1013 , 2 × 1014 , or 5 × 1014 vg / kg , or Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg followed for approximately 12 . ( A ) Anti - GAA antibody titer , ( B ) cardiac troponin I ( cTnI ) , ( C ) N - terminal pro b - type natriuretic peptide ( BNP ) . Bars show mean .

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A-C Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3) at 6×1013, 2×1014, or 5×1014 vg/kg, or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. (A) Anti-GAA antibody titer, (B) cardiac troponin I (cTnI), and (C) N-terminal pro b-type natriuretic peptide (BNP). Bars show mean and SEM.


A , B cynomolgus monkeys were treated with vehicle control ( n = 2 ) , AT845 ( n = 3 ) , or Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg followed for approximately 12 . GAA activity in ( A ) heart , quadriceps , triceps , diaphragm , ( B ) liver , brain , spinal cord , testes . Data are presented as box - - whisker plots with Tukey whiskers that show minimum , median , maximum values .

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A, B Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3), or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. GAA activity in (A) heart, quadriceps, triceps, and diaphragm, and (B) liver, brain, spinal cord, and testes. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values.


A , B Western blot of heart quadriceps lysates using anti - GAA detects both precursor GAA ( 110 kDa ) mature lysosomal GAA ( 70 to 76 kDa ) forms in muscle of NHPs treated with ( A ) AT845 ( n = 3 ) or ( B ) Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg . Each tissue was analyzed in triplicate , each respective symbol indicates use of a vehicle control heart tissue ( ^ ) or a vehicle control quadriceps tissue ( * ) . ( See also Appendix Table S3 )

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A, B Western blot analysis of heart and quadriceps lysates using anti-GAA detects both the precursor GAA (110 kDa) and the mature lysosomal GAA (70 to 76 kDa) forms in muscle of NHPs treated with (A) AT845 (n=3) or (B) Cyno-AT845 (n=3) at 2×1014 vg/kg. Each tissue was analyzed in triplicate, and each respective symbol indicates use of a vehicle control heart tissue (^) or a vehicle control quadriceps tissue (*). (See also Appendix Table S3)


( D ) At 48h after transfection of H7 plasmid , transfectants were incubated with TPCK - trypsin in presence of recombinant wtSPINK6 or PBS or a protease inhibitor AEBSF for 30 min then applied to examine HA cleavage .

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(D) At 48h after transfection of H7 plasmid, the transfectants were incubated with TPCK-trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage.


( E ) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate , A549 were inoculated with H7N9 / ah . At indicated hours post infection ( hpi ) , culture media ( supernatant ) lysate were harvested for viral load detection . Data are presented as mean SD in a representative experiment performed 3 times . * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t .

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(E) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate, A549 cells were inoculated with H7N9/ah virus. At the indicated hours post infection (hpi), culture media (supernatant) and cell lysate were harvested for viral load detection. Data are presented as mean and SD in a representative experiment performed 3 times. **, P<0.01; ***, P<0.001. Student's t test.


( A ) At 48 hours post transfection of indicated protease plasmids in triplicate , BHK21 were incubated with a fluorogenic substrate wtSPINK6 or loss - of - function mutSPINK6 or PBS for 2 hours then applied to . Data represent mean SD of triplicated wells in a representative experiment performed 3 times . * * , P < 0 . 01 . Student ' s t .

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(A) At 48 hours post transfection of the indicated protease plasmids in triplicate, BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.


( B ) At 48h after co - transfection of indicated protease plasmids H7 or H1 plasmid , BHK21 were lysed to examine HA cleavage with WB .

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(B) At 48h after co-transfection of the indicated protease plasmids and H7 or H1 plasmid, BHK21 cells were lysed to examine HA cleavage with WB.


( E ) At 48h after triple - transfection of H1 , indicated proteases wtSPINK6 or mutSPINK6 , BHK21 were lysed for detection of HA cleavage .

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(E) At 48h after triple-transfection of H1, the indicated proteases and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage.


( A ) At 36h after transfection of indicted protease wtSPINK6 plasmids or blank vector in triplicate , BHK21 were inoculated with H7N9 / ah at a MOI of 0 . 5 . - free culture media were harvested at 24 hpi for viral titration . Data represent mean SD of triplicated wells in a representative experiment performed 3 times . * * , P < 0 . 01 . Student ' s t .

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(A) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell-free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.


( B ) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate , A549 were inoculated with H1N1 / pdm at MOI of 0 . 25 . infected were incubated with recombinant wtSPINK6 or mutSPINK6 in triplicate for 24h . Culture media in each well were stored in two aliquots , one was applied to viral titration with conventional plaque ( black bars ) , other was pre - treated with TPCK - trypsin for 1 hour prior to plaque ( grey bars ) . Data represent mean SD of triplicated wells in a representative experiment performed 3 times . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t .

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(B) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars), the other was pre-treated with TPCK-trypsin for 1 hour prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( A ) Confocal imaging ( left ) shows intrinsic SPINK6 ( green ) expression in epithelial in 2D human airway organoids . Nuclei actin filaments are counterstained shown as blue purple respectively . Scale , 10 µm . Flow cytometry demonstrates HAT SPINK6 expression in human airway organoids . plots ( middle ) show representative result of one experiment performed 3 times . percentages of HAT SPINK6 positive ( right ) are shown as mean SD of 3 independent experiments . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t .

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(A) Confocal imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple respectively. Scale, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed 3 times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of 3 independent experiments. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( B ) At 24h after inoculation of H1N1 / pdm or mock inoculation in triplicate , 2D human airway organoids were harvested to detect mRNA expression of indicated genes normalized with GAPDH . Data represent mean SD of a representative experiment performed three times . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t .

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(B) At 24h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( E ) 2D airway organoids were applied to after incubation with α - SPINK6 or isotypic IgG in triplicate for 4 hours ( left ) . Data present mean SD of triplicated samples ( right ) . Data present mean SD of triplicated samples . ( F ) After inoculation of H1N1 / pdm , 2D human airway organoids were incubated with an α - SPINK6 or isotype IgG in triplicate for 24 hours ( left ) . - free culture media were harvested for viral load detection ( right ) examination of HA cleavage by WB after 10 - fold ( middle ) . Viral load data present mean SD of triplicated samples . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t .

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(E) 2D airway organoids were applied to fluorescence assay after incubation with α-SPINK6 or isotypic IgG in triplicate for 4 hours (left). Data present mean and SD of triplicated samples (right). Data present the mean and SD of triplicated samples. (F) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with an α-SPINK6 or isotype IgG in triplicate for 24 hours (left). Cell-free culture media were harvested for viral load detection (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral load data present the mean and SD of triplicated samples. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( B ) Survival rates of mice treated with wtSPINK6 mutSPINK6 was analyzed with Mantel - Cox .

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(B) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel-Cox test.


( C ) Viral load viral titer in lung homogenates of mice treated with wtSPINK6 mutSPINK6 . Data represent mean ± SD . * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t .

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(C) Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6. Data represent mean ± SD. **, P < 0.01; ***, P< 0.001. Student's t test.


( D ) Mouse lung tissues are applied to immunofluorescence to identify viral NP positive ( green ) . Nuclei are counterstained with DAPI ( blue ) . Representative confocal of - infected in indicated mice . Scale bar , 10 µm .

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(D) Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP positive cells (green). Nuclei are counterstained with DAPI (blue). Representative confocal images of virus-infected cells in the indicated mice. Scale bar, 10 µm.


( A ) association SNP rs1432689 genotypes are correlated to differential SPINK6 mRNA expression in over 1000 human lung tissues from three centers . box denotes interquartile range ; thick diamond within box are median mean respectively ; whiskers are minimum maximum open dots are outliers . Linear regression was used for eQTL data . Data of three cohorts were applied to meta - resulted in P - value of association SNP .

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(A) The association SNP rs1432689 genotypes are correlated to differential SPINK6 mRNA expression in over 1000 human lung tissues from three centers. The box denotes interquartile range; the thick line and diamond within the box are the median and mean respectively; whiskers are the minimum and maximum and open dots are outliers. Linear regression was used for eQTL data analysis. Data of three cohorts were applied to meta-analysis and resulted in the P-value of the association SNP.


( B ) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues esophagus mucosa from GTEx database . Sample sizes for each genotype are shown in x - axis . y - axis shows normalized expression of SPINK6 ( Norm . expression ) in indicated trusses . Median interquartile range are represented by a box respectively . Data distribution is colored with blue ; a wider section of violin plot indicates data on section have a higher frequency . Linear regression was used for data .

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(B) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database. Sample sizes for each genotype are shown in the x-axis. The y-axis shows the normalized expression of SPINK6 (Norm. expression) in the indicated trusses. Median and interquartile range are represented by a white line and dark box respectively. Data distribution is colored with light blue; a wider section of the violin plot indicates the data on the section have a higher frequency. Linear regression was used for data analysis.


A Electron microscopy analyses to see autophagic bodies in vacuoles under both growth nitrogen starvation conditions . Autophagic bodies typically contain ribosomes . pep4 ∆ prb1 ∆ pep4 ∆ prb1 ∆ atg2 ∆ were grown in SD to mid ‐ log phase ( OD600 = 1 ) transferred to SD ( ‐ N ) for 5 h . were examined by transmission electron microscopy as described in . Scale bar , 500 nm .

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A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm.


B - D Time ‐ dependent changes in nucleoside contents under nitrogen starvation ( B ) , rapamycin treatment ( C ) , carbon starvation ( D ) conditions . Wild ‐ type atg2Δ were grown in SD transferred to SD ( ‐ N ) , SD medium with 0 . 2 μM rapamycin , or SD ( ‐ C ) at time 0 . At indicated time points , nucleosides were analyzed by LC / MS as described in . Results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . data are means of triplicate samples . error bars represent standard deviation .

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B-D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.


A Time ‐ dependent changes in nucleoside contents under nitrogen starvation . Nucleosides in wild ‐ type , atg2Δ , atg7Δ , atg11Δ , atg17Δ , atg19Δ , atg32Δ , nvj1Δ , pep4prb1Δ under nitrogen starvation . data are means of triplicates . error bars represent standard deviation .

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A Time‐dependent changes in nucleoside contents under nitrogen starvation. Nucleosides in wild‐type, atg2Δ, atg7Δ, atg11Δ, atg17Δ, atg19Δ, atg32Δ, nvj1Δ, and pep4prb1Δ cells under nitrogen starvation. All data are means of triplicates. The error bars represent the standard deviation.


B Alkaline phosphatase ( ALP ) ( Pho8 ∆ 60 ) . Wild ‐ type , atg2 ∆ , atg11Δ , atg17Δ , atg19Δ expressing Pho8 ∆ 60 were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , lysates were prepared subjected to ALP . bars represent standard deviation of three independent experiments . * P 0 . 05 ; * * P 0 . 005 ; * * * P 0 . 0005 ( paired t ‐ , two ‐ )

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B Alkaline phosphatase (ALP) assay (Pho8∆60). Wild‐type, atg2∆, atg11Δ, atg17Δ, and atg19Δ cells expressing Pho8∆60 were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, lysates were prepared and subjected to the ALP assay. The bars represent the standard deviation of three independent experiments. *P 0.05; **P 0.005; ***P 0.0005 (paired t‐test, two‐tailed)


A - C Wild ‐ type , atg2Δ , pho8Δ , rny1Δ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , nucleosides ( A ) were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . data are means of triplicates . error bars represent standard deviation .

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A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, nucleosides (A) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


A - C Wild ‐ type , atg2Δ , pho8Δ , rny1Δ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , 5 ′ ‐ NMPs ( B ) were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . data are means of triplicates . error bars represent standard deviation .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O

A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 5′‐NMPs (B) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


A - C Wild ‐ type , atg2Δ , pho8Δ , rny1Δ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , 3 ′ ‐ NMPs ( C ) were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . data are means of triplicates . error bars represent standard deviation .

A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 3′‐NMPs (C) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


A , B Expression localization of Rny1 Pho8 . Wild ‐ type atg2Δ expressing Rny1 ‐ GFP or Pho8 ‐ GFP were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) . After 2 h of starvation , GFP ‐ tagged were observed by microscopy ( A ) . Scale bar , 5 μm .

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A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2Δ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were observed by fluorescence microscopy (A). Scale bar, 5 μm.


A , B Expression localization of Rny1 Pho8 . Wild ‐ type atg2 ∆ expressing Rny1 ‐ GFP or Pho8 ‐ GFP were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) . After 2 h of starvation , GFP ‐ tagged were analyzed by Western blot with anti ‐ GFP antibody ( B ) .

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A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2∆ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were analyzed by Western blot with anti‐GFP antibody (B).


C Detection of free . Wild ‐ type , atg2 ∆ , rny1 ∆ , rny1 ∆ atg2 ∆ grown in SD ( left ) or SD ( ‐ N ) for 2 h ( right ) were stained with FM4 ‐ 64 GR Green observed by microscopy . Scale bar , 5 μm .

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C Detection of free RNA. Wild‐type, atg2∆, rny1∆, and rny1∆atg2∆ cells grown in SD (left) or SD(‐N) for 2 h (right) were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.


A Time ‐ dependent change in nucleoside after nitrogen starvation . Wild ‐ type , atg2 ∆ , ubp3 ∆ , bre5 ∆ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . Metabolites were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . data are means of triplicates . error bars represent standard deviation .

O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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 Time‐dependent change in nucleoside levels after nitrogen starvation. Wild‐type, atg2∆, ubp3∆, and bre5∆ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. Metabolites were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.


B Detection of free within . rny1 ∆ , rny1 ∆ atg2 ∆ , rny1 ∆ ubp3 ∆ , rny1 ∆ bre5 ∆ grown in SD ( ‐ N ) for 2 h were stained with FM4 ‐ 64 GR Green observed by microscopy . Scale bar , 5 μm .

O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O

B Detection of free RNA within cells. rny1∆, rny1∆atg2∆, rny1∆ubp3∆, and rny1∆bre5∆ cells grown in SD(‐N) for 2 h were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.


B , C Nucleosides ( B ) nucleobases ( C ) within . Wild ‐ type atg2 ∆ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , were collected ; their metabolites were extracted , nucleosides nucleobases were analyzed by LC / MS . level of each nucleoside base is presented as its absolute , as described in Supplementary Methods . data are means of duplicates except wild ‐ type at 1 h .

O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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, C Nucleosides (B) and nucleobases (C) within the cells. Wild‐type and atg2∆ were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; their metabolites were extracted, and the nucleosides and nucleobases were analyzed by LC/MS. The level of each nucleoside and base is presented as its absolute concentration, as described in and Supplementary Methods. All data are means of duplicates except the wild‐type strain at 1 h.


D , E Nucleosides ( D ) nucleobases ( E ) in medium . culture medium of above cultures was filtered , nucleosides nucleobases were directly analyzed by LC / MS as described in Supplementary Methods . data are means of duplicates .

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D, E Nucleosides (D) and nucleobases (E) in the medium. The culture medium of the above cultures was filtered, and nucleosides and nucleobases were directly analyzed by LC/MS as described in and Supplementary Methods. All data are means of duplicates.


F , G Destinations of nucleosides bases . on data from ( B - E ) , level of each nucleoside base in ( open circle ) in medium ( closed circle ) of wild ‐ type were estimated . See Supplementary Methods in detail ( flow chart of calculation procedure ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G Destinations of nucleosides and bases. Based on the data from (B-E), the level of each nucleoside and base in cells (open circle) and in cells and medium (closed circle) of wild‐type strain were estimated. See Supplementary Methods in detail (flow chart of the calculation procedure).


Wild ‐ type atg1Δ of S . pombe were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , were collected ; metabolites in these were extracted analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . data are means of triplicates . error bars represent standard deviation .

O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild‐type and atg1Δ of S. pombe were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; the metabolites in these cells were extracted and analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.


B , C GOT2 can be acetylated . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T treated with NAM ( 5 mM ) / or TSA ( 0 . 5 mM ) for indicated time period . Acetylation of Flag ‐ bead ‐ purified GOT2 were determined by Western blot using a pan ‐ anti ‐ acetyllysine antibody ( α ‐ Ac ) . IB IP denote immunoblotting immunoprecipitation , respectively . Relative GOT2 acetylation ratios were calculated after normalizing against Flag .

O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O

B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.


D GOT2 acetylation enhances interaction between ectopically expressed GOT2 MDH2 . Flag ‐ GOT2 Myc ‐ MDH2 were co ‐ overexpressed in HEK293T treated without or with NAM ( 5 mM ) for indicated time period . acetylation level of Flag ‐ bead ‐ purified GOT2 association between ectopic of GOT2 MDH2 were determined by Western blot .

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D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.


E Acetylation enhances interaction between ectopically expressed GOT2 endogenous MDH2 . Flag ‐ GOT2 was co ‐ overexpressed in HEK293T treated without or with NAM ( 5 mM ) for indicated time period . acetylation level of Flag ‐ GOT2 its association with endogenous MDH2 were determined by Western blot .

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E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.


F Acetylation enhances interaction between ectopically expressed MDH2 endogenous GOT2 . Flag ‐ MDH2 was co ‐ overexpressed in HEK293T treated without or with NAM ( 5 mM ) for indicated time period . association between Flag ‐ MDH2 endogenous GOT2 was determined by Western blot .

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F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis.


H Mapping major lysine residue ( s ) of acetylation in GOT2 whose acetylation can affect interaction between GOT2 MDH2 . Putative acetylated residues were divided into six groups according to their position in structure of GOT2 . Each group of putative acetylated lysine ( K ) sites was mutated to arginine ( R ) , deacetylated mimic K ‐ to ‐ R mutants were examined for their association with ectopically expressed Myc ‐ MDH2 by Western blot .

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H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis.


I GOT2 coupled with MDH2 shows a high level of K159 acetylation . Flag ‐ GOT2 was overexpressed in HEK293T without or with co ‐ overexpression of Myc ‐ MDH2 . These transfected were treated with NAM ( 5 mM ) for 5 h . Double immunoprecipitation was performed to obtain Flag ‐ GOT2 from Myc ‐ MDH2 immunoprecipitates ( Ppt , supposed to be GOT2 bound with MDH2 ) . remaining supernatants ( Sup , supposed to be GOT2 not bound with MDH2 ) were also harvested precipitated by Flag antibody to K159 acetylation level of Flag ‐ GOT2 by Western blot .

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I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis.


J 3KQ / R mutations do not change GOT2 enzyme activity . Wild ‐ type 3K mutant GOT2 were overexpressed purified from E . coli , enzyme activity of GOT2 was determined as described in " " . Shown are average values with standard deviation ( SD ) of triplicated experiments . n . s . = not significant for indicated comparison .

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J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild‐type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in "". Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.


A , B Glucose glutamine increase GOT2 K159 acetylation GOT2 - MDH2 association in HEK293T . Flag ‐ GOT2 Myc ‐ MDH2 were overexpressed in treated with increased concentrations of glucose ( A ) or glutamine ( B ) for 6 h . GOT2 were purified by Flag beads , K159 acetylation level of GOT2 its association with Myc ‐ MDH2 were determined by Western blot .

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A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.


C , D Glucose glutamine increase GOT2 K159 acetylation in Panc ‐ 1 . Flag ‐ GOT2 was overexpressed in treated with different concentrations of glucose ( C ) glutamine ( D ) for 4 h . GOT2proteins were purified by Flag beads , K159 acetylation level of GOT2 was determined by Western blot . Relative GOT2 K159 acetylation were normalized against Flagprotein .

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C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels.


E , F of of K159 ‐ acetylated endogenous GOT2 in Panc ‐ 1 . Recombinant fully K159 ‐ acetylated GOT2 was loaded onto same gel , together with endogenous GOT2 from Panc ‐ 1 treated without or with glucose ( 12 mM ) ( E ) or glutamine ( 2 mM ) ( F ) for 4 h . GOT2 K159 acetylation were detected by Western blot . percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 .

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E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.


G , H 3K mutant GOT2 displays negligible response in changing association with MDH2 after glucose or glutamine treatment . Panc ‐ 1 with GOT2 knockdown re ‐ expression of wild ‐ type or 3K mutant GOT2 were treated with glucose ( G ) or glutamine ( H ) at indicated concentrations for 4 h . association between Flag ‐ tagged wild ‐ type or 3K mutant GOT2 endogenous MDH2 was determined by Western blot .

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G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.


A GOT2 interacts with SIRT3 , but not SIRT4 SIRT5 . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T together with HA ‐ tagged SIRT as indicated . were purified by IP with Flag beads , following Western blot to detect SIRTs with an HA antibody .

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A GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA‐tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.


B Endogenous of GOT2 SIRT3 interact with each other . SIRT3 in HEK293T was purified by IP with an anti ‐ SIRT3 antibody , following Western blot to detect GOT2 with an anti ‐ GOT2 antibody .

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B Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti‐SIRT3 antibody, following Western blot to detect GOT2 with an anti‐GOT2 antibody.


C GOT2 deacetylation is dependent on SIRT3 catalytic activity . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T together with HA ‐ tagged wild ‐ type SIRT3 a catalytically inactive mutant , SIRT3 H248Y . GOT2 were purified by Flag beads , following Western blot to detect GOT2 K159 acetylation .

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C GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with HA‐tagged wild‐type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.


D SIRT3 impairs interaction between GOT2 MDH2 . HEK293T were transfected with plasmids as indicated . GOT2 were purified by Flag beads , their interaction with Myc ‐ MDH2 was determined by Western blot .

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D SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc‐MDH2 was determined by Western blot.


E Knockdown of SIRT3 increases GOT2 K159 acetylation enhances GOT2 - MDH2 association . HEK293T with or without transient SIRT3 knockdown were transfected with plasmids as indicated . GOT2 were purified by Flag beads , K159 acetylation level of GOT2 its association with endogenous MDH2 were determined by Western blot .

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E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2-MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.


F , G Knockdown of SIRT3 diminishes effect of glucose or glutamine on changing GOT2 K159 acetylation . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T without or with transient SIRT3 knockdown . These were treated with different concentrations of glucose ( F ) glutamine ( G ) as indicated . GOT2 were purified by Flag beads , K159 acetylation level of GOT2 was determined by Western blot .

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F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.


H , I Up ‐ regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting . C57BL / 6 mice ( , 20 ; n = 2 per group ) were fed a normal chow diet or fasted overnight . Upon sacrifice , mouse liver adipose tissues were harvested , of indicated were determined by Western blot .

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H, I Up‐regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.


A malate - aspartate shuttle , such as LDH , also plays a key role in regulating NADH in Panc ‐ 1 . were treated with AOA ( 1 . 6 mM , 20 min ) , oxamate ( 5 mM , 20 min ) , EGCG ( 0 . 5 mM , 20 min ) . NADH level was measured as described in .

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A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in .


B , C GOT2 3K acetylation promotes net transfer of NADH into mitochondria . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , NADH level in cytosol ( B ) mitochondrion ( C ) was determined in extracts as described in .

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B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in .


D GOT2 3K acetylation regulates mitochondrial NADH / NAD + redox in . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , of NADH / NAD + in mitochondria of was measured as described in .

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D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in .


E GOT2 3K acetylation promotes ATP production . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , ATP production was determined in extracts as described in .

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E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in .


F GOT2 3K acetylation promotes NADPH production . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , of NADPH / NADP + was determined in extracts as described in .

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F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in .


G GOT2 3K acetylation promotes GSH production . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , GSH / GSSG was determined in extracts as described in .

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G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in .


H GOT2 3K acetylation suppresses ROS . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , ROS was determined as described in in under non ‐ stressed condition or exposed to menadione ( 50 μM for 30 min ) .

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H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min).


I GOT2 3K acetylation protects from oxidative damage . Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated were treated with increasing concentrations of menadione for 3 h as indicated , viability was determined by counting remaining adherent .

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I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.


J , K Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated were seeded in a 6 ‐ well plate , were maintained under condition of low glucose ( 0 . 5 mM , J ) or high glucose ( 12 mM , K ) . Culture medium was refreshed every day , numbers were counted every 1 - 2 days over a period of 7 days .

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J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days.


L , M Panc ‐ 1 stable in ( J , K ) were grown under low glucose ( 0 . 5 mM ) or high glucose ( 12 mM ) for 7 days . level of histone H3 phosphorylation at Ser10 was determined by Western blot ( L ) . percentages of living apoptotic were determined by flow cytometry as described in ( M ) .

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L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).


A - C GOT2 3K acetylation promotes xenograft tumorgrowth . Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated ( 2 × 106cells ) were injected subcutaneously into flanks of nude mice . volume was carefully monitored over indicated time period ( A ) . At 8 after injection , tumors from six mice were extracted , photographed , weighted ( B , C ) .

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A-C GOT2 3K acetylation promotes xenograft tumorgrowth. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins (2 × 106cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C).


D GOT2 3K acetylation promotes cellproliferation . sections from xenografts were prepared for , PCNA ‐ stained sections were analyzed by quantifying PCNA ‐ positive .

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D GOT2 3K acetylation promotes tumor cellproliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA‐stained tumor sections were analyzed by quantifying the PCNA‐positive area.


E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) adjacent normal tissue ( shown as N ) were lysed . acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot . F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) adjacent normal tissue ( shown as N ) were lysed . SIRT3protein were compared against tubulin by Western blot . G , H of GOT2 K159 acetylation SIRT3protein expression in clinical cases as described in ( E ) ( F ) .

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E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot.F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3protein levels were compared against tubulin by Western blot.G, H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in (E) and (F).


( a ) Subcellular compartments from U937 extracts were separated by sedimentation velocity on discontinuous sucrose gradient probed for indicated by immunoblotting . Numbers represent fractions from gradient ( 1 , top ; 5 , bottom ) .

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(a) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom).


( b ) Immunoblot of compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit .

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(b) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit.


( c ) Immunoblot of KDEL IRGM distribution in different fractions after purification of mitochondria as in b .

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(c) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b.


d ) Intracellular localization of IRGM analysed by confocal microscopy . were stained with antibodies against indicated or vitally with MitoTracker Red ( MTR ) where indicated . on right are merged from on left . A - L ; endogenous IRGM localization in HeLa relative to calnexin , MTR , cytochrome c COX IV . M - O ; IRGM localization in human macrophages relative to mitochondria visualized with cytochrome c antibody . Scale bars , 5 μm .

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d) Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A-L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M-O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm.


( e ) of IRGM ( green ) COX IV ( red ) as a function of along shown in inset . Image in inset corresponds to panels J - L in d . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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(e) Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J-L in d. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Purified mitochondria were left untreated , or were treated with Proteinase K ( Pr . K ) following osmotic shock ( OS ) , or solubilization of total using Triton X - 100 ( TX - 100 ) , as indicated . Samples were analysed by immunoblotting with antibodies against indicated .

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(a) Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins.


( b ) Mitochondrial preparations were subjected to disruption by freeze - thaw cycles as indicated accessibility of indicated to Proteinase K was examined by immunoblotting .

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(b) Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting.


( c ) Immunoblot of membranous organelles from U937 separated by isopycnic sucrose density - gradient centrifugation . Numbers represent fractions from gradient ( 1 , top ; 12 , bottom ) . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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(c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) were transfected with control siRNA or with siRNA specific to IRGM ( top ) DRP1 ( bottom ) for 48 h samples from these were then analysed by immunoblotting with anti - IRGM anti - DRP1 antibodies . Actin was used as a loading control .

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(a) Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control.


( b ) HeLa were treated with siRNA specific to either IRGM or DRP1 as indicated were then labelled with MTR analysed by live - confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm.


( c ) were treated with either control siRNA or with DRP1 , IRGM , ATG7 or BECN1 ( Beclin 1 ) siRNAs , labelled with MTR of with mitochondrial morphologies ranging from normal , punctiform ( dots ) elongated were quantified . Definition of mitochondrial morphologies criteria are given in Supplementary Information , Figure S2 .

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(c) Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2.


( d ) were transfected with either control siRNA or with siRNA specific to MFN1 / MFN2 , IRGM or IRGM MFN1 / MFN2 , then labelled with MTR analysed for mitochondrial morphologies . Data are means ± s . e . m . ( c , n = 3 ; d , n = 4 ) . Asterisk indicates P 0 . 05 , double asterisks indicate P 0 . 01 dagger indicates P > 0 . 05 ( t - ) .

O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial morphologies. Data are means ± s.e.m. (c, n = 3; d, n = 4). Asterisk indicates P 0.05, double asterisks indicate P 0.01 and dagger indicates P > 0.05 (t-test).


( a ) U937 were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by treatment with hIFN - γ . GFP - LC3 in were imaged by confocal microscopy . Scale bars , 5 μm . ( b ) LC3 per were quantified from treated as in a .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O

(a) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by treatment with hIFN-γ. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. (b) LC3 puncta per cell were quantified from cells treated as in a.


( c ) U937 were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by starvation . GFP - LC3 in were imaged by confocal microscopy . Scale bars , 5 μm .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(c) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by starvation. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm.


( d ) U937 were transfected with either control siRNA or siRNA specific to MFN1 / MFN2 , endogenous LC3 per were quantified .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O

(d) U937 cells were transfected with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified.


( e ) U937 were transfected with indicated siRNA infected with BCG . Autophagy was induced by starvation phagosome maturation was analysed using LysoTracker probe ( LTR ) as a reporter of acidification .

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(e) U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification.