Fig 2: Stronger inhibitory effect of E2 on AOM/DSS-induced PD-L1 expression in Nrf2 KO male mice than in WT males. (A, B) Representative Western blot images of three independent experiments performed with colon tissue from the male (A) and female mice (B). (C–H) Relative protein expression of PD-L1 (C, D), iNOS (E, F), and COX-2 (G, H) in males (C, E, G) and females (D, F, H). GAPDH was used as a loading control. The P-values obtained from the Kruskal–Wallis test is shown in the figure; *P < 0.05 for comparison between two groups (Mann–Whitney test); # P < 0.05 for comparison between males and females (Mann–Whitney test). WT, wild-type; KO, knockout; Ctrl, control; AOM, azoxymethane; DSS, dextran sodium sulfate; PD-L1, programmed death-ligand 1; iNOS, inducible nitric oxide synthase; COX-2, cyclo-oxygenase 2; Nrf2, nuclear factor erythroid 2-related factor 2; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Fig 3: STAT1 promotes expression of PD-L1 and proliferative biomarkers in HNSCC cells.a, b Gene expression of Stat1 determined by RT-qPCR, STAT1 and GAPDH protein levels, determined by western blot, and phospho STAT1 (pSTAT1) protein levels after IFN? stimulation determined by flow cytometry, in Stat1 siRNA and scrambled RNA treated a CAL27 and b UM-SCC22A cells. STAT1 and GAPDH protein levels were detected after 72 h of siRNA treatment, and western blot band intensity of STAT1:GAPDH were calculated in scramble and Stat1 siRNA-treated cells. Numbers in histogram plots represent percentage of phospho STAT1 positive cells. Controls are unstimulated cells. c, e PD-L1 expression by c CAL27 and e UM-SCC22A cells after 72 h of siRNA treatment, determined by flow cytometry. d, f Flow gating and frequencies of AnnexinV+PI- populations among d CAL27 and f UM-SCC22A cells after 72 h of siRNA treatment. h, i Gene expression of Ki67 and Mcm3, determined by RT-qPCR in h CAL27 and i UM-SCC22A cells after 48 or 72 h siRNA treatment. Data are presented as mean +/- SEM. *p value <0.05; **p value <0.01; ***p value <0.001, ****p value <0.0001 between groups.

Fig 4: IHC results of NAS-related genes (TMSB10 and VIM) and PD-L1. A Correlations between NAS and expression levels of TMSB10, VIM and PD-L1, respectively. B Representative IHC images of TMSB10 B, VIM C and PD-L1 D, respectively. Normal brain tissues (n = 3) and glioma tissues of WHO grade II (n = 3), III (n = 3) and IV (n = 3) were used. C Positive ratios of TMSB10, VIM and PD-L1 in IHC results, respectively. Bar = 50 µm

Fig 5: RAS-ROS-p38 Signaling Controls TTP Activity(A) Histograms represent peak areas from extracted ion chromatograms for non-phosphorylated and phosphorylated peptides corresponding to S52 and S178 phosphosites of mouse TTP. Myc-TTP was immunoprecipitated from CT26 Myc-TTP (tet-ON) cells 1 hr after the indicated treatment. Mean ± SD of technical triplicates. Representative of two independent biological experiments.(B) qPCR analysis of ER-KRASG12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of four independent experiments.(C) Representative flow cytometry histograms of PD-L1 surface protein expression in MCF10A ER-?MEKK3 cells treated in starvation medium for 1 day or 4 days. Data are representative of two independent experiments.(D) Flow cytometry analysis of PD-L1 surface protein expression on ER-HRASG12V MCF10A cells (24 hr) and ER-HRASG12V HKE-3 cells (48 hr) after treatment in starvation medium. Data are representative of biological duplicates.(E) qPCR analysis of CT26 cells at 2 hr or 24 hr after MK2 inhibition with PF 3644022. Mean ± SEM of two independent experiments.(F) Sequence alignments of the conserved phosphosites (highlighted red) targeted by MK2 in mouse (Mm) and human (Hs) TTP protein.(G) Western blotting of immunoprecipitations from CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs. Cells were treated with dox. for 24 hr before the addition of PMA or DMSO for 1 hr. Arrow indicates Myc-TTP. Data are representative of two independent experiments.(H) qPCR analysis of CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs, treated with dox or vehicle for 48 hr. Data represent the mean ± SEM of two independent experiments.**p < 0.005, *p < 0.05. Unpaired, two-tailed Student’s t test. Abbreviations and quantities: 4-OHT, 100 nM; NAC, N-acetyl-L-cysteine, 10 mM; PMA, 200 nM; MEK inhibitor, GSK1120212, 25 nM; MK2 inhibitor PF 3644022, 1 µM; MK2 inhibitor III, 1 µM; dox., doxycycline, 1 µg/mL. See also Figure S5.