Fig 2: Effects of EIF4G1 on NSCLC via the activation of mTOR signalling. A, Western blot analysis was used to confirm the change in mTOR signalling in A549 cells treated with shNC or shEIF4G1. B, The relative protein levels of EIF4G1 and mTOR signalling molecules. C, Immunohistochemical staining of EIF4G1 and mTOR protein expression in the tumour tissues from NSCLC patients, 1 bar = 50 μm. D, Correlation between EIF4G1 and mTOR expression in NSCLC tissues. The data are expressed as the mean ± SD of three independent experiments. *P < .05

Fig 3: USP10 Deubiquitylated EIF4G1. A The EIF4G1 expression vector and HA-UB were transfected into control and USP10 knockout cells. The cells were then treated with MG132 (40 µM) for 4 h. Immunoprecipitation was performed using FLAG beads and immunoblotting was performed as indicated. B The EIF4G1 expression vector and HA-UB were transfected into wild-type USP10 and USP10 mutant overexpressing cells. The cells were then treated with MG132 (40 µM) for 4 h, followed by immunoprecipitation using FLAG beads, and immunoblotting as indicated. Original blots are presented in Supplementary Fig. 4.

Fig 4: USP10 regulates non-small cell lung cancer cell migration in an EIF4G1-dependent manner. A Cell scratch assay for the migratory ability of control H1299 cells, USP10 knockdown cells, and USP10 knockdown plus EIF4G1 overexpression cells. B Cell scratch assay to detect the migration ability of cells overexpressing USP10 and control H1299 cells. C Transwell migration assay to detect the migration ability of control H1299 cells, USP10 knockdown cells, and USP10 knockdown plus EIF4G1 overexpression cells. D Transwell migration assay was performed to detect the migration ability of cells overexpressing USP10 and control H1299 cells. Mean ± SD, n = 3. **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: 50 μm.

Fig 5: EIF4G1 regulates LSCC tumorigenesis in vivo A, B. The expression levels of EIF4G1 in stable “knockdown” NCI-H1703 cells was performed using lentiviral vectors containing 2 shRNA specifically targeting EIF4G1. The protein expression was detected by western blot analysis. A non-silence (vector)-shRNA was used as a negative control. C A Q-PCR was used to measure the expression of EIF4G1 in tumors from BLAB/C mice. ***P < 0.005. D NCI-H1703 cells containing shEIF4G1-vector or shEIF4G1-KD lentiviral vectors were injected into nude mice (n = 18 for each group). The tumor volume was recorded every three days for a maximum period of 15 days. The data are presented as mean ± SEM. ***P < 0.005. E The tumors were harvested and weighted. The data are presented as mean ± SEM. ***P < 0.005. F The volume and weight were compared between the shEIF4G1-vector and shEIF4G1-KD G. H, E staining indicated cellular morphology. EIF4G1 protein expression was markedly decreased in xenograft tumors of the shEIF4G1-KD group compared with that noted in the shRNA-vector group