Fig 1: HMGCS1 enhances the integrated stress response (ISR) pathway and interacts with protein kinase RNA-like endoplasmic reticulum kinase (PERK). (A–C) KATO III and NCI-N87 cells were cultured in complete media containing 10% FBS or under serum deprivation (0.5% FBS) for 24 h (A). AGS, KATO III, and NCI-N87 cells were treated with 2.5 μg/mL tunicamycin (Tuni.) or an equal volume of DMSO for 6 h (B). Additionally, AGS, KATO III, and NCI-N87 cells were also transfected with HMGCS1-expressing construct (HMGCS1) or empty vector (EV) for 48 h (B,C). Whole-cell extracts of the treated cells were prepared for Western blot analysis using anti-HMGCS1, anti-p- eukaryotic translation initiation factor 2 alpha (eIF2α), anti-eIF2α, anti-activating transcription factor 4 (ATF4), anti-general control nonderepressible 2 (GCN2), anti-p-PERK, anti-PERK, and anti-GAPDH antibodies. The asterisk indicates a nonspecific band. (D) For indirect immunofluorescence staining, AGS cells seeded on coverslips were fixed and then stained with mouse anti-HMGCS1 and rabbit anti-PERK antibodies. HMGCS1 was detected by DyLight™ 488-conjugated goat anti-mouse IgG (green), and PERK was detected by DyLight™ 594-conjugated goat anti-rabbit IgG (red) under confocal microscopy. Cell nuclei were also visualized by staining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). Bar, 5 μm. (E) Whole-cell extracts of AGS cells were immunoprecipitated with anti-HMGCS1 antibody. Then, the precipitated proteins were analyzed by Western blot analysis using anti-HMGCS1 (upper) or anti-PERK (lower) antibodies. (F) HMGCS1 knockout (KO) #BF4 and #FE4 cells and negative control (NC) cells were seeded for the assays of cell growth (MTT, upper), migration (middle), and invasion (lower). Cells were treated with 2.5 μg/mL tunicamycin or an equal volume of DMSO for 6 h before assessment. Mean ± SD (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. ##, p < 0.01; ###, p < 0.001. (G) Molecular model for the nonmetabolic functions of HMGCS1 in gastric cancer progression. To enhance progression of gastric cancer cells, HMGCS1 translocates into nuclei to induce Oct4 and SOX-2 expression under stress conditions. Additionally, HMGCS1 can activate the ISR pathway to promote prosurvival signaling.