( D ) Experimental scheme for evaluation of the chromosomal aberrations is shown . shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU , then left to recover for 16h in drug - free medium and metaphases collected with colcemid . Next , cells were fixed and processed as reported in " Supplemental information " section . Dot plot shows the number of chromosomal aberrations per cell . Horizontal black lines represent the mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative Giemsa - stained metaphases of cells treated or not with 4 mM HU . Arrows indicate chromosomal aberrations .

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(D) Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU, then left to recover for 16h in drug-free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.


( E ) Experimental scheme of the Mirin aberration analysis is given . The experiment was carried out as in ( D ) but cells were pre - treated or not with 50 µM Mirin . Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells . Horizontal black lines represent the mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative Giemsa - stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU . Arrows indicate chromosomal aberrations .

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(E) Experimental scheme of the Mirin aberration analysis is given. The experiment was carried out as in (D) but cells were pre-treated or not with 50 µM Mirin. Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.


( F ) Experimental design of the chromosomal aberration assay is reported . shWRNIP1 cells were transfected with control siRNAs ( siCtrl ) or FBH1 siRNA ( siFBH1 ) . Fourth - eight hours thereafter , cells were treated or not with 4 mM HU and then left to recover for 16 h . Metaphases were collected with colcemid and prepared as reported in " Supplemental information " section . Dot plot shows the number of chromosomal aberrations per cell . Western blot shows FBH1 depletion in the cells . The membrane was probed with an anti - FBH1 . GAPDH was used as a loading control . Horizontal black lines represent the mean SE . Error bars represent standard error . ( * * , p < 0 . 01 ; two - tailed Student ' s t test ) .

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(F) Experimental design of the chromosomal aberration assay is reported. shWRNIP1 cells were transfected with control siRNAs (siCtrl) or FBH1 siRNA (siFBH1). Fourth-eight hours thereafter, cells were treated or not with 4 mM HU and then left to recover for 16 h. Metaphases were collected with colcemid and prepared as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Horizontal black lines represent the mean SE. Error bars represent standard error. (**, p < 0.01; two-tailed Student's t test).


SILAC - based proteomics data identify known ( red ) & novel ( green ) hypoxia - induced proteins in SW620 cells . One sample T - test was performed .

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SILAC-based proteomics data identify known (red) & novel (green) hypoxia-induced proteins in SW620 cells. One sample T-test was performed.


Western blotting confirmed GPRC5A as a hypoxia - induced protein in SILAC lysates .

Western blotting confirmed GPRC5A as a hypoxia-induced protein in SILAC lysates.


Validation of GPRC5A western blot data using siRNA . * Non - specific band of ~ 60kDa not depleted by GPRC5A siRNA .

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Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA.


Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells ( scale bars : 75um ) .

Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75um).


Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines .

Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines.


Basal & hypoxia - induced GPRC5A protein expression was decreased by HIF - 1 / 2α depletion .

Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion.


Depletion of HIF - 1β decreased GPRC5A protein upregulation in hypoxia .

Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia.


Hypoxia mimetic DMOG induced HIF - 1 / 2α , CA9 & GPRC5A protein expression . Dual HIF - 1 / 2α depletion reduced GPRC5A induction by DMOG .

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Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.


qRT - PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


qRT - PCR demonstrating that HIF - 1 / 2α depletion decreased GPRC5A induction during hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


ChIP / PCR analyses identify HIF - 1α a binding to the GPRC5A promoter region containing a putative optimal HRE ( error bars ± SD ) .

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ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).


Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced CA9 expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um).


Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced ; GPRC5A expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).


IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia ( strangulated colon ) . GPRC5A is co - expressed with CA9 in the colonic epithelial cells ( scale bars : 50um ) .

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IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um).


Quantitative RT - PCR analysis of mouse intestinal tissue . Gene expression was normalised to housekeeping gene Tbp . Raw data from three independent experiments ( n = 3 mice ) are shown ( error bars ± SEM ) .

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Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM).


Tg [ fli1 : eGFP ; vhl - / - ] & Tg [ fli1 : eGFP ] zebrafish embryos ( 5 days post - fertilisation ) demonstrate excessive angiogenesis & increased expression of HIF target genes ( scale bars : 100um ) .

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Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).


gprc5ba was induced in vhl mutant zebrafish embryos & fli1 : eGFP zebrafish embryos exposed to 5 % O2 ( vs normoxia ) for 24 hours ( RT - PCR ) .

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gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).


Kaplan - Meier curve following analysis of transcriptomics dataset GSE24551 . Event - free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA .

Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.


GPRC5A depletion markedly increases caspase - 3 activation / PARP cleavage during hypoxia .

GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.


Three independent siRNA sequences targeting GPRC5A induce caspase - 3 activation / PARP cleavage during hypoxia .

Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.


GPRC5A depletion reduces hypoxic cell growth / survival . Crystal violet cell assays show reduced cell growth / survival in GPRC5A depleted cells during hypoxia [ n = 3 ] .

GPRC5A depletion reduces hypoxic cell growth/survival. Crystal violet cell assays show reduced cell growth/survival in GPRC5A depleted cells during hypoxia [n=3].


Expression of an siRNA - resistant GPRC5A cDNA rescues hypoxic GPRC5A - depleted cells from apoptosis . Upper : doxycycline - induced expression GPRC5Asi1R rescues increases caspase - 3 / PARP cleavage induced by GPRC5A depletion in hypoxia . Lower : generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations .

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Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.


Caspase inhibitor QVD prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

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Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe / dead cell apoptosis assay and flow

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GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry


Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells .

Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.


Hypoxia - induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells .

Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells.


Hypoxia activated TEAD activity ( 8x GTIIC - luc reporter ) but this was reduced by GPRC5A depletion . A representative triplicate experiment is shown ( n = 3 ) .

Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3).


Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion .

Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion.


Constitutively active RhoA ( G14V ) expression restored YAP stabilisation ( Ser397 dephosphorylation ) by hypoxia in GPRC5A depleted cells .

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Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells.


The YAP / TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay ( n = 3 independent experiments ) .

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The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).


YAP knockdown was sufficient to induce caspase - 3 activation / PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion .

YAP knockdown was sufficient to induce caspase-3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion.


Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival . GPRC5A depleted cells were rescued by expression of an siRNA - resistant GPRC5A cDNA ( GPRC5Asi1R ) but this was prevented by co - depletion of YAP ( n = 3 independent experiments ) .

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Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A depleted cells were rescued by expression of an siRNA-resistant GPRC5A cDNA (GPRC5Asi1R) but this was prevented by co-depletion of YAP (n=3 independent experiments).


Expression of an siRNA - resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion . GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia - induced YAP stabilisation ( Ser397 dephosphorylation ) ; these phenotypes were reversed by YAP depletion .

Expression of an siRNA-resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia-induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion.


GPRC5A depletion attenuated hypoxia - induced BCL - XL expression .

GPRC5A depletion attenuated hypoxia-induced BCL-XL expression.


Constitutively active YAP ( S127A ) expression induced BCL - XL expression and prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

Constitutively active YAP (S127A) expression induced BCL-XL expression and prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5Asi1R expression restored BCL - XL expression and prevents the appearance of cleaved caspase - 3 induced by GPRC5A depletion in hypoxia .

GPRC5Asi1R expression restored BCL-XL expression and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.


( A ) Immunoblot analysis of NRF1 , NRF2 - α and PGC1 - α in mouse PMs infected with VSV or HSV - 1 or treated with poly ( I : C ) or ISD for the indicated time points . ACTIN was used as a loading control . Ratios of target proteins versus loading control normalized to the 0 - h time point of each condition .

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(A) Immunoblot analysis of NRF1, NRF2-α and PGC1-α in mouse PMs infected with VSV or HSV-1 or treated with poly(I:C) or ISD for the indicated time points. ACTIN was used as a loading control. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition.


( B ) qPCR analysis of Nrf1 , Gabpa and Ppargc1a mRNA in mouse PMs infected with VSV or HSV - 1 or treated with poly ( I : C ) or ISD for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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(B) qPCR analysis of Nrf1, Gabpa and Ppargc1a mRNA in mouse PMs infected with VSV or HSV-1 or treated with poly(I:C) or ISD for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( C ) Immunoblot analysis of NRF1 expression in mouse PMs transfected with p65 - specific siRNA ( si - p65 ) or control siRNA ( si - NC ) for 48 h , followed by infection with VSV or HSV - 1 for 8 h . GAPDH was used as a loading control . Ratios of target proteins versus loading control normalized to the untreated sample of each condition .

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(C) Immunoblot analysis of NRF1 expression in mouse PMs transfected with p65-specific siRNA (si-p65) or control siRNA (si-NC) for 48 h, followed by infection with VSV or HSV-1 for 8 h. GAPDH was used as a loading control. Ratios of target proteins versus loading control normalized to the untreated sample of each condition.


( E ) Immunoblot analysis of mitochondrial proteins and autophagy marker LC3 in mouse PMs transfected with NRF1 - specific siRNA ( si - NRF1 ) or control siRNA ( si - NC ) for 48 h , followed by infection with VSV or HSV - 1 for the indicated time points . ACTIN was used as a loading control . Ratios of target proteins versus loading control normalized to the untreated sample of each condition .

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(E) Immunoblot analysis of mitochondrial proteins and autophagy marker LC3 in mouse PMs transfected with NRF1-specific siRNA (si-NRF1) or control siRNA (si-NC) for 48 h, followed by infection with VSV or HSV-1 for the indicated time points. ACTIN was used as a loading control. Ratios of target proteins versus loading control normalized to the untreated sample of each condition.


( F ) Mitochondrial mass determined by mtDNA / nuclear DNA ( nDNA ) ratio in mouse PMs transfected with NRF1 - specific siRNA ( si - NRF1 ) or control siRNA ( si - NC ) for 48 h , followed by infection with VSV or HSV - 1 for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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(F) Mitochondrial mass determined by mtDNA/nuclear DNA (nDNA) ratio in mouse PMs transfected with NRF1-specific siRNA (si-NRF1) or control siRNA (si-NC) for 48 h, followed by infection with VSV or HSV-1 for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


RNA - sequencing ( RNA - seq ) analysis of gene expression in wild - type and NRF1 - KO mouse PMs ( n = 3 biological replicates per group ) infected with VSV for 8 h . ( C ) Pheatmap analysis showing downregulated DEGs encoding mitochondrial inner membrane proteins . TPM , transcripts per kilobase of exon model per million mapped reads .

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RNA-sequencing (RNA-seq) analysis of gene expression in wild-type and NRF1-KO mouse PMs (n=3 biological replicates per group) infected with VSV for 8 h. (C) Pheatmap analysis showing downregulated DEGs encoding mitochondrial inner membrane proteins. TPM, transcripts per kilobase of exon model per million mapped reads.


( D ) Mitochondrial mass determined by mtDNA / nuclear DNA ( nDNA ) ratio in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments ( D ) and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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(D) Mitochondrial mass determined by mtDNA/nuclear DNA (nDNA) ratio in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments (D) and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( F and G ) The representative curves of oxygen consumption rate ( OCR ) in wild - type and NRF1 - KO mouse PMs infected with VSV ( F ) or HSV - 1 ( G ) for 8 h ( n = 5 biological replicates per group ) . Oligomycin : 1 µM , FCCP : 4 µM , Rotenone : 1 µM . Quantification of basal and maximal OCR . Data information : Data are from at least three biological replicates and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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(F and G) The representative curves of oxygen consumption rate (OCR) in wild-type and NRF1-KO mouse PMs infected with VSV (F) or HSV-1 (G) for 8 h (n=5 biological replicates per group). Oligomycin: 1 µM, FCCP: 4 µM, Rotenone: 1 µM. Quantification of basal and maximal OCR. Data information: Data are from at least three biological replicates and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( H and I ) Electron micrographs of mitochondria in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 for 8 h . Scale bars , 500 nm ( H ) . ( I ) Quantitative analysis of mitochondrial cristae ( the ratio of cristae number to mitochondrial area ) in ( H ) ( 20 - 30 mitochondria per group , biological replicates ) . Data information : Data are from at least three biological replicates and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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(H and I) Electron micrographs of mitochondria in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 for 8 h. Scale bars, 500 nm (H). (I) Quantitative analysis of mitochondrial cristae (the ratio of cristae number to mitochondrial area) in (H) (20-30 mitochondria per group, biological replicates). Data information: Data are from at least three biological replicates and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( A ) Luciferase activity of IFN - β , NF - κB and ISRE promoter reporter in HEK293T cells transfected with empty vector or plasmids encoding incremental NRF1 - Myc for 24 h followed by infection with VSV for 8 h ( n = 4 biological replicates per group ) .

(A) Luciferase activity of IFN-β, NF-κB and ISRE promoter reporter in HEK293T cells transfected with empty vector or plasmids encoding incremental NRF1-Myc for 24 h followed by infection with VSV for 8 h (n=4 biological replicates per group).


( B and C ) Immunoblot analysis of phosphorylated and total TBK1 , p65 and STAT1 in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 ( B ) or stimulated with poly ( I : C ) or ISD ( C ) for the indicated time points . GAPDH was used as a loading control . Ratios of phosphorylated proteins versus total proteins normalized to the 0 - h or 4 - h time point of each wild - type sample .

O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(B and C) Immunoblot analysis of phosphorylated and total TBK1, p65 and STAT1 in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 (B) or stimulated with poly(I:C) or ISD (C) for the indicated time points. GAPDH was used as a loading control. Ratios of phosphorylated proteins versus total proteins normalized to the 0-h or 4-h time point of each wild-type sample.


( D ) qPCR analysis of Ifnb1 , Il6 , Tnfa and Cxcl10 mRNA in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 or stimulated with poly ( I : C ) or ISD for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ND , not detectable , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(D) qPCR analysis of Ifnb1, Il6, Tnfa and Cxcl10 mRNA in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 or stimulated with poly(I:C) or ISD for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ND, not detectable, *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( E ) ELISA quantification of IFN - β , IL6 and TNF - α secretion in wild - type and NRF1 - KO mouse PMs treated as in ( D ) ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ND , not detectable , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(E) ELISA quantification of IFN-β, IL6 and TNF-α secretion in wild-type and NRF1-KO mouse PMs treated as in (D) (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ND, not detectable, *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( A ) 8 - week - old Nrf1F / F and Nrf1MyeKO mice ( n = 3 for PBS treatment and n = 6 for virus treatment ) were intravenously infected with VSV ( 4 × 107 PFU / mouse ) or HSV - 1 ( 1 × 107 PFU / mouse ) . After 24 h , ELISA quantification of IFN - β , IL6 and TNF - α in sera . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * 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 O O O O O O O O O O O O O O O

(A) 8-week-old Nrf1F/F and Nrf1MyeKO mice (n=3 for PBS treatment and n=6 for virus treatment) were intravenously infected with VSV (4×107 PFU/mouse) or HSV-1 (1×107 PFU/mouse). After 24 h, ELISA quantification of IFN-β, IL6 and TNF-α in sera. Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( B ) qPCR analysis of Ifnb1 , Il6 and Tnfa mRNA in the lung of Nrf1F / F and Nrf1MyeKO mice infected with VSV as in A ( n = 6 per group ) . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * 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

(B) qPCR analysis of Ifnb1, Il6 and Tnfa mRNA in the lung of Nrf1F/F and Nrf1MyeKO mice infected with VSV as in A (n=6 per group). Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( C ) qPCR analysis of VSV mRNA and plaque assay of VSV titers in the lungs of Nrf1F / F and Nrf1MyeKO mice infected with VSV as in A ( n = 6 per group ) . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * 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

(C) qPCR analysis of VSV mRNA and plaque assay of VSV titers in the lungs of Nrf1F/F and Nrf1MyeKO mice infected with VSV as in A (n=6 per group). Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( E ) qPCR analysis of Ifnb1 , Il6 and Tnfa mRNA in the brains of Nrf1F / F and Nrf1MyeKO mice infected with HSV - 1 as in ( A ) ( n = 6 per group ) . ( F ) qPCR analysis of HSV - 1 UL30 mRNA and plaque assay of HSV - 1 titers in the brain of Nrf1F / F and Nrf1MyeKO mice infected with HSV - 1 as in ( A ) ( n = 6 per group ) . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * 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 O O O O 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) qPCR analysis of Ifnb1, Il6 and Tnfa mRNA in the brains of Nrf1F/F and Nrf1MyeKO mice infected with HSV-1 as in (A) (n=6 per group). (F) qPCR analysis of HSV-1 UL30 mRNA and plaque assay of HSV-1 titers in the brain of Nrf1F/F and Nrf1MyeKO mice infected with HSV-1 as in (A) (n=6 per group). Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( G ) Survival of Nrf1F / F and Nrf1MyeKO mice ( n = 10 per group ) at various times after intraperitoneal ( i . p . ) infection with HSV - 1 ( 2 × 107 PFU per mouse ) . Data information : Data are presented as mean ± s . e . m . log - rank ( Mantel - Cox ) test ( G ) were used to measure significance . * 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

(G) Survival of Nrf1F/F and Nrf1MyeKO mice (n=10 per group) at various times after intraperitoneal (i.p.) infection with HSV-1 (2×107 PFU per mouse). Data information: Data are presented as mean ± s.e.m. log-rank (Mantel-Cox) test (G) were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( A ) mtROS levels measured by MitoSOX staining and flow cytometry in wild - type and NRF1 - KO mouse PMs infected with VSV ( left ) or HSV - 1 ( right ) for the indicated time points . MFI , mean fluorescence intensity ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(A) mtROS levels measured by MitoSOX staining and flow cytometry in wild-type and NRF1-KO mouse PMs infected with VSV (left) or HSV-1 (right) for the indicated time points. MFI, mean fluorescence intensity (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( B ) DNA was isolated from digitonin extracts of wild - type and NRF1 - KO mouse PMs infected with VSV ( left ) or HSV - 1 ( right ) for the indicated time points . Cytosolic translocation of mtDNA was quantified via qPCR using the indicated primer sets . 18S rRNA served as a control ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(B) DNA was isolated from digitonin extracts of wild-type and NRF1-KO mouse PMs infected with VSV (left) or HSV-1 (right) for the indicated time points. Cytosolic translocation of mtDNA was quantified via qPCR using the indicated primer sets. 18S rRNA served as a control (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( C and D ) Wild - type and NRF1 - KO mouse PMs infected with VSV ( 8 h ) or HSV - 1 ( 4 h ) were imaged by confocal microscope after staining with anti - DNA ( DNA ) and anti - Prohibitin ( Mito ) antibodies . Scale bars , 5 µm ( C ) . ( D ) The number of cytosolic mtDNA puncta per cell was quantitated by ImageJ ( n = 30 cells per group ) . Data information : Data are from three independent experiments or representative data ( C ) and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Wild-type and NRF1-KO mouse PMs infected with VSV (8 h) or HSV-1 (4 h) were imaged by confocal microscope after staining with anti-DNA (DNA) and anti-Prohibitin (Mito) antibodies. Scale bars, 5 µm (C). (D) The number of cytosolic mtDNA puncta per cell was quantitated by ImageJ (n=30 cells per group). Data information: Data are from three independent experiments or representative data (C) and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( E ) ELISA quantification of IFN - β ( left ) and IL6 ( right ) in wild - type and NRF1 - KO mouse PMs pretreated with or without EtBr ( 450 ng / ml , 72 h ) , followed by infection with VSV or HSV - 1 for 8 h ( n = 3 biological replicates per group ) . ( F ) ELISA quantification of IFN - β ( left ) and IL6 ( right ) in wild - type and NRF1 - KO mouse PMs pretreated with or without MitoQ ( 100 nM , 2 h ) , followed by infection with VSV or HSV - 1 for 8 h ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O 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) ELISA quantification of IFN-β (left) and IL6 (right) in wild-type and NRF1-KO mouse PMs pretreated with or without EtBr (450 ng/ml, 72 h), followed by infection with VSV or HSV-1 for 8 h (n=3 biological replicates per group). (F) ELISA quantification of IFN-β (left) and IL6 (right) in wild-type and NRF1-KO mouse PMs pretreated with or without MitoQ (100 nM, 2 h), followed by infection with VSV or HSV-1 for 8 h (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( H ) qPCR analysis of Ifnb1 and Il6 mRNA in the lungs of Nrf1F / F and Nrf1MyeKO mice pretreated with MitoQ as in ( G ) and 1 h later infected i . p . with VSV ( 5 × 108 PFU / mouse ) for 24 h ( n = 6 per group ) . ( I ) qPCR analysis of Ifnb1 and Il6 mRNA in the brains of Nrf1F / F and Nrf1MyeKO mice pretreated with MitoQ as in ( G ) and 1 h later infected i . p . with HSV - 1 ( 1 × 107 PFU / mouse ) for 24 h ( n = 6 per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(H) qPCR analysis of Ifnb1 and Il6 mRNA in the lungs of Nrf1F/F and Nrf1MyeKO mice pretreated with MitoQ as in (G) and 1 h later infected i.p. with VSV (5×108 PFU/mouse) for 24 h (n=6 per group). (I) qPCR analysis of Ifnb1 and Il6 mRNA in the brains of Nrf1F/F and Nrf1MyeKO mice pretreated with MitoQ as in (G) and 1 h later infected i.p. with HSV-1 (1×107 PFU/mouse) for 24 h (n=6 per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( A ) Immunoblot analysis and Phos - tag SDS - PAGE analysis of mouse PMs infected with VSV or HSV - 1 for the indicated time points .

O O O O O 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) Immunoblot analysis and Phos-tag SDS-PAGE analysis of mouse PMs infected with VSV or HSV-1 for the indicated time points.


( C ) Interaction between TBK1 and NRF1 in HEK293T cells was detected by co - immunoprecipitation of differentially tagged proteins .

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

(C) Interaction between TBK1 and NRF1 in HEK293T cells was detected by co-immunoprecipitation of differentially tagged proteins.


( G ) Endogenous phospho - NRF1 S318 signal was detected in wild - type and NRF1 S318A mouse PMs after the infection with different viruses for 8 h . The red arrows indicate phosphorylated signals for NRF1 .

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

(G) Endogenous phospho-NRF1 S318 signal was detected in wild-type and NRF1 S318A mouse PMs after the infection with different viruses for 8 h. The red arrows indicate phosphorylated signals for NRF1.


( J ) ELISA quantification of IFN - β and IL6 secretion in wild - type and NRF1 S318A mouse PMs treated as in ( I ) ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 . Two - tailed , unpaired Student ' s t test were used to measure significance .

(J) ELISA quantification of IFN-β and IL6 secretion in wild-type and NRF1 S318A mouse PMs treated as in (I) (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01. Two-tailed, unpaired Student's t test were used to measure significance.


( L ) Survival of wild - type and NRF1 S318A mice ( n = 10 per group ) at various times after intraperitoneal infection with HSV - 1 ( 2 × 107 PFU per mouse ) . Data information : log - rank ( Mantel - Cox ) test ( L ) were used to measure significance .

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

(L) Survival of wild-type and NRF1 S318A mice (n=10 per group) at various times after intraperitoneal infection with HSV-1 (2×107 PFU per mouse). Data information: log-rank (Mantel-Cox) test (L) were used to measure significance.


Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C . elegans hypodermal mitochondria . Scale bar 10 µm .

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

Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C. elegans hypodermal mitochondria. Scale bar 10 µm.


Immunoblot comparing the cytosolic supernatant and the mitochondria - enriched pellet of isolation fractions . GFP - tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria - target sequence , the proton pump , and GFP . mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 ( mitochondria ) and actin ( cytosol ) . All blots are from the same lanes on one membrane .

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

Immunoblot comparing the cytosolic supernatant and the mitochondria-enriched pellet of isolation fractions. GFP-tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria-target sequence, the proton pump, and GFP. mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 (mitochondria) and actin (cytosol). All blots are from the same lanes on one membrane.


Representative TMRE fluorescence traces in arbitrary units ( a . u . ) before and after Δψm dissipation with FCCP . Dashed lines indicate where FCCP was added . + / - ATR traces were performed in the absence of succinate . mtON activation was continuous throughout the traces . Light green trace is from mitochondria with ATR , gray traces are without . Quantification of change in fluorescence ( ΔF ) normalized to the maximum change in fluorescence given by succinate respiration ( ΔFmax ) for each mitochondrial preparation . Data are from the maximum light dose in Fig . EV 3A . Two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0469 , + ATR succinate vs . + ATR + light p = 0 . 9978 , n = 6 mitochondrial isolations . Data show mean ± SEM .

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

Representative TMRE fluorescence traces in arbitrary units (a.u.) before and after Δψm dissipation with FCCP. Dashed lines indicate where FCCP was added. +/- ATR traces were performed in the absence of succinate. mtON activation was continuous throughout the traces. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in fluorescence (ΔF) normalized to the maximum change in fluorescence given by succinate respiration (ΔFmax) for each mitochondrial preparation. Data are from the maximum light dose in Fig. EV 3A. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0469, +ATR succinate vs. +ATR +light p = 0.9978, n = 6 mitochondrial isolations. Data show mean ± SEM.


Representative BCECF - AM 490 / 440 nm ratio trace . Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation . Dashed lines are where light or FCCP treatment occurred . Light green trace is from mitochondria with ATR , gray traces are without . Quantification of change in BCECF - AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization . Two - way ANOVA with Sidak ' s test for multiple comparisons , - ATR succinate vs . - ATR , + light * p = 0 . 0212 , + ATR succinate vs . + ATR light p = 0 . 999 , + ATR succinate vs . + ATR , - light p = 0 . 0237 , + ATR , + light vs , + ATR , - light p = 0 . 0474 , n = 3 mitochondrial isolations . Some individual points overlap . Data show mean ± SEM .

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

Representative BCECF-AM 490/440 nm ratio trace. Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation. Dashed lines are where light or FCCP treatment occurred. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in BCECF-AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization. Two-way ANOVA with Sidak's test for multiple comparisons, -ATR succinate vs. -ATR, +light *p = 0.0212, +ATR succinate vs. +ATR light p = 0.999, +ATR succinate vs. +ATR,-light p = 0.0237, +ATR, +light vs, +ATR, -light p = 0.0474, n = 3 mitochondrial isolations. Some individual points overlap. Data show mean ± SEM.


ATP levels normalized to total ATP synthesis given by succinate respiration . Data from Fig . EV 3B . Succinate data shown for comparison after normalization . Two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0011 , + ATR succinate vs + ATR light p = 0 . 5680 , n = 3 , 7 , 3 , 7 , biological replicates for each bar from left to right . Some individual points overlap . Data show mean + SEM .

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

ATP levels normalized to total ATP synthesis given by succinate respiration. Data from Fig. EV 3B. Succinate data shown for comparison after normalization. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0011, +ATR succinate vs +ATR light p = 0.5680, n = 3,7,3,7, biological replicates for each bar from left to right. Some individual points overlap. Data show mean + SEM.


O2 required to consume 50 nmoles of ADP after mtON activation . Data are the maximum illumination from Fig . EV 4D using time - matched dark conditions , One - way ANOVA * p = 0 . 013 ( - ATR , - light vs . + ATR , + light p = 0 . 010 . - ATR , + light vs . + ATR , + light p = 0 . 013 . ) - ATR , - light n = 5 , rest n = 6 mitochondrial preparations . Data show mean + SEM .

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O2 required to consume 50 nmoles of ADP after mtON activation. Data are the maximum illumination from Fig. EV 4D using time-matched dark conditions, One-way ANOVA *p = 0.013 (-ATR, -light vs. +ATR, +light p = 0.010. -ATR, +light vs. +ATR, +light p = 0.013.) -ATR, -light n = 5, rest n = 6 mitochondrial preparations. Data show mean + SEM.


Day 1 adult animals expressing mtON were exposed to 50 µM rotenone ( ETC complex I inhibitor ) for 5 hours and survival was scored . Illumination was continuous throughout toxin exposure ( see methods ) . ATR alone was protective ( - ATR , - light vs . + ATR , - light p = 0 . 016 . ) The effect of mtON activation was greater than the ATR alone effect , one - way ANOVA with Tukey ' s post hoc test * p = 0 . 01 . ( - ATR , - light vs + ATR , + light p = 0 . 0002 . - ATR , + light vs . + ATR , + light p = 0 . 0009 . n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 50 µM antimycin A ( complex III inhibitor ) and survival was scored 18 hours later . One - way ANOVA with Tukey ' s post hoc test * p = 0 . 02 ( - ATR , - light vs + ATR , + light p = 0 . 03 , - ATR , + light vs . + ATR , + light p = 0 . 01 . n = 5 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 0 . 25 M azide ( complex IV inhibitor ) for 1 hour and scored for survival 1 hour after recovering , one - way ANOVA with Tukey ' s post hoc test * p = 0 . 0002 . ( - ATR , - light vs + ATR , + light p < 0 . 0001 , - ATR , + light vs . + ATR , + light p < 0 . 0001 . n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 31 µM oligomycin A ( ATP synthase inhibitor ) and survival was scored 18 hours later . No significant differences were found by one - way ANOVA ( n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM .

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

Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.


mtON - expressing animals in a complex I mutant background ( gas - 1 ) were scored for locomotion speed in the presence of food by counting body bends per minute . mtON activation rescued the decreased locomotion of the gas - 1 mutant background , one - way ANOVA with Tukey ' s post hoc test , - ATR , + light vs . + ATR , + light * p = 0 . 0145 , + ATR , + light vs . wildtype ( WT ) * p = 0 . 0008 ( - ATR , - light vs + ATR , + light p = 0 . 0272 , + ATR , - light vs . + ATR , + light p = 0 . 0332 , all conditions vs WT , p < 0 . 001 , n = 30 , 30 , 30 , 41 , 41 animals each bar from left to right ) . Data show mean ± standard deviation .

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mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.


Top : Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status . Top bands ( ~ 62 kDa ) are phosphorylated AMPK signal , and bottom bands ( ~ 43 kDa ) are actin signal . Image is from the same membrane cut to separately probe for phosphorylated AMPK ( pAMPK ) and actin . Bottom : Densitometry analysis showing decreased pAMPK to actin ratio , as there is no known antibody directed against total AMPK in C . elegans . Phosphorylation increases in the absence of food , but low phosphorylation is preserved when mtON is activated , two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0203 , n = 3 , 3 , 3 , 4 , 4 biological replicates each bar from left to right . Data show mean + standard deviation .

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Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation.


Locomotion was assessed by counting body bends per minute . Wild type animals were compared to aak - 2 ( ok524 ) mutant animals . 2 - sample , 2 - tailed unpaired t - test * p < 0 . 0001 , wild type n = 35 , aak - 2 n = 39 animals across at least 3 days . Data show mean ± standard deviation .

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Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation.


Locomotion in response to mtON activation . Illumination was continuous throughout body bends measurement ( see methods ) . For AAK - 2 activation , animals were exposed to 1 mM AICAR for 4 hours before body bends measurement . One - way ANOVA with Tukey ' s post hoc test , - ATR , + light vs + ATR , + light * p < 0 . 0001 , + ATR , + light vs . AICAR * p < 0 . 0001 , + ATR , + light vs AICAR + light * p < 0 . 0001 . n = 36 , 39 , 37 , 46 , 36 , 36 animals each bar from left to right . Data show mean ± standard deviation .

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Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.


Survival after HR in day 1 adult animals is shown with hypoxic preconditioning ( PC , represented by diagonal stripes ) 2 - sample 2 - tailed unpaired t - test , ctrl vs . PC * p = 0 . 0035 , n = 4 , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Survival after HR in day 1 adult animals is shown with hypoxic preconditioning (PC, represented by diagonal stripes) 2-sample 2-tailed unpaired t-test, ctrl vs. PC *p = 0.0035, n = 4, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Protection ( % ) is percent survival minus percent survival of control condition . PC data calculated from panel B . FCCP final concentrations 0 . 001 , 0 . 01 , 0 . 1 , 1 nM . One - way ANOVA with Tukey ' s post hoc test , PC vs . 0 . 001 nM FCCP , * p = 0 . 0012 , 0 . 001 nM FCCP vs 0 . 01 FCCP , * p = 0 . 0142 , n = 4 , 3 , 3 , 3 , 3 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Illumination was continuous throughout PC alone ; control illumination was for the same duration under normoxic conditions ( see methods & panel A ) . Two - way ANOVA comparing + ATR vs - ATR in each group . * p = 0 . 016 , n = 11 , 11 , 4 , 4 , 6 , 6 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Illumination was continuous throughout PC alone; control illumination was for the same duration under normoxic conditions (see methods & panel A). Two-way ANOVA comparing +ATR vs -ATR in each group. *p = 0.016, n = 11,11,4,4,6,6 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


light during hypoxia . 2 sample 2 - tailed unpaired t - test , * p = 0 . 0276 , n = 7 independent experiments , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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light during hypoxia. 2 sample 2-tailed unpaired t-test, *p = 0.0276, n = 7 independent experiments, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Immunoblot of expression levels of MYC , TSC1 , TSC2 , and β - actin loading control in high MYC Burkitt ' s lymphoma ( BL ) cells compared to low MYC Hodgkin lymphoma ( HL ) cells .

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Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells.


Immunoblots showing expression levels of MYC , TSC1 , TSC2 or β - actin loading control in P493 - 6 cells treated with tetracycline for 72 hours ( + Tet ) or in untreated cells ( - Tet ) .

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Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet).


Relative TSC1 and TSC2 mRNA expression levels determined by qRT - PCR for high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells treated for 24 h with tetracycline ( mean ± st . dev . , n = 3 technical replicates ) * p < 0 . 05 ; * * p < 0 . 01 ; statistical relevance was determined by unpaired t - test ( two - tailed ) .

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Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed).


qRT - PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h - 72h ( + Tet ) . Immunoblots for 24h and 48h ( + Tet ) show S6K and phosphorylation ( P - ) of S6K as downstream mTORC1 target , and β - actin loading control . For 72 h ( + Tet ) the immunoblots show expression of MYC and phosphorylation ( P - ) of downstream mTORC1 targets S6K and S6 , and α - tubulin as loading control .

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qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control.


Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1 - specific sh - RNAs compared to scrambled control sh - RNA in P493 - 6 cells . Other blots show the expression levels of TSC2 , S6K / P - S6K , S6 / P - S6 and α - tubulin for loading control .

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Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific sh-RNAs compared to scrambled control sh-RNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6 and α-tubulin for loading control.


Immunoblots of indicated proteins in P493 - 6 cells with high MYC ( - Tet , 72 hours ) or low MYC ( + Tet , 72 hours ) levels either treated with rapamycin or solvent .

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Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.


Elevated TSC1 and MYC expression in BL ( cohort 1 ) . Example of immune staining of TSC1 , MYC , the B - cell marker CD20 , and DAPI nuclear DNA - staining in germinal centers of control lymph nodes ( upper rows ) and samples from BL patients ( lower rows ) . Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples ( see materials and methods ) ( the horizontal line shows the median , whiskers show maximum and minimum data points and the box represents the first to the third quartiles , n = 56 fields for tumor samples and n = 21 fields for control germinal centers ; scale bar = 100 μm ) .

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Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods) (the horizontal line shows the median, whiskers show maximum and minimum data points and the box represents the first to the third quartiles, n=56 fields for tumor samples and n=21 fields for control germinal centers; scale bar = 100 μm).


Quantification of immunoblot analysis of TSC expression in BL ( cohort 2 ) ( n = 13 ) compared to healthy tonsils ( n = 3 ) or reactive lymph nodes ( n = 3 ) ( mean ± st . dev . ) shown in Fig EV2B .

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Quantification of immunoblot analysis of TSC expression in BL (cohort 2) (n=13) compared to healthy tonsils (n=3) or reactive lymph nodes (n=3) (mean ± st. dev.) shown in Fig EV2B.


Left graph shows the multiplication rate of P493 - 6 ( - Tet ) cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA determined by viable cell counting 3 days after seeding of equal number of viable cells ( determined by Trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) . Right graph shows percentage of apoptotic P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or a TSC1 - specific sh - RNA determined by FACS analysis of AnnexinV / 7AAD stained cells ( mean ± st . dev . , n = 3 biological replicates ) .

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Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).


Rapamycin treatment recovers survival of TSC1 knockdown in P493 - 6 cells . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 14 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 30pM rapamycin where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).


TSC1 knockdown is synthetic lethal with MYC deregulation . U2OS - MYC - ER cells expressing either scrambled control sh - RNA or TSC1 - specific sh - RNA were treated with hydroxytamoxifen to induce MYC and rapamycin ( 100 nM ) where indicated . Percentage of apoptotic cells was determined with Annexin / 7AAD staining 4 days after MYC induction ( mean ± st . dev . , n = 3 biological replicates ) .

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TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).


Survival rate of different BL cell lines upon TSC1 knockdown . Graphs show numbers of viable cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA 3 days after seeding of equal number of viable cells ( determined by trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) .

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Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates).


Immunoblots of control - or TSC1 - shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity ( 10nM ) or titrate the activity to control levels ( 30 pM ) , and survival rate of these cells over 7 days ( mean ± st . dev . , n = 3 biological replicates ) ; ( Bl2 cells were selected for stable knockdown with Puromycin , DG75 cells without selection ) .

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Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).


Ramos cells expressing either a TSC1 - specific or a control shRNA were inoculated into NOD / SCID mice and tumor volume was measured regularly . The immunoblot shows the level of knockdown of TSC1 ( sh - TSC1b ) compared to control ( sh - contr ) , and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation . Tumor growth curves show mean ± SEM ( n = 8 / group ) . Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation . The top panels show tumors derived from Ramos cells stably expressing control sh - RNA ( sh - contr ) ; the lower panels show tumors derived from Ramos cells stably expressing TSC1 - specific sh - RNA ( sh - TSC1b ) .

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Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).


TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or TSC - specific sh - RNA , treated with 20 nM rapamycin for 12 h , or tetracycline to repress MYC for 48 h where

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

TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicated


TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated ( B ) ( mean ± st . dev . , n = 6 biological replicates ) .

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TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).


Ratio of mitochondrial to genomic DNA determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) .

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Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).


expression of cytochrome C ( CYCS ) or ATP5G1 mRNAs ( D ) determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) .

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expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).


TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner . FACS analysis of DCF - DA stained P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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

TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).


TSC1 knockdown results in increased phosphorylation of SAPK / JNK . Immunoblot of SAPK / JNK Thr183 / Tyr185 - phosphorylation in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA . α - tubulin expression serves as loading control .

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

TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.


Antioxidant treatment rescues cells from death caused by TSC1 knockdown . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 3 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 10 μM of the antioxidant BHA where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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

Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).


Accumulation of EU - labeled ( Click - It , Invitrogen ) TSC1 - mRNA over 3 h in high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells ( mean ± st . dev . , n = 3 technical replicates ) .

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Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).


Drawing of the TSC1 promoter and luciferase - reporter constructs with the predicted E - box ( CACGTG , pos . - 317 / - 322 ) indicated . Relative luciferase units ( RLU ) of co - transfection experiments using the indicated TSC1 - promoter - reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells ( mean ± st . dev . , n = 3 ) . Immunoblot shows MYC overexpression and α - tubulin as loading control .

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Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control.


TSC1 mRNA turnover determined by pulse and chase ( Click - It , Invitrogen ) over 8 h in P493 - 6 cells , either treated with tetracycline to repress MYC or left untreated ( mean ± st . dev . , n = 3 technical replicates ) .

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TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).


miR - 15a expression determined by qRT - PCR in P493 - 6 cells treated with tetracycline for 3 days ( + Tet ) to suppress MYC expression or in untreated ( - Tet ) cells ( mean ± st . dev . , n = 3 technical replicates ) .

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miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).


Immunoblots showing expression levels of TSC1 , S6K / P - S6K and α - tubulin in P493 - 6 and HEK293T cells overexpressing miR - 15a or a control miRNA . At the right miR - 15a overexpression was determined by qRT - PCR in P493 - 6 and HEK293T cells ( mean ± st . dev . , n = 3 technical replicates ) .

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Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates).


Immunoblot showing TSC1 expression , S6K / P - S6K and α ‑ tubulin in MCF - 7 cells transfected with LNA modified anti - sense miR - 15a oligos or control LNAs . At the right , successful miR - 15a knockdown determined by qRT - PCR in MCF - 7 cells is shown ( mean ± st . dev . , n = 3 technical replicates ) .

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

Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates).


Relative luciferase activities ( RLU ) derived from reporter constructs containing TSC1 - 3 ´ UTR wild type sequences or with mutated miR - 15a seed sequence binding site on co - transfection with miR - 15a in MCF - 7 cells ( mean ± st . dev . , n = 3 biological replicates ) . The drawing at the left shows the TSC1 - 3 ' UTR with the position of the miR - 15a / 16 - 1 seed sequence and used mutation indicated .

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Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated.


Rate of oxygen consumption in P493 - 6 ( - Tet ) cells ectopically expressing miR - 15a or control - miRNA vector , under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated ( mean ± st . dev . , n = 8 biological replicates ) .

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Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).


a , Polychromasia ( Wright - Giemsa stain , indicated by arrows ) and increased reticulocytes ( brilliant cresyl blue stain ) revealed by staining blood smears from 6 - week - old wild - type ( WT ) or Nix - / - mice ( scale bar , 20 µm ) .

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a, Polychromasia (Wright-Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or Nix-/- mice (scale bar, 20 µm).


b , c , Ter119 ( b ) or Mitotracker deep red ( c ) versus CD71 staining of RBCs from 3 - or 6 - week - old mice . Cell counts were also calculated ( mean ± s . e . m . ; n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.


d , Analyses of RBCs from 3 - or 6 - week - oldmice by

d, Analyses of RBCs from 3- or 6-week-oldmice by immunocytochemistry


Analyses of RBCs from 3 - or 6 - week - oldmice by transmission electron

Analyses of RBCs from 3- or 6-week-oldmice by transmission electron microscopy


a , Quantification of NHS - biotin - labelled RBCs or transferred CMFDA - labelled RBCs ( n = 3 ) .

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a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3).


b , Western blot analyses of caspases in RBCs after in vitro culture . Asterisks denote processed caspases . FL , full length .

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b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length.


c , The relative luminescence units ( RLU ) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix - / - RBCs after 12 - h culture in the presence of qVD - oph or solvent control ( DMSO ) ( n = 3 ) .

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c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3).


d , Mitotracker versus annexin V staining of RBCs after in vitro culture .

d, Mitotracker versus annexin V staining of RBCs after in vitro culture.


e , Mean fluorescent intensity ( MFI ) of ROS staining in RBCs after in vitro culture , and caspase activities in Nix - / - RBCs after 24 h culture with solvent control , ROS scavengers or qVD - oph ( n = 3 ) . BHT , butylated hydroxytoluene ; CAT , catalase ; TEM , tempol .

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e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol.


f , Haematoxylin and eosin ( HE ) staining of spleen sections of 9 - week - old wild - type and Nix - / - mice . Arrows denote iron deposits within macrophagecytoplasm . Iron deposits were also stained with Prussian blue , followed by counterstain with nuclear - fast red . Scale bar , 20 µm .

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f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm.


g , Real - time RT - PCR of erythropoietin ( Epo ) ( n = 12 ) . For all relevant panels , statistical significance of the data ( mean ± s . e . m . ) is : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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g, Real-time RT-PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P < 0.05; **P < 0.01; ***P < 0.001.


a , Ter119 + CD71 + reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 ( top row ) , 2 ( middle row ) or 4 ( bottom row ) days , followed by Mitotracker deep red and Ter119 staining .

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a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining.


b , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue . Scale bar , 20 µm .

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b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm.


c , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy . Scale bar , 5 µm . The Pearson coefficiency for LC3 and COX IV co - localization at day 0 ( mean ± s . e . m . ) is : wild type , 0 . 81 ± 0 . 001 ; Nix - / - , 0 . 44 ± 0 . 036 ( n = 35 , P = 0 . 0006 ) .

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c, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006).


d , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy . Scale bar , 0 . 5 µm .

d, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy. Scale bar, 0.5 µm.


a , Ter119 + CD71 + reticulocytes were sorted from the peripheral blood of wild - type and Nix - / - mice at day 6 after PHZ treatment . The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT - 737 , followed by staining with Mitotracker deep red and phycoerythrin - conjugated anti - Ter119 .

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a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119.


b , c , Reticulocytes were cultured with FCCP ( b ) or ABT - 737 ( c ) for 24 h and analysed by transmission electron microscopy . Scale bar , 0 . 5 µm .

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b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.


( B ) BW25113 ( ycbB , relA ' ) and its derivatives obtained by individual deletion of endopeptidase genes . BW25113 ( ycbB , relA ' ) is an abbreviated name for BW25113 ΔrelA pKT2 ( ycbB ) pKT8 ( relA ' ) .

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(B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , and its derivatives harboring plasmids encoding MepM , MepM H393A , and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg / ml ( + ceftriaxone ) or in the absence of the drug ( - ceftriaxone ) in BHI agar plates supplemented with 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively . The genes encoding MepM , MepM H393A , and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter , which is inducible by 2 , 4 - diacetylphloroglucinol ( DAPG ) . Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer .

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Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.