Pd l1

Western blotting was performed as previously described [16 (link)]. Briefly, cell lysates were prepared from parental wild type or miR-214KO PC3 and MDA-PCa-2b cells using lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Cell lysates (40 μg) were separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 °C with anti-PCNA, VEGFR2, E-Cadherin, N-Cadherin, Vimentin, PTK6, and GAPDH (Cell Signaling Technology), CD31 (Life Technologies), VEGFA (Abcam), CXCR4, SESN3, PD-L1, ALK, and SAA1 (Abclonal Technology, and the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. All primary antibodies were used at a concentration of 1:1000. Following the incubation, membranes were washed, and protein bands were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

DSF was obtained from Sigma (USA) and then dissolved in DMSO. siRNA of IRF7 (5′-TCGAGTGCTTCCTTATGGA- 3′) (RiboBio, Guangzhou, China). Anti-DNMT1, IRF7, PD-L1, granzyme B, CD3, CD4, CD8, and GAPDH Abs were obtains from ABclonal (Wuhan, China). PE-conjugated PD-L1 Ab were obtained from BioLegend (San Diego, CA, USA). The DNMT Activity/Inhibition Assay Ultra Kit (Colorimetric) was purchased from EpiGentek (USA). The anti-PD-1 blocking Ab were obtained from Innovent Biologics (China).