G . Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations . Positive control shows that prazosin binds to membrane preparations of yeast expressing 1 - AR . PRZ : prazosin . CRZ : cirazoline .

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G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline.


H . Immunoblotting for phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) and total ERK1 / 2 following prazosin treatment for 30 min . Prazosin induces ERK1 / 2 phosphorylation in GICs .

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H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.


I . SRE - luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 ( 10 μM ) , an inhibitor of the ERK - activating kinase MEK . U0126 prevented prazosin - induced SRE - luciferase activation in GICs .

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I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.


J . Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 ( 10 μM ) , an inhibitor of the ERK - activating kinase MEK . U0126 did not counteract prazosin - induced GIC death . Blockade of prazosin - induced ERK1 / 2 phosphorylation by U0126 was confirmed by immunoblotting ( insert ) . PRZ : prazosin .

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J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.


A . PKCδ expression analysis in two patient - derived GICs and NSCs by immunoblotting . GICs express higher levels of PKCδ than NSCs . kDa : kilodaltons .

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A. PKCδ expression analysis in two patient-derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons.


B . PKCδ by immunostaining ( green ) . Nuclei were stained with DAPI ( blue ) . Enhanced PKCδ punctate nuclear expression in prazosin - treated ( PRZ , 5µM ) GICs , as compared to vehicle - treated GICs . PRZ : prazosin . Scale bar : 10 µm .

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B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm.


C . Total PKCδ ( PKCδ 78kDa ) and cleaved PKCδ ( PKCδ 41kDa ) expression analysis in GICs by immunoblotting . Prazosin ( PRZ ) induces PKCδ cleavage into a 41kDa active fragment . 2 µM rottlerin ( Rott ) , a PKCδ inhibitor , inhibits prazosin - induced PKCδ cleavage . V : vehicle . kDa : kilodaltons .

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C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.


D . Pro - caspase - 3 ( Pro - CASP3 ) and activated caspase - 3 ( CASP3 ) expression analysis in GICs by immunoblotting . 2 µM rottlerin ( Rott ) inhibits prazosin ( PRZ ) - induced caspase - 3 activation . V : vehicle . kDa : kilodaltons .

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D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons.


E . Inhibition of PKCδ by rottlerin prevents , in a concentration - dependent manner , prazosin - induced GIC death . Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Inhibition of PKCδ by the specific anti - PKCδ RACK peptide δV1 . 1 ( 10 μM ) counteracts prazosin - induced GIC death . Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1 . 1 . * P < 0 . 005 for Prazosin 1 and 5 µM and P < 0 . 001 for Prazosin 10 µM by two - tailed unpaired Student ' s t - test , n = 4 .

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F. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4.


G . Decreased PKCδ expression using shRNA counteracts prazosin - induced GIC death . Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h . * P < 0 . 005 by two - tailed unpaired Student ' s t - test , n = 4 .

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G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4.


H . Prazosin inhibits AKT phosphorylation in GICs . LY294002 ( LY , 30 µM ) , an inhibitor of the PI3K / AKT pathway , was used as positive control . Terazosin , which does not affect GIC viability , does not alter AKT phosphorylation ( see the corresponding cell viability counting in Fig EV4C ) . Phosphorylated AKT ( P - AKT ) and total AKT ( AKT ) expression analysis in GICs by immunoblotting . V : vehicle . kDa : kilodaltons .

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H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.


I . Prazosin ( 10 µM ) does not inhibit AKT phosphorylation in NSCs . Analysis by immunoblotting . V : vehicle . kDa : kilodaltons .

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I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


J . β - catenin expression , a downstream target of AKT , is decreased in prazosin - treated GICs , as opposed to NSCs . Analysis by immunoblotting . V : vehicle . kDa : kilodaltons .

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J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


K . L . Inhibition of PKCδ using rottlerin ( Rott , 2 µM ) or δV1 . 1 ( 10 µM ) counteracts prazosin inhibition of AKT phosphorylation . Analysis by immunoblotting . V : vehicle . kDa : kilodaltons .

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K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


B . Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA . All mice were treated with prazosin ( PRZ ) for 45 days . C . Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth . Fold change in total flux represents the ratio : total flux after treatment / total flux prior treatment . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - test .

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B. Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA. All mice were treated with prazosin (PRZ) for 45 days.C. Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth. Fold change in total flux represents the ratio: total flux after treatment/total flux prior treatment. *P=0.0007, n=8, two-sided Mann-Whitney U-test.


Heatmap of RNA - Seq analysis of differentially expressed lncRNAs ( ≥ 2 - fold change and FDR < 0 . 05 ) in mesenchymal ( MES ) 1123 and proneural ( PN ) 528 GSCs . GSCs were pre - cultured for 16 h in DMEM / F12 medium with EGF ( 2 ng / ml ) and bFGF ( 2 ng / ml ) and then followed by co - culturing with or without 20 - μg / ml TGF - β1 for 3 h .

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Heatmap of RNA-Seq analysis of differentially expressed lncRNAs (≥ 2-fold change and FDR< 0.05) in mesenchymal (MES) 1123 and proneural (PN) 528 GSCs. GSCs were pre-cultured for 16 h in DMEM/F12 medium with EGF (2 ng/ml) and bFGF (2 ng/ml) and then followed by co-culturing with or without 20-μg/ml TGF-β1 for 3 h.


Representative images of LINC00115 expression in clinical GBM tumors and adjacent tissue using RNAscope analysis . Images are representative of two independent experiments . Scale bars : 50 μm .

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Representative images of LINC00115 expression in clinical GBM tumors and adjacent tissue using RNAscope analysis. Images are representative of two independent experiments. Scale bars: 50 μm.


Kaplan - Meier analysis of patients with high versus low LINC00115 - expressing GBM tumors from B . Median survival ( in months ) : low , 13 . 9 ; high , 8 . 1 . Black bars , censored data . Data information : , * , p < 0 . 05 , by log - rank test .

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Kaplan-Meier analysis of patients with high versus low LINC00115-expressing GBM tumors from B. Median survival (in months): low, 13.9; high, 8.1. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.


Expression levels of LINC00115 are higher in LGG ( low grade gliomas ) versus normal brain tissues and GBM versus LGG . Expression data of LINC00115 were downloaded from the REMBRANDT dataset . Data are presented as mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 , by two - tailed t test .

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Expression levels of LINC00115 are higher in LGG (low grade gliomas) versus normal brain tissues and GBM versus LGG. Expression data of LINC00115 were downloaded from the REMBRANDT dataset. Data are presented as mean ± SEM. **, p < 0.01, ***, p < 0.001, by two-tailed t test.


E to G . Kaplan - Meier analysis of patients with high versus low LINC00115 - expressing Grade III and GBM tumors from the REMBRANDT ( E ) or the TCGA ( G ) 26 [ ] , and GBM from GSE83300 50 [ ] ( F ) datasets . Median survival ( in months ) in E : low , 20 . 2 ; high , 15 . 9 ; in F : low , 19 . 4 ; high , 13 . 0 ; in G : low , 33 . 2 ; high , 25 . 4 . Black bars , censored data . Data information : , * , p < 0 . 05 , by log - rank test .

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E to G. Kaplan-Meier analysis of patients with high versus low LINC00115-expressing Grade III and GBM tumors from the REMBRANDT (E) or the TCGA (G) 26[], and GBM from GSE83300 50[] (F) datasets. Median survival (in months) in E: low, 20.2; high, 15.9; in F: low, 19.4; high, 13.0; in G: low, 33.2; high, 25.4. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.


qRT - PCR analysis of LINC00115 knockdown using two different shRNAs , shL115 - 1 and shL115 - 2 in 1123 and 528 GSCs . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , by One - way

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qRT-PCR analysis of LINC00115 knockdown using two different shRNAs, shL115-1 and shL115-2 in 1123 and 528 GSCs. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA


Effects of LINC00115 knockdown on GSC proliferation ( B ) data are representative of three independent experiments . Error bars , ± SD . * , by two - tailed t - test in B .

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Effects of LINC00115 knockdown on GSC proliferation (B) data are representative of three independent experiments. Error bars, ± SD. *, by two-tailed t-test in B.


C . Effects of LINC00115 knockdown on neuro - like sphere formation ( C ) . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA

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C. Effects of LINC00115 knockdown on neuro-like sphere formation (C). Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA in


D . Representative bioluminescence images of brains with indicated 528 GSC tumor xenografts expressing shC , shL115 - 1 , or shL115 - 2 at 45 days after implantation . Images represent results of 5 mice per group of 2 independent experiments . E . Quantification of the bioluminescence activity in D .

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D. Representative bioluminescence images of brains with indicated 528 GSC tumor xenografts expressing shC, shL115-1, or shL115-2 at 45 days after implantation. Images represent results of 5 mice per group of 2 independent experiments. E. Quantification of the bioluminescence activity in D.


F . Representative hematoxylin and eosin ( H & E ) - stained images of mouse brain sections with GSC1123 xenografts expressing shC , shL115 - 1 , or shL115 - 2 . Images represent results of five mice per group of two independent experiments . Scale bars : 1 mm . G . Quantification of tumor volume in F .

F. Representative hematoxylin and eosin (H&E)-stained images of mouse brain sections with GSC1123 xenografts expressing shC, shL115-1, or shL115-2. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. G. Quantification of tumor volume in F.


H . Immunofluorescence ( IF ) analysis of Nestin expression in 1123 GSC control and LINC00115 knockdown xenograft tumors from F . Images are representative of two independent experiments . Scale bars : 50 μm .

H. Immunofluorescence (IF) analysis of Nestin expression in 1123 GSC control and LINC00115 knockdown xenograft tumors from F. Images are representative of two independent experiments. Scale bars: 50 μm.


I . Kaplan - Meier analysis of animal implantation with 1123 / shC , 1123 / shL115 - 1 or 1123 / shL115 - 2 GSCs . n = 10 . Data information : * * , p < 0 . 01 , by log - rank test .

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I. Kaplan-Meier analysis of animal implantation with 1123/shC, 1123/shL115-1 or 1123/shL115-2 GSCs. n = 10. Data information: **, p < 0.01, by log-rank test.


B and C . MS2 - RIP followed by qRT - PCR analysis of endogenous microRNAs association with LINC00115 . 12XMS2 empty vector , LINC00115 - 12XMS2 , or LINC00115 - mut - 12XMS2 with Flag - tagged MS2 were co - transfected into 1123 GSCs . Data information data are representative of three independent experiments . Error bars , ± SD . , * * * , p < 0 . 001 , by two - tailed t - test .

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B and C. MS2-RIP followed by qRT-PCR analysis of endogenous microRNAs association with LINC00115. 12XMS2 empty vector, LINC00115-12XMS2, or LINC00115-mut- 12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs. Data information data are representative of three independent experiments. Error bars, ± SD. , ***, p < 0.001, by two-tailed t-test.


E . RNA pull - down and qRT - PCR assays of LINC00115 binding with miR - 200s . GSC1123 cell lysates were incubated with biotin - labeled LINC00115 . Data information data are representative of three independent experiments . Error bars , ± SD . p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA .

E. RNA pull-down and qRT-PCR assays of LINC00115 binding with miR-200s. GSC1123 cell lysates were incubated with biotin-labeled LINC00115. Data information data are representative of three independent experiments. Error bars, ± SD. p < 0.05, **, p < 0.01, by One-way ANOVA.


F . Luciferase activity analysis of 1123 GSCs co - transfected with miR - 200s and luciferase reporters containing LINC00115 WT , mutant , or empty vector ( EV ) . Data information : data are representative of three independent experiments . Error bars , ± SD . p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA .

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F. Luciferase activity analysis of 1123 GSCs co-transfected with miR-200s and luciferase reporters containing LINC00115 WT, mutant, or empty vector (EV). Data information: data are representative of three independent experiments. Error bars, ± SD. p < 0.05, **, p < 0.01, by One-way ANOVA.


A and B . Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA - Seq dataset ( A ) and REMBRANDT GBM array dataset ( B ) . * * * , p < 0 . 001 , by two - tailed t - test .

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A and B. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (A) and REMBRANDT GBM array dataset (B). ***, p < 0.001, by two-tailed t-test.


C . LINC00115 knockdown reduced ZEB1 mRNA expression in 1123 and 528 GSCs . Data information : , data are representative of three independent experiments . Error bars , ± SD . * * , p < 0 . 01 , by One - way

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C. LINC00115 knockdown reduced ZEB1 mRNA expression in 1123 and 528 GSCs. Data information: , data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by One-way ANOVA


D . Effects of LINC00115 knockdown on ZEB1 , vimentin , and E - cadherin protein expression . Data information : data are representative of three independent experiments . Error bars , ± SD . * * ,

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D. Effects of LINC00115 knockdown on ZEB1, vimentin, and E-cadherin protein expression. Data information: data are representative of three independent experiments. Error bars, ± SD. **,


E . Effects of re - expression of shRNA - resistant LINC00115 WT ( L115 - WT ) , miR - 200 binding mutant ( L115 - mut ) with or without miR - 200b on ZEB1 signaling activation . Data information data are representative of three independent experiments . Error bars , ± SD .

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E. Effects of re-expression of shRNA-resistant LINC00115 WT (L115-WT), miR-200 binding mutant (L115-mut) with or without miR-200b on ZEB1 signaling activation. Data information data are representative of three independent experiments. Error bars, ± SD.


F . Neuro - like sphere formation assay of effects of re - expression of shRNA - resistant LINC00115 WT and mutant with or without miR - 200b . Data information : data are representative of three independent experiments . Error bars , ± SD . * * , p < 0 . 01 , by two - tailed t - test in F .

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F. Neuro-like sphere formation assay of effects of re-expression of shRNA-resistant LINC00115 WT and mutant with or without miR-200b. Data information: data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by two-tailed t-test in F.


G . Kaplan - Meier analysis of animal implantation with 1123 GSCs with indicated modifications . n = 10 . Statistical analysis was performed by log - rank test .

G. Kaplan-Meier analysis of animal implantation with 1123 GSCs with indicated modifications. n = 10. Statistical analysis was performed by log-rank test.


H and I . GSEA of EMT signature genes using ranked gene expression changes in TGF - β1 - treated 1123 / shLINC00115 GSCs versus the control ( H ) or in 1123 GSCs with ectopic expression of LINC00115 or an empty vector ( EV ) ( I ) . NES , normalized enrichment score .

H and I. GSEA of EMT signature genes using ranked gene expression changes in TGF-β1-treated 1123/shLINC00115 GSCs versus the control (H) or in 1123 GSCs with ectopic expression of LINC00115 or an empty vector (EV) (I). NES, normalized enrichment score.


LINC00115 knockdown reduced ZNF596 mRNA expression . Data information : In data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by One - way

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LINC00115 knockdown reduced ZNF596 mRNA expression. Data information: In data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, by One-way ANOVA


LINC00115 knockdown reduced ZNF596 protein ( D ) expression . Data information : data are representative of three independent experiments . Error bars , ± SD .

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LINC00115 knockdown reduced ZNF596 protein (D) expression. Data information: data are representative of three independent experiments. Error bars, ± SD.


E . Effects of exogenous ZNF596 expression on LINC00115 knockdown - inhibited ZNF596 protein expression . Data information : data are representative of three independent experiments . Error bars , ± SD .

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E. Effects of exogenous ZNF596 expression on LINC00115 knockdown-inhibited ZNF596 protein expression. Data information: data are representative of three independent experiments. Error bars, ± SD.


F . ZNF596 overexpression had no effect on LINC00115 expression . Data information data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by One - way

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F. ZNF596 overexpression had no effect on LINC00115 expression. Data information data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, by One-way ANOVA


G . Expression of exogenous ZNF596 rescued LINC00115 knockdown - inhibited cell proliferation . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by two - tailed t - test in G .

G. Expression of exogenous ZNF596 rescued LINC00115 knockdown-inhibited cell proliferation. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05 , by two-tailed t-test in G.


H . Representative H & E staining images of mouse brain sections with 1123 / shC and 1123 / shL115 GSC tumor xenografts with empty vector ( EV ) or ectopic ZNF596 expression . Images represent results of five mice per group of two independent experiments . Scale bars : 1 mm . I . Quantification of tumor size in H .

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H. Representative H&E staining images of mouse brain sections with 1123/shC and 1123/shL115 GSC tumor xenografts with empty vector (EV) or ectopic ZNF596 expression. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. I. Quantification of tumor size in H.


J and K . Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA - Seq dataset ( J ) and REMBRANDT GBM array dataset ( K ) . * * , p < 0 . 01 , by two - tailed t - test .

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J and K. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (J) and REMBRANDT GBM array dataset (K). **, p < 0.01, by two-tailed t-test.


Expression of shRNA - resistant LINC00115 WT but not mutant or empty vector ( EV ) rescued LINC00115 shRNA - inhibited ZNF596 mRNA ( A ) expression in 1123 GSCs .

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Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 mRNA (A) expression in 1123 GSCs.


Expression of shRNA - resistant LINC00115 WT but not mutant or empty vector ( EV ) rescued LINC00115 shRNA - inhibited ZNF596 protein ( B ) expression in 1123 GSCs .

Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 protein (B) expression in 1123 GSCs.


D . MS2 - RIP followed by qRT - PCR for endogenous microRNAs association with ZNF596 . 12XMS2 empty vector , ZNF596 - 5 ' - UTR - WT - 12XMS2 , or ZNF596 - 5 ' - UTR - mut - 12XMS2 with Flag - tagged MS2 were co - transfected into 1123 GSCs .

D. MS2-RIP followed by qRT-PCR for endogenous microRNAs association with ZNF596. 12XMS2 empty vector, ZNF596-5'-UTR-WT-12XMS2, or ZNF596-5'-UTR-mut -12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs.


F . Luciferase activity in 1123 GSCs co - transfected with miR - 200s and luciferase reporters containing ZNF596 5 ' - UTR WT , mutant , or empty vector ( EV ) .

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F. Luciferase activity in 1123 GSCs co-transfected with miR-200s and luciferase reporters containing ZNF596 5'-UTR WT, mutant, or empty vector (EV).


GSEA of EZH2 - targeted signature genes using ranked gene expression changes in 1123 GSCs with ZNF596 sgRNA or a control sgRNA . NES , normalized enrichment score .

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GSEA of EZH2-targeted signature genes using ranked gene expression changes in 1123 GSCs with ZNF596 sgRNA or a control sgRNA. NES, normalized enrichment score.


qRT - PCR analysis of effect of ZNF596 knockout on EZH2 expression . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , by One - way

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qRT-PCR analysis of effect of ZNF596 knockout on EZH2 expression. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by One-way ANOVA


ZNF596 knockout impaired EZH2 expression , its downstream H3K27me3 , and STAT3 phosphorylation in 1123 and 528 GSCs . Data information , data are representative of three independent experiments . Error bars , ± SD .

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ZNF596 knockout impaired EZH2 expression, its downstream H3K27me3, and STAT3 phosphorylation in 1123 and 528 GSCs. Data information , data are representative of three independent experiments. Error bars, ± SD.


LINC00115 regulated ZNF596 / EZH2 / STAT3 signaling . 1123 and 528 GSCs were treated with or without EZH2 inhibitor GSK343 ( 3 μM ) for 6 Data information : data are representative of three independent experiments . Error bars , ± SD .

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LINC00115 regulated ZNF596/EZH2/STAT3 signaling. 1123 and 528 GSCs were treated with or without EZH2 inhibitor GSK343 (3 μM) for 6 Data information: data are representative of three independent experiments. Error bars, ± SD.


LINC00115 and ZNF596 regulate EZH2 promoter activity . Data information : data are representative of three independent experiments . Error bars , ± SD . , by two - tailed t -

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LINC00115 and ZNF596 regulate EZH2 promoter activity. Data information: data are representative of three independent experiments. Error bars, ± SD. , by two-tailed t-test


ChIP - qPCR analysis of ZNF596 binding with the EZH2 promoter . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , by One - way

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ChIP-qPCR analysis of ZNF596 binding with the EZH2 promoter. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by One-way ANOVA


EZH2 inhibitor GSK343 impaired ZNF596 overexpression - reversed and LINC00115 knockdown - inhibited neuro - like sphere formation . 1123 and 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox ( 2 μg / ml ) and / or EZH2 inhibitor GSK343 ( 1 μM ) for 7 days . Data information : data are representative of three independent experiments . Error bars , ± SD . by two - tailed t -

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EZH2 inhibitor GSK343 impaired ZNF596 overexpression-reversed and LINC00115 knockdown-inhibited neuro-like sphere formation. 1123 and 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox (2 μg/ml) and/or EZH2 inhibitor GSK343 (1 μM) for 7 days. Data information: data are representative of three independent experiments. Error bars, ± SD. by two-tailed t-test


Growth kinetics of subcutaneous xenograft tumor with indicated genetic modifications after treatment with or without Dox and / or GSK343 . Tumor - bearing mice ( five mice per group ) were fed with 10 % sucrose water with or without 2 mg / ml Dox and combined with treatment with or without GSK343 ( 5 mg / kg body weight from Monday to Friday via intraperitoneal injection ) for three weeks beginning five days post - xenograft transplantation . Data were from three independent experiments with five mice per group . Data information : * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , by two - tailed t -

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Growth kinetics of subcutaneous xenograft tumor with indicated genetic modifications after treatment with or without Dox and/or GSK343. Tumor-bearing mice (five mice per group) were fed with 10% sucrose water with or without 2 mg/ml Dox and combined with treatment with or without GSK343 (5 mg/kg body weight from Monday to Friday via intraperitoneal injection) for three weeks beginning five days post-xenograft transplantation. Data were from three independent experiments with five mice per group. Data information: *, p < 0.05, **, p < 0.01, ***, p < 0.001 , by two-tailed t-test


Kaplan - Meier analysis of animal implantation with GSC1123 with indicated modifications . n = 10 . Statistical analysis was performed by log - rank test .

Kaplan-Meier analysis of animal implantation with GSC1123 with indicated modifications. n = 10. Statistical analysis was performed by log-rank test.


Representative images of RNAscope for LINC00115 and IHC for ZEB1 and ZNF596 expression in two GBM specimens , Sample # 1 ( positive ) and Sample # 2 ( negative ) . Scale bar , 50 μm . Images are representative of two independent experiments .

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Representative images of RNAscope for LINC00115 and IHC for ZEB1 and ZNF596 expression in two GBM specimens, Sample #1 (positive) and Sample #2 (negative). Scale bar, 50 μm. Images are representative of two independent experiments.


Correlation of detected expression between ZEB1 or ZNF596 , and LINC00115 from a separate cohort of a total of 75 paraffin - embedded clinical GBM samples . Statistical analysis was performed by Chi - square test .

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Correlation of detected expression between ZEB1 or ZNF596, and LINC00115 from a separate cohort of a total of 75 paraffin-embedded clinical GBM samples. Statistical analysis was performed by Chi-square test.


Kaplan - Meier survival analysis of patients with GBM tumors expressing high or low levels of LINC00115 with high or low levels of ZEB1 or ZNF596 expression . Median survival ( in months ) : LINC00115hi / ZEB1hi , 5 . 4 ; LINC00115lo / ZEB1lo , 27 . 9 ; LINC00115hi / ZNF596hi , 4 . 2 ; LINC00115lo / ZNF596lo , 23 . 8 . Statistical analysis was performed by log - rank test .

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Kaplan-Meier survival analysis of patients with GBM tumors expressing high or low levels of LINC00115 with high or low levels of ZEB1 or ZNF596 expression. Median survival (in months): LINC00115hi/ZEB1hi, 5.4; LINC00115lo/ZEB1lo, 27.9; LINC00115hi/ZNF596hi, 4.2; LINC00115lo/ZNF596lo, 23.8. Statistical analysis was performed by log-rank test.


Representative H & E and immunohistochemical stainings of CK5 / 6 and p63 in pulmonary and head - and - neck squamous - cell carcinomas . Scale bar : 500 µm .

Representative H&E and immunohistochemical stainings of CK5/6 and p63 in pulmonary and head-and-neck squamous-cell carcinomas. Scale bar: 500 µm.


Heatmap showing somatic mutations in defined pathways and HPV status for all HNSCC and SQCLC cases analysed . ( Blue , no mutation / no HPV detected ; red , mutation / HPV detected ; white , no data available )

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Heatmap showing somatic mutations in defined pathways and HPV status for all HNSCC and SQCLC cases analysed. (Blue, no mutation/ no HPV detected; red, mutation/HPV detected; white, no data available)


Comparison of mutation rates in HNSCC ( n = 279 ) and SQCLC ( n = 178 ) cases from TCGA . Genes exhibiting a mutation rate > 0 . 25 in both cancer types are labelled .

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Comparison of mutation rates in HNSCC (n=279) and SQCLC (n=178) cases from TCGA. Genes exhibiting a mutation rate >0.25 in both cancer types are labelled.


Representative histogram showing the SILAC ratio distribution between the Super - SILAC standard and SQCLC ( left ) and HNSCC ( right ) samples .

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Representative histogram showing the SILAC ratio distribution between the Super-SILAC standard and SQCLC (left) and HNSCC (right) samples.


Boxplot showing the numbers of quantified proteins , derived from 44 SQCLC and 30 HNSCC tissue samples . The central line in the boxes represents the median number of proteins over all samples , upper and lower borders of boxes correspond to 25 % and 75 % quantiles . Whiskers indicate minimum and maximum .

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Boxplot showing the numbers of quantified proteins, derived from 44 SQCLC and 30 HNSCC tissue samples. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum.


Distribution of MS - based protein signal intensities in all 74 tissue samples of the training cohort .

Distribution of MS-based protein signal intensities in all 74 tissue samples of the training cohort.


Principal - component analysis of SQCLC ( n = 44 ; blue ) and HNSCC ( n = 30 ; red ) protein expression . Shown are the first two principal components , accounting for 8 . 51 % and 8 . 03 % of the total variance in the data , respectively .

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Principal-component analysis of SQCLC (n=44; blue) and HNSCC (n=30; red) protein expression. Shown are the first two principal components, accounting for 8.51% and 8.03% of the total variance in the data, respectively.


Volcano plot adjusted p values for differential protein expression to averaged normalised SILAC ratios of two replicates . Red ( higher expression in HNSCC ) and blue ( higher expression in SQCLC ) dots indicate significantly regulated proteins ( p < 0 . 05 ) .

Volcano plot adjusted p values for differential protein expression to averaged normalised SILAC ratios of two replicates. Red (higher expression in HNSCC) and blue (higher expression in SQCLC) dots indicate significantly regulated proteins (p < 0.05).


Two - class comparison of genetic dependencies from a publically available genome - scale CRISPR - Cas9 screen of HNSCC ( 12 cell line ) versus SQCLC ( 10 cell lines ) identified a subset of differentially expressed proteins that were also differential dependencies in these tumor types . The x - axis represents the effect size of the mean difference of dependency scores in HNSCC compared to SQCLC cell lines . Positive effect size indicates a greater mean dependency in HNSCC ; negative effect size indicates a greater mean dependency in SQCLC . The y - axis represents the statistical significance of differential enrichment calculated as - log10 ( p - value ) from a two - sided t - test . The p - values used for this plot are uncorrected for multiple hypothesis testing . Highlighted in green are genetic dependencies that were also identified as differentially expressed proteins in our study .

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Two-class comparison of genetic dependencies from a publically available genome-scale CRISPR-Cas9 screen of HNSCC (12 cell line) versus SQCLC (10 cell lines) identified a subset of differentially expressed proteins that were also differential dependencies in these tumor types. The x-axis represents the effect size of the mean difference of dependency scores in HNSCC compared to SQCLC cell lines. Positive effect size indicates a greater mean dependency in HNSCC; negative effect size indicates a greater mean dependency in SQCLC. The y-axis represents the statistical significance of differential enrichment calculated as -log10(p-value) from a two-sided t-test. The p-values used for this plot are uncorrected for multiple hypothesis testing. Highlighted in green are genetic dependencies that were also identified as differentially expressed proteins in our study.


Immunohistochemical analysis of the expression of HMGCS - 1 ( p = 0 . 0014 ) , FTL ( p = 0 . 0001 ) ( C ) , LGALS7 ( p = 0 . 0001 ) and FAM83H ( p = 0 . 0001 ) ( D ) in an independent cohort of 212 SQCLC and 343 HNSCC cases . Scale bar indicates 100µm . Shown are mean values and standard deviation . Statistical significance was assessed using Wilcoxon - Mann - Whitney test .

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Immunohistochemical analysis of the expression of HMGCS-1 (p=0.0014),FTL (p=0.0001) (C),LGALS7 (p=0.0001) and FAM83H (p=0.0001)(D) in an independent cohort of 212 SQCLC and 343 HNSCC cases. Scale bar indicates 100µm. Shown are mean values and standard deviation. Statistical significance was assessed using Wilcoxon-Mann-Whitney test.


Boxplot showing the number of quantified proteins derived from 19 SQCLC , 19 HNSCC and 51 squamous - cell lung tumours of unknown origin derived from HNSCC patients . The central line in the boxes represents the median number of proteins over all samples , upper and lower borders of boxes correspond to 25 % and 75 % quantiles . Whiskers indicate minimum and maximum .

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Boxplot showing the number of quantified proteins derived from 19 SQCLC, 19 HNSCC and 51 squamous-cell lung tumours of unknown origin derived from HNSCC patients. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum.


Representative H & E and immunohistochemical stainings of CK5 / 6 and p63 in squamous - cell lung tumours of unknown origin derived from HNSCC patients . Scale bar : 500 µm .

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Representative H&E and immunohistochemical stainings of CK5/6 and p63 in squamous-cell lung tumours of unknown origin derived from HNSCC patients. Scale bar: 500 µm.


Kaplan - Meier analysis of overall survival ( OS ) in which all patients with lung tumours were grouped according to the clinical classification ( left , median survival SQCLC : 55 months ; metHNSCC : 31 months ) or proteomic classification ( right , median survival SQCLC : 55 months ; metHNSCC : 8 months ) of the tumour as metHNSCC or SQCLC . The p value is from a log - rank test . Kaplan - Meier analysis of overall survival ( OS ) in which only the patients that were independent from the training cohort were grouped according to the clinical classification ( left , median survival SQCLC : 84 months ; metHNSCC : 35 months ) or proteomic classification ( right , median survival SQCLC : 84 months ; metHNSCC : 5 months ) of the tumour as metHNSCC or SQCLC . The p value is from a log - rank test .

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Kaplan-Meier analysis of overall survival (OS) in which all patients with lung tumours were grouped according to the clinical classification (left, median survival SQCLC: 55 months; metHNSCC: 31 months) or proteomic classification (right, median survival SQCLC: 55 months; metHNSCC: 8 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test. Kaplan-Meier analysis of overall survival (OS) in which only the patients that were independent from the training cohort were grouped according to the clinical classification (left, median survival SQCLC: 84 months; metHNSCC: 35 months) or proteomic classification (right, median survival SQCLC: 84 months; metHNSCC: 5 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test.


( A - B ) Hemoglobin ( A ) and hematocrit ( B ) levels of healthy subjects and cachectic cancer patients presenting a body weight loss superior to 10 % of initial body weight ( 19 healthy subjects , 17 cachectic patients ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(A-B) Hemoglobin (A) and hematocrit (B) levels of healthy subjects and cachectic cancer patients presenting a body weight loss superior to 10% of initial body weight (19 healthy subjects, 17 cachectic patients). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C ) TFR1 mRNA levels in muscle biopsies from cancer - patients of late stage cachexia ( 19 healthy subjects , 17 cachectic patients with at least 10 % total body weight loss ) Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(C) TFR1 mRNA levels in muscle biopsies from cancer-patients of late stage cachexia (19 healthy subjects, 17 cachectic patients with at least 10% total body weight loss) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) TFR1 mRNA levels in the gastrocnemius of mice subjected to iron deprivation by feeding with an iron deficient diet ( IDD ) combined to a phlebotomy ( PHL ) ( n = 5 - 6 ) Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(F) TFR1 mRNA levels in the gastrocnemius of mice subjected to iron deprivation by feeding with an iron deficient diet (IDD) combined to a phlebotomy (PHL) (n=5-6) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( G ) Cross - sectional area of skeletal muscle fiber transfected with shSCR ( scramble ) and shTFR1 ( n = 3 - 4 ) and representative picture of a shTFR1 transfected fiber . Scale bar = 50µm . ( H ) Cross - sectional area of skeletal muscle fibers transfected with TFR - pHuji ( n = 4 ) and representative picture of a TFR - pHuji transfected fiber . Scale bar = 50µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(G) Cross-sectional area of skeletal muscle fiber transfected with shSCR (scramble) and shTFR1 (n=3-4) and representative picture of a shTFR1 transfected fiber. Scale bar=50µm. (H) Cross-sectional area of skeletal muscle fibers transfected with TFR-pHuji (n=4) and representative picture of a TFR-pHuji transfected fiber. Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( L ) Reprensentative pictures and diameter measurements of human myoblast - derived myotubes after 48h treatment with DFO 100µM ( n = 3 ) . Scale bar = 50µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(L) Reprensentative pictures and diameter measurements of human myoblast-derived myotubes after 48h treatment with DFO 100µM (n=3). Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C ) TFR1 protein expression and densitometric quantification in mouse gastrocnemius ( n = 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(C) TFR1 protein expression and densitometric quantification in mouse gastrocnemius (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( E ) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification ( n = 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ 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

(E) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( H ) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification ( n = 4 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

(H) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification (n=4). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( I - J ) ICP - MS quantification of total ( I ) and protein - bound ( J ) iron in mouse quadriceps ( n = 4 - 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ 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 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

(I-J) ICP-MS quantification of total (I) and protein-bound (J) iron in mouse quadriceps (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( A ) ICP - MS quantification of mitochondrial iron in mouse quadriceps ( n = 6 - 8 ) . ( B ) Gastrocnemius heme content quantified by fluorescent heme assay ( n = 4 - 6 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(A) ICP-MS quantification of mitochondrial iron in mouse quadriceps (n=6-8). (B) Gastrocnemius heme content quantified by fluorescent heme assay (n=4-6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C - D ) mRNA levels of mitochondrial iron importer MFRN2 ( C ) and the rate limiting enzyme of heme synthesis ALAS2 ( D ) normalized to 18s in mouse gastrocnemius ( n = 4 - 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ 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

(C-D) mRNA levels of mitochondrial iron importer MFRN2 (C) and the rate limiting enzyme of heme synthesis ALAS2 (D) normalized to 18s in mouse gastrocnemius (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) Succinate Dehydrogenase activity staining in transversal sections of mouse gastrocnemius and corresponding intensity quantification ( n = 3 - 4 ) . Scale bar = 50µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ 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

(F) Succinate Dehydrogenase activity staining in transversal sections of mouse gastrocnemius and corresponding intensity quantification (n=3-4). Scale bar= 50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( B ) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate . ( n = 3 )

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 O O O

(B) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate. (n=3)


( G - H ) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain ( G ) or human myoblast - derived myotubes ( H ) after 48h treatment with C26 CM and ferric citrate ( n = 3 per condition ) . Scale bars = 50 and 100 µm , respectively . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± SEM . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 CM - treated group .

O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain (G) or human myoblast-derived myotubes (H)after 48h treatment with C26 CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.


( B ) Kaplan - Meier depicting the survival of C26 - tumor bearing mice after I . V . injection of saline or iron every 5 days post C26 - injection ( 3 - month - old Balb / C , n = 8 - 11 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by chi - square test for the survival curves Data are mean ± SEM . ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier depicting the survival of C26-tumor bearing mice after I.V. injection of saline or iron every 5 days post C26-injection (3-month-old Balb/C, n=8-11). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by chi-square test for the survival curves Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


Immunofluorescent staining of myosin heavy chain fast ( green ) and slow ( red ) of transversal sections of gastrocnemius ( midbelly ) with corresponding frequency distribution Scale bar = 100µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± SEM . ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

Immunofluorescent staining of myosin heavy chain fast (green) and slow (red) of transversal sections of gastrocnemius (midbelly) with corresponding frequency distribution Scale bar=100µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( I - K ) mRNA levels of Murf 1 ( I ) , Atrogin 1 ( J ) , and REDD1 ( K ) normalized to GAPDH in gastrocnemius ( n = 5 - 14 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction , Data are mean ± SEM . ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(I-K) mRNA levels of Murf 1 (I), Atrogin 1 (J), and REDD1 (K) normalized to GAPDH in gastrocnemius (n=5-14). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction, Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( B ) Aconitase activity of quadriceps lysates normalized to protein content ( n = 3 - 7 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aconitase activity of quadriceps lysates normalized to protein content (n=3-7). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( E ) Representative Western blot and densitometric quantification of phospho - AMPK and total AMPK ( stripped and re - blotted ) in the gastrocnemius of C26 - tumor bearing mice after iron carboxymaltose supplementation ( n = 6 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Western blot and densitometric quantification of phospho-AMPK and total AMPK (stripped and re-blotted) in the gastrocnemius of C26-tumor bearing mice after iron carboxymaltose supplementation (n=6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( F - H ) mRNA levels of ACOT 1 ( F ) , MCD ( G ) , and CPT1B ( H ) normalized to GAPDH in gastrocnemius ( n = 5 - 10 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

(F-H) mRNA levels of ACOT 1 (F), MCD (G), and CPT1B (H) normalized to GAPDH in gastrocnemius (n=5-10). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( A - B ) Grip force of dominant or non - dominant arm in iron - deficient cancer patients , expressed in percentage of baseline force ( A ) and absolute values normalized to height ( B ) before / after single dose of iron carboxymaltose ( 15mg / kg , 7 subjects ) . Data information : For all data , n represents In graph ( A ) , the boxes represent the range of values with the median value being the central band and whiskers the 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 O O O B-CONTROLLED_VAR I-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

(A-B) Grip force of dominant or non-dominant arm in iron-deficient cancer patients, expressed in percentage of baseline force (A) and absolute values normalized to height (B) before/after single dose of iron carboxymaltose (15mg/kg, 7 subjects). Data information: For all data, n represents In graph (A), the boxes represent the range of values with the median value being the central band and whiskers the SEM.


( A ) Bacterially purified 6xHis - rRv2626c and analyzed by Coomassie blue staining ( left ) or immuno blotting ( IB ) with αHis ( right ) .

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(A) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right).


( B ) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay .

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(B) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay.


( C ) Fluorescence confocal images showing in BMDMs treated with Rv2626c ( 2 . 5 μg / ml ) for 30 min , fixed , immunostained with antibodies for His ( Alexa 488 ) and DAPI . Scale bar , 10 μm .

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(C) Fluorescence confocal images showing in BMDMs treated with Rv2626c (2.5 μg/ml) for 30 min, fixed, immunostained with antibodies for His (Alexa 488) and DAPI. Scale bar, 10 μm.


( D and E ) BMDMs from WT mice were treated with LPS ( 100 ng / ml ) or rRv2626c for indicated times ( D ) . BMDMs from WT , TLR2 - / - , TLR4 - / - , MyD88 - / - , TRIF - / - , IRAK1 - / - , TRAF6 - / - , TBK1 - / - mice were treated with 2 . 5 μg / ml rRv2626c or rVector for 18 h ( E ) . Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) BMDMs from WT mice were treated with LPS (100 ng/ml) or rRv2626c for indicated times (D). BMDMs from WT, TLR2-/-, TLR4-/-, MyD88-/-, TRIF-/-, IRAK1-/-, TRAF6-/-, TBK1-/- mice were treated with 2.5 μg/ml rRv2626c or rVector for 18 h (E). Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


( F ) BMDMs were treated with 2 . 5 μg / ml rRv2626c for 1 h , harvested and then subjected to IB for αIκBα and His . αActin was used as a loading control .

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(F) BMDMs were treated with 2.5 μg/ml rRv2626c for 1 h, harvested and then subjected to IB for αIκBα and His. αActin was used as a loading control.


BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml LPS ( A for indicated times . ( A Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A for indicated times. (A Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml Pam2CSK4 ( B ) B ) Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


( C ) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT , MAPK ( ERK , p38 , JNK ) , IκBα , total forms of IκBα . αActin was used as a loading control ( left ) . The densitometric values were calculated from the ratios of protein levels relative to actin ( right ) .

(C) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT, MAPK (ERK, p38, JNK), IκBα, total forms of IκBα. αActin was used as a loading control (left). The densitometric values were calculated from the ratios of protein levels relative to actin (right).


( A ) Identification of TLR2 , IRAK1 , RIP1 , TRAF6 , MAPK11 and TOLLIP as endogenous binding partners of rRv2626c . THP - 1 cell lysates were incubated with His tagged - rRv2626c or rVector and then IP with His - agarose beads . Binding partners were confirmed by silver staining and mass spectrometric analysis . The red - colored letters indicate the peptides identified from mass spectrometry analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Identification of TLR2, IRAK1, RIP1, TRAF6, MAPK11 and TOLLIP as endogenous binding partners of rRv2626c. THP-1 cell lysates were incubated with His tagged-rRv2626c or rVector and then IP with His-agarose beads. Binding partners were confirmed by silver staining and mass spectrometric analysis. The red-colored letters indicate the peptides identified from mass spectrometry analysis.


( B ) 293T cells were co - transfected with Flag - TRAF6 and Myc - Rv2626c and IP with αFlag or αMyc . WCLs were used for IB with αFlag , αMyc or αActin .

O O O O O O O O O O O O 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) 293T cells were co-transfected with Flag-TRAF6 and Myc-Rv2626c and IP with αFlag or αMyc. WCLs were used for IB with αFlag, αMyc or αActin.


( C ) THP - 1 and BMDMs were stimulated with rRv2626c ( 2 . 5 μg / ml ) for the indicated times , followed by IP with αHis ( up ) or αTRAF6 ( down ) and IB with αTRAF6 , αHis , and αActin .

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(C) THP-1 and BMDMs were stimulated with rRv2626c (2.5 μg/ml) for the indicated times, followed by IP with αHis (up) or αTRAF6 (down) and IB with αTRAF6, αHis, and αActin.


( D ) Immunostaining of BMDMs treated with 2 . 5 μg / ml rRv2626c for 30 min . and then immunolabeled with antibody to His - rRv2626c ( Alexa Fluor 488 ) or TRAF6 ( Alexa Fluor 568 ) . DAPI ( blue ) stained nuclei .

(D) Immunostaining of BMDMs treated with 2.5 μg/ml rRv2626c for 30 min. and then immunolabeled with antibody to His-rRv2626c (Alexa Fluor 488) or TRAF6 (Alexa Fluor 568). DAPI (blue) stained nuclei.


( A ) Schematic diagram of the structures of Rv2626c ( up ) . 293T cells were co - transfected with Flag - TRAF6 together with GST - vector or GST - Rv2626c and its mutant constructs , subjected to GST pulldown , followed by IB with αFlag . WCLs were used for IB with αGST , αFlag or αActin ( down ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic diagram of the structures of Rv2626c (up). 293T cells were co-transfected with Flag-TRAF6 together with GST-vector or GST-Rv2626c and its mutant constructs, subjected to GST pulldown, followed by IB with αFlag. WCLs were used for IB with αGST, αFlag or αActin (down).


( B ) At 12 hr post - transfection with mammalian Myc - Rv2626c constructs together with Flag - TRAF6 and 293T cells treated with several Tat - CBS2 peptide for 12 h ( 10 µM ) for 6h , subjected to IP with αMyc , followed by IB with αFlag . WCLs were used for IB with αMyc , αFlag or αActin .

(B) At 12 hr post-transfection with mammalian Myc-Rv2626c constructs together with Flag-TRAF6 and 293T cells treated with several Tat-CBS2 peptide for 12 h (10 µM) for 6h, subjected to IP with αMyc, followed by IB with αFlag. WCLs were used for IB with αMyc, αFlag or αActin.


( C ) Schematic diagram of the structures of TRAF6 ( up ) . 293T cells were co - transfected with Myc - Rv2626c together with GST - vector or GST - TRAF6 and its mutant constructs , subjected to GST pulldown , followed by IB with αMyc . WCLs were used for IB with αGST , αMyc or αActin ( down ) .

(C) Schematic diagram of the structures of TRAF6 (up). 293T cells were co-transfected with Myc-Rv2626c together with GST-vector or GST-TRAF6 and its mutant constructs, subjected to GST pulldown, followed by IB with αMyc. WCLs were used for IB with αGST, αMyc or αActin (down).


( D ) 293T cells were co - transfected with Flag - TRAF6 , Myc - Rv2626c as indicated doses and HA - ubiquitin ( left ) , HA - K48 - linked ubiquitin ( middle ) , HA - K63 - linked ubiquitin ( right ) and then IP with αFlag , followed by IB with αHA . WCLs were used for IB with αFlag , αMyc , or αActin .

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(D) 293T cells were co-transfected with Flag-TRAF6, Myc-Rv2626c as indicated doses and HA-ubiquitin (left), HA-K48-linked ubiquitin (middle), HA-K63-linked ubiquitin (right) and then IP with αFlag, followed by IB with αHA. WCLs were used for IB with αFlag , αMyc, or αActin.


( E ) BMDMs were pretreated with 2 . 5 μg / ml rRv2626c for 1 h , and stimulated with 100 ng / ml LPS for 30 min , followed by IP with αTRAF6 , IB with ubiquitin , K48 - linked ubiquitin , or K63 - linked ubiquitin . WCLs were used for IB with αHis , αTRAF6 , or αActin .

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(E) BMDMs were pretreated with 2.5 μg/ml rRv2626c for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αTRAF6, IB with ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin. WCLs were used for IB with αHis, αTRAF6, or αActin.


( B ) Bacterially purified 6xHis - rRv2626c and analyzed by Coomassie blue staining ( left ) or immuno blotting ( IB ) with αHis ( right ) .

(B) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right).


( C ) BMDMs were incubated with rRv2626c - CA , rRv2626c - DN , or rVehicle for the indicated times and then cell viability measured with MTT assay .

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(C) BMDMs were incubated with rRv2626c-CA, rRv2626c-DN, or rVehicle for the indicated times and then cell viability measured with MTT assay.


( D ) BMDMs were pretreated with rRv2626c - WT , rRv2626c - CA , or rRv2626c - DN for 1 h , and stimulated with 100 ng / ml LPS for 18 h . Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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(D) BMDMs were pretreated with rRv2626c-WT, rRv2626c-CA, or rRv2626c-DN for 1 h, and stimulated with 100 ng/ml LPS for 18 h. Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


( E ) Myc - Rv2626c - expressing Raw264 . 7 or THP - 1 cells were pretreated with rRv2626c - WT ( 2 . 5 μg / ml ) , rRv2626c - CA ( 10 ng / ml ) , rRv2626c - DN ( 2 . 5 μg / ml ) , for 1 h , and stimulated with 100 ng / ml LPS for 30 min , followed by IP with αMyc or αTRAF6 , IB with αMyc and ubiquitin . WCLs were used for IB with αMyc , αTRAF6 , αHis or αActin .

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

(E) Myc-Rv2626c-expressing Raw264.7 or THP-1 cells were pretreated with rRv2626c-WT (2.5 μg/ml), rRv2626c-CA (10 ng/ml), rRv2626c-DN (2.5 μg/ml), for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αMyc or αTRAF6, IB with αMyc and ubiquitin. WCLs were used for IB with αMyc, αTRAF6, αHis or αActin.


( A - C ) Schematic of the CLP model treated with rRv2626c - CA or its mutants ( upper ) . The survival of mice was monitored for 10 days ; mortality was measured for n = 25 mice per group ( lower ) . Statistical differences compared with the rVector - treated mice are indicated ( log - rank test ) .

(A-C) Schematic of the CLP model treated with rRv2626c-CA or its mutants (upper). The survival of mice was monitored for 10 days; mortality was measured for n=25 mice per group (lower). Statistical differences compared with the rVector-treated mice are indicated (log-rank test).


( E ) representative hematoxylin and eosin ( H & E ) staining of the lung , liver and spleen ( left ) from 10 mice per group . Histopathology scores were obtained from H & E stained were determined at 20 h in CLP mice were treated with rRv2626c - CA or its mutants . Scale bar , 100 μm .

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(E) representative hematoxylin and eosin (H&E) staining of the lung, liver and spleen (left) from 10 mice per group. Histopathology scores were obtained from H&E stained were determined at 20 h in CLP mice were treated with rRv2626c-CA or its mutants. Scale bar, 100 μm.


( F ) Splenocytes were used for IP with αHis or αTRAF6 , followed by IB with αHis or αUb . WCLs were used for IB with αTRAF6 , αHis or αActin .

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(F) Splenocytes were used for IP with αHis or αTRAF6, followed by IB with αHis or αUb. WCLs were used for IB with αTRAF6, αHis or αActin.


( G ) In vivo imaging using IVIS spectrum - chromatography CT system . Intraperitoneal administration of rRv2626c - CA / Cy5 . 5 was carried out at intervals of 0 , 6 , 12 , and 18 h in a CLP - induced sepsis model . Pharmacokinetics and biodistribution were observed in spleen , liver , lung , and kidney tissues .

(G) In vivo imaging using IVIS spectrum-chromatography CT system. Intraperitoneal administration of rRv2626c-CA/Cy5.5 was carried out at intervals of 0, 6, 12, and 18 h in a CLP-induced sepsis model. Pharmacokinetics and biodistribution were observed in spleen, liver, lung, and kidney tissues.


( H ) The percentage of F4 / 80 + macrophages cells and rRv2626c - CA - His were found in the spleen , liver and lung using Fluorescence - activated cell sorting analysis in the background of CLP - induced sepsis .

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(H) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His were found in the spleen, liver and lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.


( A ) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c - CA or its mutants ( n = 10 mice per group ) .

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(A) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c-CA or its mutants (n=10 mice per group).


( B ) The percentage of F4 / 80 + macrophages cells and rRv2626c - CA - His or its mutants were found in the spleen or lung using Fluorescence - activated cell sorting analysis in the background of CLP - induced sepsis .

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(B) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His or its mutants were found in the spleen or lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.


( C ) CD86 , iNOS , CD163 and Arg1 mRNA levels were determined by real - time PCR .

(C) CD86, iNOS, CD163 and Arg1 mRNA levels were determined by real-time PCR.


( D ) Expression of SR - A , FcR , TLR2 , TLR4 , NRP1 or CXCR2 were measured by flow cytometry analysis .

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(D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis.


A . Significantly recurrently mutated genes identified by MutSigCV analysis based on whole - exome sequencing data of 25 STAD patients ' samples .

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A. Significantly recurrently mutated genes identified by MutSigCV analysis based on whole-exome sequencing data of 25 STAD patients' samples.


B . Distribution of PWWP2B mutations identified in 25 STAD patients . Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset ( Network , 2014 ) . Distribution of PWWP2B mutations identified in a large - scale colorectal adenocarcinoma genomic study .

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B. Distribution of PWWP2B mutations identified in 25 STAD patients. Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset (Network, 2014). Distribution of PWWP2B mutations identified in a large-scale colorectal adenocarcinoma genomic study.


E . Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations .

E. Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations.


B . The interaction between exogenous PWWP2B and UHRF1 . The indicated plasmids were transfected into 293T cells . After 24 hours , the transfected cell lysates were immunoprecipitated ( IP ) using an anti - Myc antibody and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

B. The interaction between exogenous PWWP2B and UHRF1. The indicated plasmids were transfected into 293T cells. After 24 hours, the transfected cell lysates were immunoprecipitated (IP) using an anti-Myc antibody and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


C . The interaction between endogenous PWWP2B and UHRF1 . Immunoprecipitation reactions were performed using rabbit IgG or anti - PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

C. The interaction between endogenous PWWP2B and UHRF1. Immunoprecipitation reactions were performed using rabbit IgG or anti-PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


E . Myc - UHRF1 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with the anti - Myc antibody and then immunoblotted with the indicated antibodies .

E. Myc-UHRF1 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with the anti-Myc antibody and then immunoblotted with the indicated antibodies.


G . SFB - PWWP2B WT and either Myc - UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti - FLAG and then immunoblotted with the indicated antibodies .

G. SFB-PWWP2B WT and either Myc-UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti-FLAG and then immunoblotted with the indicated antibodies.


I Cell lysates of 293T cells transfected with Myc - UHRF1 expression vector were incubated with 2 of purified GST , GST - PWWP2B - N , or GST - PWWP2B - C recombinant proteins for 1 h at 4 ° C . After pull - down with GST - beads , the bound complexes were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) and subjected to Western blot analysis using the indicated antibodies ( upper panel ) . The Coomassie blue stain shows the purified GST fusion proteins .

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I Cell lysates of 293T cells transfected with Myc-UHRF1 expression vector were incubated with 2 of purified GST, GST-PWWP2B-N, or GST-PWWP2B-C recombinant proteins for 1 h at 4 °C. After pull-down with GST-beads, the bound complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using the indicated antibodies (upper panel). The Coomassie blue stain shows the purified GST fusion proteins.


Cell lysates of 293T expressing Myc - UHRF1 WT or Myc - UHRF1 - D4 mutants were incubated with 2 ug of purified GST or GST - PWWP2B - N for 1 h at 4 ° C . After pull - down with GST - beads , the bound complex was analyzed by Western blots .

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Cell lysates of 293T expressing Myc-UHRF1 WT or Myc-UHRF1-D4 mutants were incubated with 2 ug of purified GST or GST-PWWP2B-N for 1 h at 4 °C. After pull-down with GST-beads, the bound complex was analyzed by Western blots.


E . Comparison of the translocation kinetics for GFP - UHRF1 and mCherry - PWWP2B to the sites of DNA damage . HeLa cells expressing both GFP - UHRF1 and mCherry - PWWP2B were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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E. Comparison of the translocation kinetics for GFP-UHRF1 and mCherry-PWWP2B to the sites of DNA damage. HeLa cells expressing both GFP-UHRF1 and mCherry-PWWP2B were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


F . HeLa cells were transfected with GFP - PWWP2B WT or deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( top panel ) . The cells with positive GFP expression on the laser stripes are presented in the bar graph ( bottom panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation .

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F. HeLa cells were transfected with GFP-PWWP2B WT or deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (top panel). The cells with positive GFP expression on the laser stripes are presented in the bar graph (bottom panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation.


H . GFP - PWWP2B - A2A3 deletion mutant translocates to the DNA damage sites . HeLa cells were transfected with GFP - tagged deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( left panel ) . The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation for the cells transfected with each expression plasmid .

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H. GFP-PWWP2B-A2A3 deletion mutant translocates to the DNA damage sites. HeLa cells were transfected with GFP-tagged deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (left panel). The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation for the cells transfected with each expression plasmid.


I - J . HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP - PWWP2B - A2A3 ( I ) and GFP - PWWP2B WT ( J ) expression plasmid . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points . The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel .

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I-J. HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP-PWWP2B-A2A3 (I) and GFP-PWWP2B WT (J) expression plasmid. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points. The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel.


The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel . K . HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP - UHRF1 expression plasmids . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel. K. HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP-UHRF1 expression plasmids. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


D - G . Quantification of radiation - induced RAD51 , BRCA1 , and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs . All data were obtained from over cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 , BRCA1 , and 53BP1 foci formation . HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( D and E ) , BRCA1 ( F ) , or 53BP1 ( G ) antibodies . The cells in S phase were identified by Cyclin A staining . ( E ) The siPWWP2B - resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA - mediated PWWP2B depletion . IR - induced RAD51 foci formation was examined by immunofluorescence .

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D-G. Quantification of radiation-induced RAD51, BRCA1, and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs. All data were obtained from over cells (open circles), and the mean values are shown as red bars (upper panel). Immunofluorescence analysis of irradiation-induced RAD51, BRCA1, and 53BP1 foci formation. HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (D and E), BRCA1 (F), or 53BP1 (G) antibodies. The cells in S phase were identified by Cyclin A staining. (E) The siPWWP2B-resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA-mediated PWWP2B depletion. IR-induced RAD51 foci formation was examined by immunofluorescence.


A . Immunoblot analysis of RAD51 , BRCA1 , or 53BP1 protein levels in PWWP2B - depleted HeLa cells .

A. Immunoblot analysis of RAD51, BRCA1, or 53BP1 protein levels in PWWP2B-depleted HeLa cells.


B . RPA phosphorylation at the indicated time points in response to IR exposure . HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4 / S8 phosphorylation and total RPA .

B. RPA phosphorylation at the indicated time points in response to IR exposure. HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4/S8 phosphorylation and total RPA.


C - D . Quantification of radiation - induced RPA2 or S4 / S8 phosphorylated RPA foci formation in PWWP2B - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RPA2 or S4 / S8 phosphorylated RPA2 foci formation . HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RPA2 ( C ) , or S4 / S8 phosphorylation - specific RPA2 ( D ) antibodies ( bottom panel ) .

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C-D. Quantification of radiation-induced RPA2 or S4/S8 phosphorylated RPA foci formation in PWWP2B-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RPA2 or S4/S8 phosphorylated RPA2 foci formation. HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RPA2 (C), or S4/S8 phosphorylation-specific RPA2 (D) antibodies (bottom panel).