Fig. 4

Nano-DOX induced PD-L1 in NSCLC cells through reinforced activation of the HMGB1/RAGE/NF-κB pathway. A–H Pharmacological blocking of the HMGB1/RAGE/NF-κB pathway suppressed PD-L1 induction by Nano-DOX but not DOX in the in vitro A549 cells. I–N HMGB1 induced PD-L1 in the in vitro A549 cells, which was repressed by blocking of the HMGB1/RAGE/NF-κB pathway. O Nano-DOX and DOX increased cell surface RAGE. Exogenously given HMGB1 was negative of this effect. P, Q Blocking of NF-κB repressed PD-L1 induction by Nano-DOX but not DOX. Exogenously given HMGB1 also increased protein level of RAGE. Cell surface PD-L1 and RAGE were assayed by FACS analysis of immunofluorescent staining and protein levels thereof were assayed by western blotting. R Nano-DOX treatment led to increased immunohistological staining of RAGE and activated NF-κB in subcutaneous xenografts of Lewis cells in mice. FACS histogram geometric means were used to quantify mean fluorescence intensity (MFI). Values were means ± SD (n = 3, *p < 0.05). EP is an inhibitor of HMGB1 secretion. GA both neutralizes HMGB1’s cytokine activity and suppresses its secretion. FPS-ZM1 is a high-affinity inhibitor of RAGE. PDTC is a selective inhibitor of NF-κB. Drug concentration was 2 μg/mL for DOX and Nano-DOX in the in vitro experiments and treatment duration was 24 h. Representative FACS dot plots for E–H and L–P were provided in Additional file 1: Figure S4. Effect of EP alone on surface PD-L1 expression in A549 cells is shown in Additional file 1: Figure S4 E’