Figure 3.

Reoxygenation restored teniposide-induced cGAS-STING activation in human HCC cells. (A) Hep3B and Huh7 cells were cultured under either a normoxic (21% O₂) or a hypoxic (1% O₂) condition for 18 hours and the cells were then transferred to normoxic (21% O₂) culture for 1 hour, 2 hours, 4 hours and 8 hours, respectively; the cellular protein expression of HIF-1α and cGAS was detected by immunoblotting at indicated time points and β-actin was used as a loading control. (B–D) Hep3B and Huh7 cells were treated with teniposide at each IC50, followed by hypoxic culture for 18 hours, and the cells were then transferred to normoxic culture for another 6 hours, and the cellular protein expression of p-IRF3 and p-P65 was detected by immunoblotting (B), the supernatant IFN-β was measured by ELISA (C), and the mRNA expression of CCL5 and CXCL10 was measured by RT-qPCR (D). (E–G) Hep3B and Huh7 cells transduced with HIF1A-shRNA or scramble-shRNA were treated with teniposide at each IC50, followed by either normoxic or hypoxic culture for 24 hours and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting (E), the supernatant IFN-β was measured by ELISA (F), and the mRNA expression of CCL5 and CXCL10 was measured by RT-qPCR (G). Data in (A), (B) and (E) are representative of three independent experiments. Data in (C), (D), (F) and (G) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; HCC, hepatocellular carcinoma; HIF-1α, hypoxia inducible factor 1α; IC50, 50% inhibitory concentration; IFN-β, interferon β; IRF3, interferon regulatory factor 3; Rel. expression, relative expression; RT-qPCR, real time quantitative PCR; sh-SCR, short hairpin RNA-scrambled; Teni, teniposide.