fig5

Figure 5. Fra-1 stabilizes G6PD protein expression levels and activates the PPP metabolic pathway by inhibiting the G6PD ubiquitin-proteasome pathway. (A and B) Overexpression of Fra-1 in GC cells AGS and HGC27, followed by treatment with the protein synthesis inhibitor CHX for various durations, and detection of G6PD protein expression levels using western blot assay; (C and D) Treatment of GC cells AGS and HGC27 with CHX for various durations, with the addition of the proteasome pathway inhibitor MG132 in the experimental group, followed by detection of G6PD protein expression levels using western blot assay; (E and F) Silencing of Fra-1 in GC cells AGS and HGC27, followed by treatment with MG132, and detection of G6PD protein expression levels using western blot assay; (G and H) Co-transfection of Fra-1 plasmid and ubiquitin plasmid with HA tag (HA-Ub) into GC cells AGS and HGC27, followed by treatment with MG132, and detection of G6PD ubiquitination using immunoprecipitation assay; (I and J) Detection of cell proliferation ability in GC cells AGS and HGC27 after silencing Fra-1, silencing Fra-1 while overexpressing G6PD, and treatment with CDDP for 24 h using the CCK8 Cell Proliferation Detection Kit; (K-N) Detection of cell proliferation ability in GC cells AGS and HGC27 after silencing Fra-1, silencing Fra-1 while overexpressing G6PD, and treatment with CDDP (10 μM) for 24 h using the EDU cell proliferation detection kit. All experiments were performed with three technical replicates. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. Fra-1: Fos-related antigen-1; G6PD: glucose-6-phosphate dehydrogenase; PPP: pentose phosphate pathway; GC: gastric cancer; CHX: cycloheximide; CDDP: cisplatin; CCK8: cell counting kit-8; cell counting kit-8; EDU: 5-ethynyl-2’-deoxyuridine.