Fig 2: ALAHM increased HGF expression by binding to the transcription factor AUF1After overexpression or knockdown of ALAHM in L02 cells, HGF expression levels of mRNA and protein were detected by real-time RT-PCR and western blot, respectively (A and B). The differential expression of ALAHM in the cytoplasm and nucleus of L02 cells was detected by real-time RT-PCR, which showed that ALAHM was preferentially localized in the cytoplasm (C). RNA FISH was performed to verify the localization of ALAHM in L02 cells and confirmed that expression was mainly localized in the cytoplasm (D). A structure diagram of ALAHM is shown in (E), which was obtained from RNAfold Web Server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). The results of RNA pull-down and RIP experiments are shown in (F) and (G), respectively. Bar graphs represent ratios plotted as mean ± SD of three separate experiments. Compared with the control group, ∗∗p < 0.01.

Fig 3: 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 4: Purification of CM1021. A. Elution of CM1021 from protein A column. Inset shows the course of the entire binding and elution. B. Chromatography of the protein A eluate on Superdex 200. C. SDS PAGE of purified protein ±2.5 mM TCEP. D. SDS PAGE of purified protein ±2.5 mM TCEP plus 80 °C for 10 min. E. Immunoblot of partially reduced and fully reduced CM1021 against an antibody to the K1 fragment of HGF. F. Immunoblot of partially and fully reduced CM1021 against an antibody to human IgG1 Fc.

Fig 5: The HGF/MET signalling pathway contributes to resistance to alectinib in ALK-positive lung cancer cells.a The concentrations of HGF in the H3122 and H2228 cell culture supernatants with or without alectinib (50 nmol/L) or crizotinib (100 nmol/L) administration were determined by ELISA. The data are presented as the mean ± SD, and the experiment was repeated three times. *p < 0.05; **p < 0.001. b H3122 and H2228 cells were treated with alectinib (50 nmol/L) or crizotinib (100 nmol/L) for the indicated amounts of time, and MET phosphorylation was measured by western blotting. c, d H3122 and H2228 cells were transfected with HGF, and the expression of HGF and the phosphorylation levels of MET were determined by western blotting. Vec negative control vector. e Cell proliferation in crizotinib-treated and alectinib-treated cells with or without HGF was examined by MTT assay; the IC50 values for crizotinib and alectinib were calculated. The data are presented as the mean ± SD, and the experiment was repeated three times. **p < 0.001. Cri crizotinib, Ale alectinib, Vec negative control vector. f H3122-AR1-6 cells are resistant to alectinib. The alectinib IC50 values of H3122 and H3122-AR cells were detected by MTT assay. The data are presented as the mean ± SD from three independent experiments. **p < 0.001 compared with H3122 cells. g Detection of HGF in the cell culture supernatants of H3122 and H3122-AR cells by ELISA. The data are presented as the mean ± SD from three independent experiments. Asterisks (*) indicates p < 0.05 compared with parental H3122 cells; **p < 0.001 compared with parental H3122 cells. h Immunoblotting analysis of ALK, MET, and the phosphorylation of MET in H3122 and H3122-AR cell lines.