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. 2020 Apr 29;30(7):590–601. doi: 10.1038/s41422-020-0315-8

Fig. 2. PD-L1 is a cell cycle dependent protein and regulates sister chromatid cohesion in TNBC.

Fig. 2

a PD-L1 level fluctuates during the cell cycle. Target proteins were detected by Western blot in MDA-MB-231 cells at indicated time points after double thymidine block. b Immunostaining of PD-L1 in interphase cells. Wildtype (WT) or PD-L1 knockout (KO) MDA-MB-231 cells were permeabilized and fixed using the indicated methods and cells were then stained with anti-PD-L1 antibody. PFA + Triton X-100: cells were fixed with 3% paraformaldehyde followed by 0.5% Triton X-100. Triton X-100 + PFA: cells were treated with 0.5% Triton X-100 first to remove most cytoplasmic/membrane proteins, and followed by fixation with 3% paraformaldehyde. Nucleus was stained with DAPI. Scale bar: 10 µm. c Detection of PD-L1 in different fractions of control and PD-L1 knockout MDA-MB-231 cells. Cytoplasmic/membrane (C/M) and nuclear (N) fraction. d Cytoplasmic/membrane (C/M) and nuclear (N) fractions of PD-L1 from different cancer cell lines. SE short exposure; LE long exposure. e Cytoplasmic/membrane (C/M) and nuclear (N) fractions of MDA-MB-231 cells with or without PNGase F treatment. Arrows indicate PD-L1 bands. f, g Loss of PD-L1 promotes multinucleation. f Representative nuclear staining by DAPI in MDA-MB-231 cells three days post-infection with control shRNA virus or PD-L1 shRNA virus. Scale bar: 10 µm. g Quantification of multinucleated cells was performed. Data are presented as means ± SD, and were independently replicated three times. Statistical significance was calculated using Student’s t test. **P < 0.01, ***P < 0.001. h, i PD-L1 is required for sister chromatid cohesion. h Representative metaphase spreads showing MDA-MB-231 cells with normal, partial loss and total loss of sister chromatid cohesion. Scale bar: 5 µm. i Quantification of different sister chromatid status in control and PD-L1 knockdown cells. Data are presented as means ± SD, independently replicated three times with similar results. j FISH assays were performed in MDA-MB-231 cells expressing control shRNA, PD-L1 shRNA, SCC1 shRNA or Sororin shRNA. myb gene probe was used and the distance between paired FISH signals was measured and quantified. Bar: 5 µm. k Western blots showing the knockdown efficiency for the experiments in j.