Fig. 3: THEMIS2 was associated with MET and promoted its phosphorylation, and MET was required for THEMIS2-mediated sphere formation and CSC marker expression.

A The protein levels of p-MET, MET, and THEMIS2 in MDA-MB-231 cells transfected with control or THEMIS2 siRNA and with or without HGF (30 ng/mL) stimulation were analyzed by immunoblotting. Actin was used as the internal control. B The protein levels of p-MET, MET, and THEMIS2 in MDA-MB-231cells transfected with control or pCMV6-THEMIS2 vector and with or without HGF (30 ng/mL) stimulation were similarly analyzed. Actin was used as an internal control. C MDA-MB-231 cells were transfected with control or THEMIS2 siRNA for 8 h, serum starved for 24 h, and then stimulated with 30 ng/mL HGF for the indicated times. D Immunofluorescence staining images of MET (green) and THEMIS2 (red) as indicated by the arrows, shown under confocal microscopy. Nuclei were counterstained with DAPI (blue). E MDA-MB-231-IV2 (IV2) cells lysates were immunoblotted directly or subjected to IP with the indicated antibodies first, followed by blotting with the indicated antibodies. IgG served as the negative control. F Protein–protein interactions between THEMIS2 and MET using Duolink PLA. The close proximity (<40 nm) of the two proteins is indicated by red fluorescent dots. Nuclei were counterstained with DAPI (blue). G Protein expression levels of ALDH1, CD133 p-MET, MET and THEMIS2 in Hs578T and BT549 cells transfected with MET-siRNAs or the indicated plasmids were analyzed by immunoblotting. Actin was used as the internal control. H Ectopic expression of THEMIS2 reversed the inhibition of the sphere-forming abilities due to MET knockdown. The histograms represent means ± SEs from three independent experiments (*P < 0.05; **P < 0.01).