Fig 2: p65 activates the transcription of PDL1. (A) Correlation between p65 and PDL1 in LUAD predicted in the GEPIA system; (B) IHC assay for the PDL1 expression in the clinically collected LUAD tissues and adjacent normal tissues (n = 47); (C) a positive correlation of RNF182 and p65 expression in the LUAD tissues (n = 47); (D) binding peaks of p65 at PDL1 promoter region analyzed by the Cistrome Data Browser database; (E) binding sequence of p65 with PDL1 obtained from the JASPAR database; (F) abundance of PDL1 promoter fragments precipitated by the p65 antibody in LUAD cells after RNF182 or p65 overexpression (n = 3); (G) luciferase activity of the PDL1-WT and PDL1-MUT reporter vectors in 293T cells after RNF182 or p65 overexpression (n = 3). Differences were analyzed by the paired t test (B) or two-way ANOVA (F, G). In panel (C), Pearson's correlation analysis was performed. *p < .05. ANOVA, analysis of varience; IHC, immunohistochemistry; LUAD, lung adenocarcinoma; PDL1, programmed death ligand 1; RNF182, RING finger protein 182.

Fig 3: The RNF182/p65 affects immunosuppression in LUAD cells by regulating PDL1. (A) RT-qPCR and WB analysis for the mRNA and protein levels of PDL1 in A549 and NCI-H1975 cells (n = 3); (B) cytotoxicity in cancer cells after 24-h coculture with CD8+ T cells analyzed by LDH release (n = 3); (C, D) ELISA kits for the concentrations of IFN-? (C) and IL-2 (D) in the supernatant of cocultured cells (n = 3); (E) volume of xenograft tumors in mice on Week 1, 2, 3 and 4 after A549 cell implantation (n = 5); (F) weight of xenograft tumors in mice on Week 4 (n = 5). Differences were analyzed by the one-way ANOVA (F) or two-way ANOVA (A–E). *p < .05. ANOVA, analysis of varience; ELISA, nzyme-linked immunosorbent assay; IFN-?, interferon-?; IL-2, interleukin-2; LDH, lactate dehydrogenase; LUAD, lung adenocarcinoma; PDL1, programmed death ligand 1; RNF182, RING finger protein 182; RT-qPCR, reverse transcription quantitative polymerase chain reaction; WB, western blot.

Fig 4: Overexpression of p65 rescues the malignant phenotype of cancer cells. (A) WB analysis for the protein level of p65 in A549 and NCI-H1975 cells after oe-p65 transfection (n = 3); (B) CCK-8 assay for the proliferation of the LUAD cells (n = 3); (C) number of the colonies formed by the LUAD cells (n = 3); (D) transwell assays for the migration and invasion capacities of the LUAD cells (n = 3); (E) flow cytometry for the apoptosis of the LUAD cells (n = 3). Differences were analyzed by the two-way ANOVA (A–E). *p < .05. ANOVA, analysis of varience; CCK-8, cell counting kit-8; LUAD, lung adenocarcinoma; RNF182, RING finger protein 182; WB, western blot.

Fig 5: RNF182 restoration suppresses malignant phenotype of LUAD cells. (A) RT-qPCR and WB analysis for the mRNA and protein levels of RNF182 in A549 and NCI-H1975 cells after oe-RNF182 transfection (n = 3); (B) CCK-8 assay for the proliferation of the LUAD cells (n = 3); (C) number of the colonies formed by the LUAD cells (n = 3); (D) transwell assays for the migration and invasion capacities of the LUAD cells (n = 3); (E) flow cytometry for the apoptosis of the LUAD cells (n = 3). Differences were analyzed by the two-way ANOVA (A–E). *p < .05. ANOVA, analysis of varience; CCK-8, cell counting kit-8; LUAD, lung adenocarcinoma; RNF182, RING finger protein 182; RT-qPCR, reverse transcription quantitative polymerase chain reaction; WB, western blot.