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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: RNF31 represses cell progression and immune evasion via YAP/PD-L1 suppression in triple negative breast Cancer

Fig. 6

RNF31 represses PD-L1 expression and immune evasion by suppressing Hippo/YAP axis. A Western blot detecting of YAP protein level in MDAMB231 cell. B ChIP assay displaying YAP interaction with a potential enhancer located 13-kb upstream from the transcription start site of PD-L1 gene. Lysates were prepared from MDAMB231 cell transfected with Myc-YAP. YAP–TEAD interacting sequence located 13-kb upstream of PD-L1 gene was amplified by PCR after immunoprecipitation using IgG or anti-Myc antibody. C ChIP-qPCR data displaying relative levels of the 13-kb upstream enhancer sequence in the ChIP assay. D Relative luciferase activity of Myc or Myc-YAP in MDAMB231 cells transfected with luciferase vector containing the 13-kb upstream enhancer. E and I Western blot detecting of PD-L1 and RNF31 expression in MDAMB231 cell with indicated treatment. F and J RT–qPCR results of PD-L1 mRNA level with indicated treatment. G-H and K-L The cell-membrane localized PD-L1 in MDAMB231 cell with indicated treatment were analyzed by Flow cytometry using PD-L1 antibody. M Immunofluorescence imaging of PD-L1 (Red) and DAPI (blue) in MDAMB231 cell with indicated treatment, scale bar 20 μm. N-T Flowchart of In vivo experiments (N). Endpoint tumor images (O). Endpoint tumor weight (P). Endpoint tumor volume (Q). Cells digested from indicate tumor tissues in BALB/c mice were stained with anti-CD45 antibody and subjected to flow cytometric analyses (R). Immunohistochemical staining was performed on tumor sections with anti-CD8 antibody. Representative images are shown. Scale bars, 50 μm (S-T). The results are representative of 3 independent experiments in panel A-M. The results are representative of 6 independent experiments in panel N-S. β-actin was engineered to the internal reference for Western blot. The data are presented as mean ± SDs. **P < 0.01, ***P < 0.001 (Student’s t test)

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