Fig. 2: ZNF598 regulates quality control of stalled translation of mRNAs encoding mitochondria-targeted proteins. | Nature Communications

Fig. 2: ZNF598 regulates quality control of stalled translation of mRNAs encoding mitochondria-targeted proteins.

From: Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis

Fig. 2

a Cell viability assay of control and ZNF598 KO HEK293T cells treated with rotenone, CCCP, TG and BFA for 24 h. b Immunoblots of GFP-P2A-Flag-K20-P2A-mCherry stall reporter expression in control (pLK0.1), ZNF598 KO, and ASCC3 KD HEK293T cells. c Flow cytometry analysis of GFP and RFP in normal, ZNF598 KO, and ASCC3 KD HEK293T cells expressing Dox-inducible GFP-P2A-Flag-K20-P2A-mKate2 after 1 μg/ml Dox incubation for 24 h. d Immunoblots showing effect of GFP-ZNF598 OE on GFP-P2A-Flag-K20-P2A-mCherry stall reporter expression in control (pLK0.1) and ASCC3 KD cells. e Immunoblots showing effect of GFP-ZNF598 and FLAG-ASCC3 OE on GFP-P2A-Flag-K20-P2A-mCherry stall reporter expression in control and ZNF598 KO cells. Asterisk indicates FLAG-ASCC3, and arrow indicates FLAG-Cas9 protein. f Immunoblots showing expression of GFP-P2A-Flag-K20-P2A-mCherry cytosol stall reporter and MTSGFP-K20-P2A-mKate2 mitochondrial stall reporter in control and ZNF598 KO cells. The ratio of arrested protein (AP) to full-length protein (FP) and mKate2 to actin were shown. g Immunoblots of MTS-GFP-K20-P2A-mKate2 stall reporter expression in ZNF598 KO, and ASCC3 KD HEK293T cells. For anti-GFP blot, the ratio of arrested GFP-K20 protein (AP) to full length protein (FP) is shown. h Immunostaining of GFP and mKate2 in normal and ZNF598 KO U2OS cells after transfecting with MTSGFP-K20-P2A-mKate2 for 24 h. i Immunoblots of GFP and mKate2 in mitochondria isolated from normal and ZNF598 KO HEK293T cells after transfecting with MTSGFP-K20-P2A-mKate2 for 24 h. j Effect of Flag-ZNF598 OE on MTS-GFP-K20 stall reporter expression in HeLa cells with or without CCCP treatment (6 hr). k Effect of ZNF598 KO on MTS-GFP-K20-P2A-mKate2 stall reporter expression in HeLa cell with or without CCCP or CCCP + ATP treatment. Data are representative of at least three biologically independent experiments (mean ± SD), n = 5 in (a), n = 3 in (c). Representative blot or image is from at least three independent experiments with similar results. p values in a were calculated by two-way ANOVA (Sidak’s test). p values in (c) were calculated by one-way ANOVA (Tukey’s test). Source data are provided as a Source Data file.

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