Fig 1.
PD1 and PD-L1 are highly expressed in lung cancer cells.
A, qPCR measuring PD1 and PD-L1 expression in fresh-frozen human lung normal or cancer tissues (n = 30/group). Data represent the mean + SD, *P < 0.05. B and C, qPCR (B) and Western blot (C) analysis assessing PD1 and PD-L1 expression in human normal and lung cancer cell lines. Data represent three independent experiments.
Fig 2.
PD1/PD-L1 signaling sustains the survival and proliferation of cisplatin resistant cells.
A, Resistant and parental H69 cells were treated with indicated doses of cisplatin and subjected to CCK8 assays. Note: CR, Cisplatin resistance. B, The H69R and H82R cells were growing in drug-free medium for 72 hours. The qPCR or Western blot was used to measure the RNA (left panel) or protein (right panel) expression of PD1 or PD-L1. Data represent three independent experiments, and are the mean ±SD, *P < 0.05. Note: PA, Parental; CR, Cisplatin resistance. C, H69R and H82R cells were transfected with PD-L1 shRNA or control vectors for 24 hours, and then exposed to 3 μM of cisplatin for additional 24 hours. Western blot (left panel) was used to measure PD-L1 protein expression, but CCK8 assays for the cell proliferation. Note: Con, Control vectors; shP, PD-L1 shRNA; Cis, Cisplatin. In CCK8 assays, the experiments are done two times independently with 8 replicates. *P < 0.05, **P < 0.01.
Fig 3.
DNMT1 and KIT are the upstream regulators of PD-L1 signaling in cisplatin resistant cells.
A, The H69R and H82R cells were growing in drug-free medium for 72 hours. The qPCR (left panel) or Western blot (right panel) analysis was used to assess the expression of DNMT1 and KIT. B and C, H69R (B) and H82R (C) cells were transfected with DNMT1 or KIT shRNA for 48 hours and subjected to the qPCR (left panel) or Western blot (right panel) analysis for the expression of indicated genes. D, H69R and H82R cells were transfected with DNMT1 or KIT shRNA for 24 hours, and then DNMT1 or KIT expression vectors were introduced for additional 24 hours. The expression of PD-L1 was determined by qPCR. Note: PA, Parental; CR, Cisplatin resistance; Con, Control vectors; shD, DNMT1 shRNA; shK, KIT shRNA; D, DNMT1 expression vectors; K, KIT expression vectors. Data represent three independent experiments, and are the mean ± SD, *P < 0.05, ** P < 0.01.
Fig 4.
DNMT1 or KIT knockdown induces growth arrest in H69R and H82R cells.
A and B, H69R and H82R cells were transfected with KIT (A) or DNMT1 (B) shRNA or control vectors for 24 hours, and then exposed to 3 μM of cisplatin for additional 24 hours. C and D, H69R and H82R cells were transfected with KIT (C) or DNMT1 (D) shRNA or control vectors for 24 hours, and then further transfected with PD-L1 shRNA vectors for additional 24 hours. E, The PD-L1 shRNA-transfected cells were subjected to Western blot analysis for PD-L1 expression. All the transfected cells were subjected to CCK8 assays; The gene knockdown was confirmed by Western blot; The experiments were done two times independently with 8 replicates; *P < 0.05, **P < 0.01. Note: Con, Control vectors; shP, PD-L1 shRNA; shD, DNMT1 shRNA; shK, KIT shRNA; Cis, Cisplatin.