Fig 2: Kaplan-Meier curves (correlation) for patients with ESCC treated by RT, according to COX-2, XRCC1 and RASSF1 expression. (A) Correlation between OS and COX-2 expression. (B) Correlation between OS and XRCC1 expression. (C) Correlation between OS and RASSF1 expression. (D) Correlation between PFS and RASSF1 expression. (E) Correlation between OS and RASSF1 positive/XRCC1 negative expression. (F) Correlation between PFS and RASSF1 positive/XRCC1 negative expression. (G) Correlation between OS and tumor response. (H) Correlation between PFS and tumor response. COX-2, cyclooxygenase-2; XRCC1, X-ray repair cross complementing group 1; RASSF1, ras association domain family 1; ESCC, esophageal squamous cell carcinoma; RT, radiotherapy; OS, overall survival; PFS, progression-free survival.

Fig 3: ATF4 occupies the RASSF1 promoter and regulates its expression. (A) Schema of the RASSF1 promoter with the localization of putative ATF4‐binding site. (B) Luciferase activity in HEK293FT cells co‐transfected with pRASSF1‐Luc and pRK‐ATF4 or pRK‐ATF4 ΔC (1–275) (n = 2 biological replicates). The results were expressed as relative luciferase activity normalized with the total protein concentration. P‐value was calculated with an unpaired two‐tailed t‐test. (C) Soluble chromatin from HEK293FT cells was precipitated with anti‐ATF4 antibody or rabbit IgG (n = 3 biological replicates). The final DNA samples were amplified by qPCR with primers for the RASSF1 promoter listed in Table 2. The results were expressed as the percentage to the input DNA. P‐value was calculated with an unpaired two‐tailed t‐test. Data represent the mean ± SEM. *P < 0.05, **P < 0.01.

Fig 4: Endoplasmic reticulum stress induced by (A) tunicamycin (Tun) or (B) thapsigargin (Thap) in HEK293FT cells upregulates RASSF1 and ATF4 expression, and the effects were reduced by ISRIB addition. HEK293FT cells were treated by tunicamycin (5 μg·mL−1) or thapsigargin (3 μm) with or without ISRIB (500 nm) addition, and the relative gene expression was detected by RT‐qPCR using primers listed in Table 2 and normalized with GAPDH expression (n = 3 biological replicates). Ctrl, control; Thap, thapsigargin treatment; Tun, tunicamycin treatment. P‐values were calculated with a one‐way ANOVA followed by the Tukey's multiple comparison test. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Endoplasmic reticulum stress induced by tunicamycin (Tun) in HEK293FT cells upregulates RASSF1, RASSF1A, RASSF1C, ATF4, and BBC3 expression. HEK293FT cells were treated with tunicamycin (5 μg·mL−1), and the relative gene expression was detected by RT‐qPCR using primers listed in Table 2 and normalized with GAPDH expression (n = 3 biological replicates). Ctrl, control; Tun, tunicamycin treatment. P‐values were calculated with an unpaired two‐tailed t‐test. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, nonsignificant.