PMC

ACTG1 affects apoptosis of HCC cells.
Notes: (A) Overexpression of ACTG1 suppressed apoptosis. (B) ACTG1 knockdown improved apoptosis (*P<0.05, ***P<0.005) (n=5). (C) Expression of Bax/cleaved PARP/cleaved caspase-3 was downregulated/upregulated when ACTG1 was overexpressed/knocked down.
Abbreviations: HCC, hepatocellular carcinoma; ACTG1, actin gamma 1; NC, negative control.

ACTG1 influences aerobic glycolysis in HCC cells.
Notes: (A, B) Glucose uptake was enhanced/decreased when ACTG1 was overexpressed/knocked down. (C, D) Lactate production was enhanced/decreased when ACTG1 was overexpressed/knocked down. (E, F) Extracellular acidification rate (ECAR) was enhanced/decreased when ACTG1 was overexpressed/knocked down. (G, H) Expression of GLUT1 was upregulated/downregulated when ACTG1 was overexpressed/knocked down. Other proteins showed no significance. *P<0.05, **P<0.01, ***P<0.005.
Abbreviations: HCC, hepatocellular carcinoma; ACTG1, actin gamma 1; NC, negative control; GLUT1, glucose transporter 1.

RRAD regulates aerobic glycolysis in HCC cells.
Notes: (A, B) Glucose uptake was enhanced/decreased when RRAD was knocked down/overexpressed. (C, D) Lactate production was enhanced/decreased when RRAD was knocked down/overexpressed. (E, F) ECAR was enhanced/decreased when RRAD was knocked down/overexpressed. (G, H) Expression of GLUT1 was upregulated/ downregulated when RRAD was knocked down/overexpressed. Other proteins showed no significance. *P<0.05, **P<0.01, ***P<0.005.
Abbreviations: RRAD, Ras-related associated with diabetes; ECAR, extracellular acidification rate; HCC, hepatocellular carcinoma; GLUT1, glucose transporter 1; NC, negative control.

ACTG1 acts as a functional downstream effector of RRAD in HCC cells.
Notes: (A–E) Glucose uptake assay/lactate production assay/extracellular acidification rate (ECAR) measurement/CCK-8 assay/apoptosis assay in SK-Hep-1 cells transfected with NC/RRAD lentivirus and co-transfected with lentiviruses encoding the RRAD and ACTG1. (F) The mRNA level of ACTG1 was downregulated when RRAD was overexpressed. (G) Expression of proteins related to cell cycle/apoptosis/aerobic glycolysis in SK-Hep-1 cells by Western blotting. **P<0.01, ***P<0.005.
Abbreviations: HCC, hepatocellular carcinoma; ACTG1, actin gamma 1; NC, negative control; RRAD, Ras-related associated with diabetes; CCK, cell counting kit.

Proteins that interact with RRAD.
Notes: (A) Proteins that predicted to interact with RRAD on STRING (Szklarczyk D, Franceschini A, Wyder S, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015;43(Database issue):D447–D452). (B) Expression of P53 after overexpression of RRAD and expression of RRAD after overexpression of P53 by Western blotting. (C) The Venn diagram of the number of proteins after Co-IP assays and LC-MS/MS analysis. (D) GST (glutathione-S-transferase) pull-down assay for ACTG1/EF1A1 with RRAD.
Abbreviations: RRAD, Ras-related associated with diabetes; Co-IP, co-immunoprecipitation; LC-MS/MS, liquid chromatography–tandem mass spectrometry; ACTG1, actin gamma 1; NC, negative control.

Effect of ACTG1 on the proliferation and cell cycle of HCC cells.
Notes: (A, B) Cell viability was enhanced/decreased when ACTG1 was overexpressed/knocked down. (C, D) EdU assays showed that cell proliferation was enhanced/ decreased when ACTG1 was overexpressed/knocked down. Arrows indicate cells with high viability. All the cells can be identified by Hoechst in blue and cells with high viability can be identified by EdU in red. Cells with high viability are pink when merged. (E, F) Cell cycle analysis for SK-Hep-1 cells treated with NC or ACTG1 lentivirus/ Huh7 cells transfected with NC or ACTG1 shRNAs. (G) Expression of cyclin A2/D1/E1 and CDK2/4 was upregulated/downregulated when ACTG1 was overexpressed/ knocked down. *P<0.05, ***P<0.005.
Abbreviations: HCC, hepatocellular carcinoma; ACTG1, actin gamma 1; NC, negative control.

RRAD inhibits tumor growth through ACTG1 in vivo and RRAD/ACTG1 is associated with prognosis of patients.
Notes: (A) Tumors removed from nude mice (n=5 biological replicates). (B, C) Mean tumor weight/volume showed that the tumor from the lv-RRAD cells were smaller, and could be rescued by ACTG1 (**P<0.01, ***P<0.001). (D) Expression of GLUT1, RRAD, and ACTG1 in tumor removed from nude mice. Magnification ×200. (E, F) Survival analysis of HCC patients by Kaplan–Meier plots and log-rank tests. Patients were categorized by high and low expression of RRAD/ACTG1. (G) Survival analysis of HCC patients by Kaplan–Meier plots and log-rank tests (four groups): high RRAD and high ACTG1; high RRAD and low ACTG1; low RRAD and high ACTG1; low RRAD and low ACTG1.
Abbreviations: HCC, hepatocellular carcinoma; ACTG1, actin gamma 1; NC, negative control; RRAD, Ras-related associated with diabetes; GLUT1, glucose transporter 1.