Fig. 1

Identification of HuR as a regulator of PD-L1. a Venn diagram depicting the number of upregulated and downregulated targets identified in two HuR CRISPR/Cas9 knockout clones compared to sgCtrl. b Heatmap representation of enriched pathways in MDA-MB-231 parental cells, sgCtrl, and two HuR knockout clones. c Western blot analysis of HuR and PD-L1 protein level in HuR knockout MDA-MB-231, MIAPaCa2, HeLa cells, and doxycycline-inducible HuR Tet-off system in HeLa cells. d ELISA detection of IL-2 production in MDA-MB-231-Jurkat cell co-culture system (n = 3). e RNP-IP analysis of relative enrichment of PD-L1 transcripts in HuR-immunoprecipitation in MDA-MB-231, H460, and A549 cells (n = 2). f. Schematic representation of CD274 (PD-L1 mRNA) structure. UTR, untranslated region; E, exon. PD-L1 AREs sequences used for RNA pulldown assay were underlined in red. g. Representative Western blot of HuR protein in the pull-downed complex by PD-L1 RNA probes in MDA-MB-231 cells. h. PD-L1 mRNA decay in MDA-MB-231 cells at 0, 0.5, 1, 2, 4, 8, and 16 h post-treatment with 5 μg/mL of Actinomycin D. Two biological repeats for each group. Data are presented as mean ± SEM. One-way ANOVA (d). Two-way ANOVA (e). n.s.: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001