Fig 2: Changes in B7H5 expression in mutant and wild-type NSCLC cell lines after EGFR siRNA interference. (A-D) Reverse transcription-quantitative PCR and western blot analysis of B7H5 and EGFR expression following transfection with an EGFR siRNA in NSCLC cells. (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC9. (E) Immunofluorescence analysis B7H5 (green colour) and EGFR (red colour) expression following transfection with EGFR siRNA in NSCLC cells (magnification, ×200). **P<0.01, ***P<0.001 vs. negative control. siRNA, small interfering RNA; NSCLC, non-small cell lung cancer.

Fig 3: B7H5 affects the killing effect of gefitinib combined with PBMCs in EGFR-mutated NSCLC cells. (A and B) Cell Counting Kit-8 assay and cytotoxicity test models of mixed cell culture to determine the cell viability and killing effect of PBMCs after treatment with or without B7H5 siRNA in (A) NCI-H358 and (B) NCI-H1299 cells. (C) Expression of B7H5 in NCI-H358 and NCI-H1299 cells transfected with or without B7H5 siRNA by immunofluorescence analysis (magnification, ×200; B7H5 expression: Green colour). (D and E) The mixed cell culture toxicity test model was used to test the efficacy of gefitinib against EGFR-mutated NSCLC cells prior to and after B7H5 siRNA interference in (D) HCC827 and (E) PC-9 cells. (F) Expression of B7H5 in HCC827 and PC-9 cells transfected with or without B7H5 siRNA by immunofluorescence analysis (magnification, ×200). (G) Killing effect of gefitinib combined with PBMCs with and without transfection with B7H5 siRNA on EGFR-mutated NSCLC cells in HCC827 and PC-9 cells. *P<0.05 vs. Negative control (A-E); *P<0.05 vs. control (G). siRNA, small interfering RNA; E/T, effector/target; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer.

Fig 4: Expression of B7H5 following treatment with Gefitinib in NSCLC cells. (A-D) Reverse transcription-quantitative PCR and western blot analysis of B7H5 expression after treatment with gefitinib in NSCLC cells (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC-9. (E) Immunofluorescence determined B7H5 expression (green colour) following treatment with or without gefitinib in NSCLC cells (magnification, ×200). **P<0.01 vs. control. NSCLC, non-small cell lung cancer.

Fig 5: Tissue microarray analysis of differences in expression of B7H5 in cytoplasm between NSCLC and adjacent non-cancer tissue. The expression of B7H5 was determined by (A) reverse transcription-quantitative PCR and (B and C) western blot analysis with (B) representative blots and (C) quantitative protein levels provided. (D) Immunohistochemical analysis of the expression difference of B7H5 in the cytoplasm and nuclear of lung cancer and paracancerous tissues. (E) Immunohistochemical analysis of the expression difference of B7H5 in the cytoplasm and nuclear of lung cancer and paracancerous tissues (scale bars, 100 and 50 µm in upper panel and magnified windows below, respectively). (F) Overall survival of patients with NSCLC represented by Kaplan-Meier curves. *P<0.05; ***P<0.001. NSCLC, non-small cell lung cancer; T, tumor tissue; N, normal tissue; WT, wild-type; Mu, mutant; Cum, cumulative.