Fig. 3: Nuclear PD-L1 interacts with cohesion complex.

a, b PD-L1 interacts with cohesin complex. a Doxycycline-inducible PD-L1 widetype (WT) (+/+) and knockout (KO) (−/−) MDA-MB-231 cells were harvested and fractionated. The nuclear fraction was incubated with monoclonal anti-PD-L1 antibody or corresponding IgG. The immunoprecipitates were blotted with indicated antibodies. b Cells were blocked with thymidine and transfected with indicated siRNAs. After releasing from the second round of thymidine block, cells were released into S phase in the presence of MG132 for 3 h. Cells were harvested and nuclear fractions were processed for immunoprecipitation. c PD-L1 is involved in the regulation of cohesion in the presence of WAPL. MDA-MB-231 cells expressing control shRNA, PD-L1 shRNA, WAPL shRNA, or a combination of PD-L1 and WAPL shRNAs were fixed and dropped on coverslips. Percentages of cells with different sister chromatid cohesion status were quantified. Data are presented as means ± SEM, and were independently replicated three times. d–f PD-L1 YSR-like motif (YKR) is essential for PD-L1 binding with PDS5B in vivo, and competes with WAPL for binding to PDS5B in vitro. PD-L1 wild-type (WT), PD-L1 AIA mutant (mutating both FGF-like motifs to AIA), and PD-L1 AKE mutant (mutating YKR to AKE) plasmids were transfected into cells and nuclear fractions were isolated and incubated with (d) HA-conjugated sepharose beads of (e) protein A beads conjugated with anti-PDS5B antibody. The immunoprecipitates were blotted with indicated antibodies. f Left panel: in vitro competition of binding affinity of PDS5B (purified from insect cells) and purified WAPL (1–30)-GST and PD-L1 (purified from nuclear fraction of MDA-MB-231 cells). Right panel: in vitro interactions between PD1 and wildtype (WT) or AKE mutant PD-L1 purified from cytoplasmic/membrane fraction (C/M) of MDA-MB-231 cells. g–j PD-L1 YSR-like motif is essential for sister chromatid cohesion and cell proliferation. myb gene distance between paired FISH signals (g), and colony formation (i) were measured in MDA-MB-231 cells infected with control shRNA, PD-L1 shRNA, PD-L1 shRNA plus WT shRNA resistant PD-L1 overexpression construct, or PD-L1 shRNA plus shRNA resistant AKE mutant PD-L1 overexpression construct. Scale bar: 10 µm. Data are presented as means ± SEM, and were independently replicated three times with similar results. Statistical significance was calculated using Student’s t-test. **P < 0.01, ***P < 0.001. h Protein levels in cells used for assays in (g and i) were detected by Western blot with indicated antibodies. j B16F10 cells expressing control shRNA (shCtrl), mouse Pd-l1 shRNA (shPd-l1), Pd-l1 shRNA plus shRNA resistant mouse wildtype Pd-l1 (shPd-l1 + Pd-l1 wt), or mouse Pd-l1 shRNA plus shRNA resistant ake mutant Pd-l1 (shPd-l1 + Pd-l1 ake) were injected into C57BL/6 mice. Mice were treated with 5 mg/kg mouse Pd-l1 blocking antibody or corresponding IgG. The tumor growth was monitored by measuring with a caliper every 3–4 days. Data are presented as means ± SEM (n = 5). Two-way ANOVA was used for the comparisons. **P < 0.01, ***P < 0.001.