Fig 2: GPAT3 inhibits apoptosis of HCC cells by TAG-initiated p65/BCL2 signaling pathway. (A, B) After treatment with sorafenib (MHCC97H: 10 μM; Hep3B: 6μM) for 72h, cell apoptosis ratios of NC- and GPAT3 OE- MHCC97H or Hep3B cells (A), WT- and sgGPAT3- MHCC97H SR or Hep3B SR cells (B) were determined by FCs. (C) CN, SR-WT, and SR-sgGPAT3 MHCC97H cells were treated with 80 μM TAG (54:3-FA 18:1) or NaOA (80 μM), followed by staining with Bodipy 558/568 C12 and analysis using flow cytometry. The apoptosis ratios of MHCC97H cells treated with sorafenib (10 μM) in combination with 80 μM TAG (54:3-FA 18:1) or NaOA were also determined using flow cytometry. (D) NC- and GPAT3 OE- MHCC97H and Hep3B cells were treated with or without sorafenib (MHCC97H: 10 μM; Hep3B: 6μM) for 72 h. The total cell lysates were extracted and subjected to WB with indicated antibodies. (E) WT- and sgGPAT3- MHCC97H SR and Hep3B SR cells were treated with sorafenib (MHCC97H: 10 μM; Hep3B: 6μM) and TAG for 72 h. The total cell lysates were extracted and subjected to WB with indicated antibodies. (F) NC- and GPAT3 OE- MHCC97H and Hep3B cells were treated with sorafenib (MHCC97H: 10 μM; Hep3B: 6μM) for 72 h, combined with or without BAY 11-7082 (10 μM), the total cell lysates were extracted and subjected to WB with indicated antibodies. (G) NC- and GPAT3 OE- MHCC97H and Hep3B cells were treated with sorafenib (MHCC97H: 10 μM; Hep3B: 6μM), with/without BAY 11-7082 (3 μM) or ABT-199 (3 μM) for 72 h. Cell apoptosis ratios were determined by FCs. Data are expressed as means ± SEM (n = 3). **p < 0.01, ***p < 0.001. ns represents no significant difference.

Fig 3: GPAT3 is directly regulated by the transcription factor STAT3. (A) Prediction of GPAT3 promoter related transcription factors in TF target, TF and PROMO. (B) WB experiment to detect the expression of STAT3 and its phosphorylation of total protein in CN- and SR-MHCC97H and Hep3B cells. (C) WB was applied to detect the expression of STAT3 and its phosphorylation of nucleoprotein in CN- and SR-MHCC97H or Hep3B cells. (D, E) Protein levels of STAT3 (D), mRNA levels of GPAT3 (E) were examined after 24 h of siRNA transfection targeting STAT3. (F, G) Schematic diagram of binding sites between GPAT3 promoter and STAT3 (F), ChIP experiment on STAT3 antibody binding to MHCC97H and Hep3B cells (G). (H) After 24 h of siRNA transfection targeting STAT3, MHCC97H and Hep3B-SR cells were stained with Bodipy 558/568 C12, and then measured by FCs. (I) Following 24 hours of siRNA transfection targeting STAT3, MHCC97H and Hep3B-SR cells were treated with varying concentrations (1, 2, 4, 8, 16, 32, 64 μM) of sorafenib for 72 hours. The IC50 was assessed using a CCK-8 assay. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: GPAT3 inhibitor reverses the sorafenib resistance of HCC. (A, B) After treatment with N-Ethylmaleimide (10 μM) (A) or FSG67 (30 μM) (B) for 24 h, MFI of Bodipy 558/568 C12 in MHCC97H SR and Hep3B SR cells were examined by FCs. (C, D) MHCC97H SR and Hep3B SR cells were treated with sorafenib (MHCC97H SR: 10 μM; Hep3B SR: 6μM) for 24 h, combined with or without NEM (10 μM) or FSG67 (30 μM). Cell deaths were estimated by CCK-8 assay (C) and apoptosis were estimated by FCs (D). (E-G) Sensitivity of MHCC97H SR cells to sorafenib (2 mg/kg, once every 3 days), NEM (1 mg/kg, once every 2 days) and FSG67 (1 mg/kg, once every 2 days) were evaluated in vivo. Tumor entity view (E), curves of tumor growth (F) and immunohistochemical (IHC) analysis for p65 and Oil Red O staining (G) were displayed, and statistical analysis of IOD of p65, Oil red O staining (right panel). Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. ns represents no significant difference.

Fig 5: GPAT3 deficiency reduces the lipid content and improves the sensitivity of SR cells to sorafenib. (A) GPAT3 expressions in CN-, SR wildtype (SR-WT), SR GPAT3 knockdown (SR-sgGPAT3) and MHCC97H and Hep3B cells determined by WB. (B) TAG contents in SR-WT and SR-sgGPAT3 MHCC97H or Hep3B cells were examined by UHPLC-MS/MS, and TAG area ratio of these cells was shown on the right. (C, D) CN, SR-WT and SR-sgGPAT3 MHCC97H and Hep3B cells were stained by Bodipy 558/568 C12, then were determined by confocal microscopy (C) and FCs (D). Lipid droplets were represented in red, cell nucleus was labeled with DAPI (blue). (E) The IC50 values for CN, SR-WT, and SR-sgGPAT3 in MHCC97H and Hep3B cells were determined using the CCK-8 assay after treatment with sorafenib at various concentrations (1, 2, 4, 8, 16, 32, 64 μM) for 72 hours. (F) After treatment with/without sorafenib (MHCC97H: 10 μM; Hep3B: 6μM) for 72 h, cell death ratios of CN, SR-WT and SR-sgGPAT3 MHCC97H cells were determined by FCs. (G-I) Sensitivity of CN, SR-WT, SR-sgGPAT3 MHCC97H cells to sorafenib (2 mg/kg, once every 3 days) were evaluated in vivo. Tumor entity view (G), curves of tumor growth (H) and immunohistochemical (IHC) analysis for GPAT3 and Oil Red O staining (I) were shown. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. ns represents no significant difference.