Fig. 6

Effect of TSPAN3 on β1 integrin recycling. A Coimmunoprecipitation assays showing the interaction between exogenous tetraspanin 3 (TSPAN3/T3) and endogenous Rab11a in A549 cells. A549 cells were transfected with pCMV6-Myc-FLAG-TSPAN3 constructs. After 48 h of transfection, cell lysates were immunoprecipitated with anti-FLAG antibodies or control IgG and subjected to western blotting with anti-Rab11a and anti-FLAG antibodies. B Coimmunoprecipitation assay showing the interaction between endogenous TSPAN3 and Rab11a in A549 cells C Immunofluorescence showing the localization of TSPAN3 (TRITC) with Rab11a (FITC) in the cytoplasm of A549 cells. Magnification ×400. D Western blot indicating that the interaction between TSPAN3 and Rab11a does not depend on the LEL domain of TSPAN3. After 48 h of transfection for wild-type GFP-TSPAN3 or TSPAN3 mutant, cell lysates were immunoprecipitated with the anti-GFP antibody; the presence of Rab11a in the anti-GFP-precipitated complex was examined through western blotting with the anti-Rab11a antibody. E Immunofluorescence showing the colocalization of TSPAN3 (TRITC) and Rab11a (FITC) in A549 cells at different time points after nocodazole (NZ) washout (magnification ×400). F Pearson correlation coefficient of the colocalization between Rab11a and β1 integrin at 60 min after NZ washout. G Immunofluorescence showed that TSPAN3 knockdown (si-T3) resulted in reduced colocalization of β1 integrin and Rab11a (60 min after NZ washout) and the surface expression of β1 integrin (120 min, indicated by arrowheads). Magnification ×400. H Immunofluorescence showed that TSPAN3 knockdown (si-T3) resulted in increased colocalization of β1 integrin and LAMP1 at random time without NZ washout. Magnification ×400. I Pearson correlation coefficient of the colocalization between LAMP1 and β1 integrin at random time without NZ washout. J Western blot indicating that Rab11a knockdown attenuated the TSPAN3-induced increase in β1 integrin, p-FAK (Tyr397), p-MEK, p-ERK, cyclin B1, and cyclin D1 expression. GAPDH served as the loading control (n = 3 independent experiments). K Schematic of the mechanism of TSPAN3 regulating β1 integrin intracellular recycling, TSPAN3 promotes β1 integrin intracellular recycling by facilitating β1 integrin sorting into Rab11a recycling endosomes, which reduces β1 integrin lysosomal degradation and enables more rapid resurface of integrin