Fig. 2

Niclosamide inhibits HuR nucleocytoplasmic shuttling. a MTT-based cytotoxicity assay of niclosamide on different cancer cells. b ICC staining of HuR in MDA-MB-231 cells treated with DMSO or 1 µM niclosamide (NIC) after 48 h. Utilizing DAPI stains cells nuclei blue. The white row indicates cytoplasmic HuR. Original magnification 40X; scale bar 100 µm. c Representative Western blot of HuR and PD-L1 protein in parental MDA-MB-231 cells. α-tubulin is used as the loading control. NIC, niclosamide. d Quantified relative level of cytoplasmic HuR in MDA-MB-231 cells (n = 2). Linear regression and correlation matrix between cytoplasmic HuR and PD-L1 relative band intensity. e Representative Western blot of HuR and PD-L1 protein levels in MDA-MB-231 sgCtrl cells. α-tubulin is used as the loading control. f Quantified relative level of HuR in MDA-MB-231 sgCtrl cells (n = 2). Linear regression and correlation matrix between cytoplasmic HuR and PD-L1 relative band intensity. Representative Western blot of HuR and PD-L1 protein levels in MDA-MB-231 HuR KO1 (g), and KO2 (i). Quantified relative level of cytoplasmic HuR and PD-L1 in MDA-MB-231 HuR KO1 (h), and KO2 (j) (n = 2). k PD-L1 mRNA decay in MDA-MB-231 cells at 0, 0.5, 1, 2, 4, 8, and 16 h post-treatment with 1.0 μM niclosamide or DMSO and 5 μg/mL of Actinomycin D. Two biological repeats for each group. Data are presented as mean ± SEM. Two-way ANOVA (d, f, h, j, k). n.s.: no significance. Single linear regression and Pearson correlation coefficient (d, f). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001