Fig. 4: TKS4/5 promote shedding of MT1-MMP.
From: Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding
a HeLa cells were co-transfected with TKS4-GFP or TKS5-GFP together with mCh-MT1-MMP wt or ΔC, lysed and subjected to WB. b Quantification of WB from (a) representing the relative intensities of the 34 kDa cleavage product of MT1-MMP from n = 3 independent experiments, mean +/− SEM. One sample two-sided t-test. c Illustration showing autocatalytic and non-autocatalytic cleavage of mCh-MT1-MMP and the resulting fragments. Created with BioRender.com. d HeLa cells were co-transfected with mCh-MT1-MMP wt and GFP, TKS4-GFP wt or the double mutant of TKS4 (µµ). Total cell lysates were analysed by WB, one representative WB is shown in Supplementary Fig. 7b. The graph shows the relative intensities of the 34 kDa cleavage product of MT1-MMP. Data is from n = 6 independent experiments, mean +/− SD. One sample two-sided t-test. e Rescue experiment: HeLa cells were depleted for TKS4 and then transfected with GFP, TKS4-GFP or an siRNA resistant version of TKS4-GFP (TKS4-GFPsiRES) together with mCh-MT1-MMP. Depletion of TKS4 reduces the amount of cleaved MT1-MMP, while overexpression of TKS4 increases it (one representative WB is shown in Supplementary Fig. 7c). An siRNA resistant construct of TKS4 rescues the siRNA-mediated effect on cleavage. The graph shows the relative intensities of the 34 kDa cleavage product of MT1-MMP. Data is from n = 3 independent experiments, mean +/− SEM. One sample two-sided t-test. f MDA-MB-231-TKS5-GFP-mCh-MT1-MMP wt cells were transfected with siRNA targeting TKS4 and TKS5 for 4 days. The whole cell lysate (WCL) and the spin-column concentrated conditioned medium (CM) were analysed by WB with anti-mCh (red) and anti-MT1-MMP-catalytic domain (green) antibodies. Representative of 4 WB. * MT1-MMP variants likely comprising processed forms of MT1-MMP and mCh-MT1-MMP (immature, posttranslationally modified or cleaved). Illustration created with BioRender.com. g MDA-MB-231-MT1-MMP (untagged) or MDA-MB-231-TKS5-GFP-MT1-MMP (untagged) cells were transfected with siRNA targeting TKS4 and TKS5 for 4 days. The spin-column concentrated CM was analysed by WB with an anti-MT1-MMP-catalytic domain antibody. Graph shows the quantification of the 50–52 kDa band from CM of the indicated cell lines. Data from n = 3 independent experiments, mean +/−SD. One sample two-sided t-test.
