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. 2024 Oct 7;13:RP96445. doi: 10.7554/eLife.96445

Figure 5. ZFP36L1 positively regulates PD-L1 by activating HDAC3 mRNA decay.

Figure 5.

(A) Word cloud of predicted ZFP36L1 target genes. (B) The mRNA expression of predicted target genes in MGC803 cell overexpressing ZFP36L1 (n=5). (C) HDAC3 protein expression in ZFP36L1 knockdown cells. (D) Effect of HDAC3 on CD274 promoter activity in 293T using dual-luciferase assay (n=3). (E) Correlation between changes of histone H3K27 acetylation and PD-L1 protein expression in MKN45 cells overexpressing HDAC3. (F) Chromatin immunoprecipitation (ChIP) assay showing the histone H3K27 acetylation levels of CD274 promoter regions in MKN45 cells overexpressing HDAC3 (n=3). (G) Correlation between changes of histone H3K27 acetylation and ZFP36L1 protein expression. (H) The H3K27ac and SPI1 enrichment of ZFP36L1-E1C regions using ChIP assay (n=3). (I, J) PD-L1 protein expression in simultaneous ZFP36L1 and HDAC3 overexpression cells. (M) HDAC3 mRNA decay in ZFP36L1 knockdown and overexpression cells after actinomycin D treatment. (N) ZFP36L1 mRNA-binding level by RNA-binding protein immunoprecipitation (n=3). (O) ZFP36L1 mRNA-binding site in AU-rich element (ARE) of 3ʹUTR confirmed using dual-luciferase assay (n=6). (P) CCCH-type zinc finger domain of ZFP36L1 protein binding to HDAC3 mRNA confirmed using RNA pull-down assay. ARE, adenylate uridylate- (AU-) rich element; 3ʹUTR, 3ʹ untranslated region. ***, p<0.001; **, p<0.01; *, p<0.05; ns, p≥0.05. (B, F) Wilcoxon rank sum test, (D) one-way ANOVA post hoc Tukey HSD test, (H) t-test, (N) Welch’s t-test, and (O) Kruskal-Wallis with Dunn’s test were used for statistical analysis.

Figure 5—source data 1. PDF file containing original western blots for Figure 5.
Figure 5—source data 2. Original files for western blot analysis displayed in Figure 5.