Fig. 2: NPM1 binds to PD-L1 promoter and upregulates PD-L1 expression in TNBC cells. | Nature Communications

Fig. 2: NPM1 binds to PD-L1 promoter and upregulates PD-L1 expression in TNBC cells.

From: NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer

Fig. 2

a Fragments of PD-L1 promoter in the luciferase reporter plasmids. b Activity of the different fragments of PD-L1 promoter was determined by dual-luciferase assay. n = 3 independent experiments. c Schematic diagram of the streptavidin-agarose pull-down assay. d Proteins from the pull-down assay were separated by polyacrylamide gel eletrophoresis and revealed by sliver staining. The protein band marked in red rectangle was cut out to be analyzed by mass spectrometry. Three biological replicate samples were used. e Binding of NPM1 to PD-L1 promoter was examined by ChIP-qPCR. n = 3 independent experiments. f PD-L1 promoter activity in MDA-MB-231 cells transiently transfected with control siRNA (nc), NPM1 siRNAs (si-NPM1-1, si-NPM1-2), control plasmid for NPM1(vector), or NPM1 overexpression plasmid (NPM1 oe) was measured by dual-luciferase assay. g, h PD-L1 mRNA levels (g), NPM1 and PD-L1 protein expression (h) in MDA-MB-231 and HCC1806 cells with transient NPM1 knockdown or overexpression was detected. i Jurkat cells were co-cultured with MDA-MB-231 or HCC1806 cells in a 1:4 ratio for 48 h, and IL-2 secreted was detected by ELISA. j Primary T cells were co-cultured with MDA-MB-231 and HCC-1806 cells in a 1:30 ratio for 24 h. IFN-γ producing T cells were detected by flow cytometry. k Western blot analysis of NPM1 and PD-L1 expression in MDA-MB-231 cells with NPM1 knockdown and/or PD-L1 overexpression. l, m MDA-MB-231 cells with NPM1 knockdown and/or PD-L1 overexpression were co-cultured with Jurkat cells (l) or primary T cells (m). Data were presented as mean ± s.d. of three independent experiments. f Right panel, g 2th and 4th panel were analyzed by two-side Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. f Left panel, g 1th and 3th panel, i, j were analyzed by one-way ANOVA + two-side Dunnett test, **P < 0.01, ***P < 0.001, ****P < 0.0001. l, m were analyzed by one-way ANOVA + two-side Tukey test, **P < 0.01, ****P < 0.0001; ns not significantly different. Source data are provided as a Source Data file.

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