Fig 2: Metformin blocked the interaction of MET and Gab1 and thereby inhibited the phosphorylation of Gab1.a H3122/Vec and H3122/HGF cells pretreated with alectinib (50 nmol/L) and/or metformin (5 mmol/L) for 48 h were lysed and then subjected to denaturing Co-IP with M2 beads followed by western blot analysis. Similar results were obtained in three independent experiments. b H2228/Vec and H2228/HGF cells pretreated with alectinib (500 nmol/L) and/or metformin (5 mmol/L) for 48 h were lysed and then subjected to denaturing Co-IP with M2 beads followed by western blot analysis. Similar results were obtained in three independent experiments. c H3122/HGF cells were treated with alectinib (50 nmol/L), JNJ-38877605 (10 nmol/L) and/or metformin (5 mmol/L) for 48 h. Cell lysates were harvested and subjected to denaturing Co-IP with M2 beads followed by western blot analysis. Similar results were obtained in three independent experiments. d H3122 cells were transfected with p-Gab1Y627A-lentivirus, p-Gab1Y627D-lentivirus, wild-type p-Gab1-lentivirus, and corresponding empty vector, and the phosphorylation levels of Gab1 were detected by western blot analysis. e H3122 cells were transfected with p-Gab1Y627A-lentivirus, p-Gab1Y627D-lentivirus, and wild-type p-Gab1-lentivirus and then treated with HGF (50 ng/mL) and alectinib (50 nmol/L) with or without metformin (5 mmol/L) for 48 h. Cell lysates were harvested and subjected to denaturing Co-IP with M2 beads followed by western blot analysis. Similar results were obtained in three independent experiments. Ale alectinib, Metf metformin, METi MET selective inhibitor JNJ-38877605, Vec negative control vector, NC empty vector, WT wild-type p-Gab1.

Fig 3: Metformin reversed HGF-induced alectinib resistance via inhibiting Gab1 activity.a H3122/Vec cells, H3122/HGF cells and H3122/HGF cells pretreated with alectinib (50 nmol/L) and/or metformin (5 mmol/L) for 48 h were lysed and subjected to western blot analysis to evaluate the phosphorylation of MET, MET, phosphorylation of Gab1 and Gab1. GAPDH was used as an internal control. Similar results were obtained in three independent experiments. b H2228/Vec cells, H2228/HGF cells and H2228/HGF cells pretreated with alectinib (500 nmol/L) and/or metformin (5 mmol/L) for 48 h were lysed and subjected to western blot analysis for phosphorylation of MET, MET, phosphorylation of Gab1 and Gab1. GAPDH was used as an internal control. Similar results were obtained in three independent experiments. c H3122 cells were treated with 50 nmol/L alectinib, 50 ng/mL HGF and metformin with the indicated doses (48 h) and for the indicated times (5 mmol/L), and the phosphorylation levels of Gab1 were assessed by western blotting. Similar results were obtained in three independent experiments. d Gab1-specific siRNAs were introduced into H3122 and H2228 cells. After incubation for 48 h, the cells were lysed, and Gab1 protein was detected by western blot analysis. e Negative control, Gab1-specific siRNA#1 or Gab1-specific siRNA#3 were introduced into H3122 and H2228 cells. The indicated cells (control, treated with 50 ng/mL HGF or treated with 50 ng/mL HGF plus 5 mmol/L metformin) were incubated with the indicated doses of alectinib for 48 h, and the IC50 values of indicated cells for alectinib were detected by MTT assay. The data are presented as the mean ± SD from three independent experiments. **p < 0.001 compared with homologous Si-NC cells treated with HGF (50 ng/mL). f, g The indicated cells were incubated with HGF (50 ng/mL), with or without alectinib (50 nmol/L for H3122-Si-NC and H3122-SiGab1#1, 500 nmol/L for H2228-Si-NC and H2228-SiGab1#1) and metformin (5 mmol/L) for 48 h. The cells were then lysed, and the indicated proteins were detected by western blot analysis. Similar results were obtained in three independent experiments. Ale alectinib, Metf metformin, Con control, NC negative control.

Fig 4: Phosphorylation profiles of Met signaling pathway by wild-type and mutant NK1 or HGF proteins. Serum-starved EHMES-1 cells were stimulated with the recombinant proteins and 5% fetal bovine serum for 10 min. Phosphorylated individual residues of Met receptors, Gab1, Akt, and Erk were detected by Western blotting. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was monitored to ensure equal loading. This experiment was performed once.