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

Figure 4. SPI1 binds to the ZFP36L1-SE region and drives the regulation of PD-L1.

(A) Schematic of transcription factor (TF) motif enrichment in ZFP36L1-E1. (B) Kaplan-Meier survival plot of SPI1 in The Cancer Genome Atlas (TCGA). (C) The mRNA expression of ZFP36L1 after TFs plasmid transfection (n=3). (D) The ZFP36L1 protein expression in cell lines overexpressing SPI1. (E) Correlation between SPI1 and PD-L1 mRNA expression in TCGA. (F) PD-L1 protein expression in simultaneous SPI1 overexpression and ZFP36L1 knockdown cells. (G) Prediction of SPI1-BRD4-P300 binding on the STRING website. Co-immunoprecipitation between (H) exogenous SPI1 and BRD4 in 293T cells, or (I) endogenous SPI1 and BRD4 in MGC803. (J) SPI1 directly interacts with BRD4 in vitro by GST pull-down experiment. (K) ZFP36L1-E1 binding of different TFs detected using dual-luciferase assay (n=3). (L) SPI1 enriched regions in ZFP36L1-E1 detected by chromatin immunoprecipitation (ChIP) assay (n=3). (M) Different binding sites of SPI1 in ZFP36L1-E1 detected using dual-luciferase assay (n=6). (N) Wild-type and motif-deletion mutant E1C binding of SPI1 detected using dual-luciferase (n=5). ***, p<0.001; **, p<0.01; *, p<0.05; ns, p≥0.05. (C ,K) One-way ANOVA post hoc Tukey HSD test, (L) t-test, (M) Welch’s t-test, and (N) Welch’s ANOVA with a Games-Howell post hoc test were used for statistical analysis.

Figure 4—source data 1. PDF file containing original western blots for Figure 4.
Figure 4—source data 2. Original files for western blot analysis displayed in Figure 4.

Figure 4.

Figure 4—figure supplement 1. Transcriptional regulation of PD-L1 by SPI1.

Figure 4—figure supplement 1.

(A) Transcription factor binding sites in ZFP36L1-SE based on chromatin immunoprecipitation (ChIP)-seq data from the Signaling Pathways Project. (B) The PD-L1 protein expression in simultaneous SPI1 overexpression and ZFP36L1 knockdown cells.