Fig 2: Rspo2 suppresses oxLDL-induced ER stress and ROS production. (A) Expression of the ER stress marker, PERK, was assessed using western blotting in THP-1 cells incubated with the indicated concentrations of oxLDL (0-100 µg/ml) for 24 h. The expression of GAPDH was used as a loading control. (B) Expression of PERK was assessed in THP-1 cells alone or in those transfected with pc-basic or pc-rspo2, and treated with or without oxLDL (100 µg/ml) for 24 h. The expression of GAPDH served as a loading control. (C) THP-1 cells transfected with pc-basic or pc-rspo2 and treated with or without oxLDL (100 µg/ml) for 24 h, were labeled with 2,7-dichlorofluorescein diacetate dye to assess the production of ROS. The labeled cells produced fluorescent signal proportional to the ROS levels, and this signal was recorded using an Olympus fluorescence microscope. *P<0.05, comparison indicated by brackets. oxLDL, oxidized low density lipoprotein; Rspo, R-spondin; ER, endoplasmic reticulum; ROS, reactive oxygen species; PERK, protein kinase RNA-like ER kinase; siRNA, small interfering RNA.

Fig 3: Targeting PERK inhibits apoptotic cell death in radio-resistant GC cells subjected to SSA in combination with 2 Gy. (A–F) PERK shRNA stable AGSR cell lines were established after AGSR cells were transfected with PERK shRNA. These cells were exposed to SSA (10 µ?), 2 Gy, and SSA/2 Gy, and cell viability, cell cytotoxicity, caspase-3 activity, intracellular ROS release, and intracellular Ca2+ release were determined along with Western blot analyses examining the levels of p-PERK, PERK, CHOP, and cleaved caspase-3; *, p < 0.05. ß-actin was used as a protein loading control.

Fig 4: Fisetin induces ER stress in liver cancer cells. (A) HepG2 and Hep3B cells were treated with fisetin (100 µM) for the indicated times, and an intracellular Ca2+ assay was performed. * = p < 0.05. (B) The ER stress markers ATF4 and CHOP were measured by RT-PCR. Fold changes to target genes were normalized to ß-actin. (C) The ER stress markers CHOP, PERK, eIF2a, p-eIF2a, p-PERK, ATF4, and GRP78 were measured by a western blot assay. ß-actin was used as the protein loading control. (D) HepG2 and Hep3B cells were treated with fisetin (100 µM) for the indicated times, and then the exosomes (30 µg) were collected from the cell supernatant. Total exosomes were determined by western blotting using the exosome marker CD63 and the ER stress marker GRP78. (E–H) Cell viability, LDH activity, caspase-3 activity, intracellular Ca2+, and ER stress-related protein (cleaved caspase-3, p-eIF2a, ATF4, PERK, CHOP, eIF2a, and p-PERK) levels were measured in the thapsigargin (TG; 3 µM, 24 h) and fisetin (100 µM, 24 h)-treated HepG2 and Hep3B cells. * = p < 0.05. Data are representative of three experiments.

Fig 5: Nox4 regulates ER stress and cell death in SSA-treated GC cells. (A) AGS and MKN-74 cells were treated with SSA (10 µM) for the indicated times, and the cells were stained with DCFDA (20 µM) and analyzed with a microplate reader. (B–F) AGS and MKN-74 cells were pre-treated with NAC (10 mM) and DPI (10 µM) for four hours and then treated with SSA (10 µM) for 24 h. Cell viability, cell cytotoxicity, ROS release, and Ca2+ release were determined using WST-1, LDH, intracellular ROS, and intracellular Ca2+ assays; *, p < 0.05. ER stress markers (p-PERK, PERK, p-eIF2a, eIF2a, ATF4, and CHOP) were determined by Western blot assay. ß-actin was used as a protein loading control. (G–I) After AGS and MKN-74 cells were transfected with Nox4 siRNA, they were treated with SSA (10 µM, 24 h), and cell viability, cytotoxicity, and Nox4 and CHOP levels were determined; *, p < 0.05. ß-actin was used as a protein loading control.