Fig 2: Comparison of PDL1 expression under normoxia and hypoxia. Tumor cells were exposed to 4Gy physical dose photon, proton, or carbon-ion radiation. The expression level of PDL1 on the tumor cell surface was detected by flow cytometry 48 h after irradiation. Representative flow cytometry images of each group are shown in the half-offset histograms. The horizontal axis represents the fluorescence intensity of PDL1-PE, and the vertical axis represents the number of cells. The fold change of the MFI for each group relative to the control group (0Gy) is shown in the bar charts. Results are presented as mean ± s.d. Each experiment was repeated at least three times. Statistical significance of each irradiated group relative to the control group (0Gy) was indicated by asterisks. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: BBR directly binds to and inhibits CSN5 activity. (A) The 293T or 293T expressing Flag-CSN5 cell lysates were incubated with BBR-biotin at 4 °C overnight, the lysates were used for streptavidin–agarose pull-down assays, and the precipitates were resolved by IB for CSN5. (B) The recombinant Flag–CSN5 proteins were incubated with BBR–biotin for 1 h at 37 °C, followed by IB with biotin (upper band) or Flag (lower band). (C) The recombinant Flag–CSN5 protein was incubated with BBR–biotin in the absence or presence of indicated concentration of unlabelled BBR for 1.5 h at 37 °C, and the mixtures were IB for biotin or flag. (D) SPR analysis of the binding between BBR and CSN5. Recombinant human CSN5 protein was immobilized on an activated CM5 sensor chip, BBR was then flowed across the chip. (E) CSN5 activity in an in vitro deubiquitination assay. The activity was measured by 7-amino-4-methylcoumarin (AMC) released from the fluorogenic substrate, ubiquitin–AMC (n = 3). ∗∗P < 0.01 and ∗∗∗P < 0.001 compared with DMSO group. (F) IB analysis of the P53 and P27 levels in H460 cells treated with indicated concentration of BBR for 24 h. (G and H) H460 cells were transfected with siRNA control, siRNA targeting CSN5 for 24 h (G), or transfected with 2 μg empty vector, 2 μg Flag–CSN5 for 24 h (H), followed by BBR (10 μmol/L) treatment for 24 h, the PD-L1 expression level was determined by IB. Data shown are mean value of three independent experiments±standard error of mean (SEM).

Fig 4: BBR decreases PD-L1 expression in NSCLC cells. (A) Chemical structure of BBR. (B) IB analysis of PD-L1 expression in H460 cells treatment with the indicated natural compounds (10 μmol/L) for 24 h. (C) and (D) H460, H1975, H358 and HCC827 cells were treated with 10 μmol/L BBR for the indicated times (C), or treated with different concentrations of BBR for 24 h (D), PD-L1 and CD47 expression levels were detected by IB. (E) A549 or H1299 cells were pre-treated with 10 μmol/L BBR for 2 h, followed by IFN-γ (5 ng/mL) stimulation for 24 h, PD-L1 and IDO1 expression levels were detected by IB. (F) H157 cells were treated with BBR (5 and 10 μmol/L) for 24 h, the plasma membrane PD-L1 was detected by flow cytometry. Statistic of PD-L1 mean fluorescence intensity (MFI) is shown in right (n = 3). Data shown are mean value of three independent experiments±standard error of mean (SEM). ∗∗P < 0.01, ∗∗∗P < 0.001 compared with DMSO group.

Fig 5: Comparison of baseline PDL1 expression under normoxia and hypoxia. The expression of PDL1 on the tumor cell surface was detected by flow cytometry. The MFI of each tumor cell line under normoxia and hypoxia is shown in the histogram. Results are presented as mean ± s.d. Each experiment was repeated at least three times. Statistical significance of the difference between cells under normoxia and hypoxia are indicated by asterisks. *p < 0.05, ***p < 0.001.