Figure 9. ELAVL1 increases the... | Rockefeller University Press

Figure 9.

$ELAVL1 increases the translational efficiency of caspase-8. (A and B) HeLa cells were treated with TNF, IFNγ, or TNF/IFNγ for 24 h (A) or 36 h (B). Cell lysates were separated into nucleus and cytosolic components. Subcellular localization of ELAVL1 was analyzed by immunoblotting (n = 3). (C and D) HeLa ELAVL1 KO cells were infected with lentivirus express Flag-ELAVL1, Flag-ELAVL1 mutant, or the empty vector. Then cells were lysed and subjected to RNA IP using anti-FLAG antibody. Relative enrichment of CASP8 (C) and CYLD (D) mRNA levels were measured by qPCR (n = 3). The mRNA levels in HeLa cells were set as 1. One-way ANOVA was performed: ****P < 0.0001 (C); NS (D). (E) HeLa cells (parental, ELAVL1 KO) were fractionated into 10 gradient fractions by sucrose gradient centrifugation. Polysome fraction analysis of the distribution of CASP8 mRNAs was measured by qPCR (mean value from two independent experiments). (F and G) HCT-116 cells (parental, ELAVL1 KO) were fractionated into 10 gradient fractions by sucrose gradient centrifugation. Polysome fraction analysis of the distribution of CASP8 (F) or CYLD (G) mRNAs was measured by qPCR (mean value from two independent experiments). (H and I) HeLa (H, n = 4) or HCT-116 (I, n = 3) cells (parental, ELAVL1 KO) were treated with CHX for the indicated time. The expression of indicated proteins was analyzed by immunoblotting. (J) The biochemistry mechanism model of TNF/IFNγ-induced cell death. IFNγ activates IFNGR1/JAK1/STAT1/IRF1 pathway, which then induces the transcription of CYLD/CASP8/CASP7. When CASP8 mRNA translocates from nucleus to cytosol, ELAVL1 promotes the mRNA stability and translation of CASP8. Finally, increased expression of caspase-8 and CYLD can synergistically promote TNF-mediated caspase-8 activation and cell death. Data represent the mean value ± SD from n biological replicates, or a representative immunoblot from n independent experiments. Source data are available for this figure: SourceData F9.$

ELAVL1 increases the translational efficiency of caspase-8. (A and B) HeLa cells were treated with TNF, IFNγ, or TNF/IFNγ for 24 h (A) or 36 h (B). Cell lysates were separated into nucleus and cytosolic components. Subcellular localization of ELAVL1 was analyzed by immunoblotting (n = 3). (C and D) HeLa ELAVL1 KO cells were infected with lentivirus express Flag-ELAVL1, Flag-ELAVL1 mutant, or the empty vector. Then cells were lysed and subjected to RNA IP using anti-FLAG antibody. Relative enrichment of CASP8 (C) and CYLD (D) mRNA levels were measured by qPCR (n = 3). The mRNA levels in HeLa cells were set as 1. One-way ANOVA was performed: ****P < 0.0001 (C); NS (D). (E) HeLa cells (parental, ELAVL1 KO) were fractionated into 10 gradient fractions by sucrose gradient centrifugation. Polysome fraction analysis of the distribution of CASP8 mRNAs was measured by qPCR (mean value from two independent experiments). (F and G) HCT-116 cells (parental, ELAVL1 KO) were fractionated into 10 gradient fractions by sucrose gradient centrifugation. Polysome fraction analysis of the distribution of CASP8 (F) or CYLD (G) mRNAs was measured by qPCR (mean value from two independent experiments). (H and I) HeLa (H, n = 4) or HCT-116 (I, n = 3) cells (parental, ELAVL1 KO) were treated with CHX for the indicated time. The expression of indicated proteins was analyzed by immunoblotting. (J) The biochemistry mechanism model of TNF/IFNγ-induced cell death. IFNγ activates IFNGR1/JAK1/STAT1/IRF1 pathway, which then induces the transcription of CYLD/CASP8/CASP7. When CASP8 mRNA translocates from nucleus to cytosol, ELAVL1 promotes the mRNA stability and translation of CASP8. Finally, increased expression of caspase-8 and CYLD can synergistically promote TNF-mediated caspase-8 activation and cell death. Data represent the mean value ± SD from n biological replicates, or a representative immunoblot from n independent experiments. Source data are available for this figure: SourceData F9.