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. 2017 Dec 19;47(6):1083–1099.e6. doi: 10.1016/j.immuni.2017.11.016

Figure 1.

Figure 1

Cell-Intrinsic Upregulation of PD-L1 through Oncogenic RAS Signaling

(A) Western blotting analysis of ER-KRASG12V type II pneumocytes treated with 4-OHT in starvation medium. Phospho-ERK and phospho-AKT was measured over time to monitor RAS pathway activation. Data are representative of two independent experiments.

(B) qPCR analysis of ER-KRASG12V type II pneumocytes treated with 4-OHT or IFN-γ in starvation medium. Mean ± SEM of biological duplicates (n = 2) from the experiment described in (A).

(C) Representative flow cytometry histogram of PD-L1 surface protein expression in ER-KRASG12V type II pneumocytes treated in starvation medium for 48 hr. Data are representative of two independent experiments.

(D) Western blotting analysis of RAS signaling following 5 hr treatment with the KRASG12C inhibitor ARS853. Phospho-ERK and phospho-AKT signal reflect RAS pathway activity. Data are representative of two independent experiments.

(E) qPCR analysis following 5 hr treatment with the KRASG12C inhibitor ARS853 (10 μM). Mean ± SEM of biological duplicates (n = 2) from the experiment described in (D).

(F) Flow cytometry analysis of PD-L1 surface protein expression in H358 cells treated with ARS853 (10 μM) for 48 hr. Mean ± SEM of biological triplicates.

(G) Flow cytometry analysis of PD-L1 surface protein expression in ER-KRASG12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of two independent experiments.

(H) qPCR analysis from the experiment described in (G). Mean ± SEM of biological triplicates pooled from two independent experiments.

(I) qPCR analysis of H358 cells treated for 24 hr. Mean ± SEM of two independent experiments.

(J) qPCR analysis of H358 cells treated with PMA for 3 hr following a 30 min pre-treatment with DMSO or MEK inhibitor. Mean ± SD of two independent experiments.

Abbreviations and quantities are as follows: MFI, mean fluorescence intensity; EtOH, ethanol vehicle; 4-OHT, 100 nM; IFN-γ, 20 ng/mL; MEK inhibitor GSK1120212, 25 nM; PI3K inhibitor GDC-0941, 500 nM; PMA, 200 nM. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, n.s., not significant. Unpaired, two-tailed Student’s t tests. See also Figure S1.