Fig. 6

Pharmacological inhibition of MAP4 kinases with P/12k increases CLSTN1 surface expression and accumulation in cell-cell contacts. (A) Upper: IB of cell fractionation analysis of CLSTN1 subcellular localization in UW228 cells. Antibodies against proteins indicated to the right of the panels were used. CE: cytoplasmic extraction; ME: membrane extraction; NE: nuclear extraction. Lower: Quantification of relative protein expression levels in the different compartments. (B) FC analysis of CLSTN1 surface expression on UW228 cells treated with 0.5 µM P/12k for 24h. (C) Quantification of relative CLSTN1 surface expression from FC analysis shown in B and estimation plot of n = 3 independent experiments. (D) IFA analysis of endogenous CLSTN1 expression in DMSO-treated (24h) UW228-LA-EGFP-mCherry-Nuc cells co-cultured with iNHA. Red: CLSTNA1, green: actin (LA-EGFP), blue: Nuclei (mCherry-Nuc). Inverted greyscale images of the green-fluorescent signal (LA-EGFP, middle) and the red-fluorescent signal (CLSTN1, right) are shown. The scale bar is 20 µm. The letter “A” indicates the positions of iNHA cells. Magnifications in a) and b) indicate cell-cell contacts between tumor cells. (E) IFA analysis of CLSTN1 expression of UW228-LA-EGFP-mCherry-Nuc cells co-cultured with iNHAs and treated for 24 h with 0.5 µM P/12k. Red: CLSTN1, green: actin (LA-EGFP), blue: Nuclei (mCherry-Nuc). The letters “A” indicate the positions of iNHAs. Arrows indicate tumor-iNHA contacts. Magnifications in c) and d) indicate cell-cell contacts between tumor cells. (F) Magnifications of tumor-iNHA cell-cell contacts in DMSO- (e and f) or – P/12k- treated samples (g and h). The letter “A” indicates the positions of iNHA cells. (G) Left IFA images: Representative images of heterotypic cell-cell contacts. Dotted squares indicate representative measuring zones across cell-cell contacts. Four representative plots of the calculated % of the total measured grey value of the whole area at the positions indicated on the x-axis are shown to the right of the IFA images. Right: Plot of the % total grey values of a 10 µm long line across all detected cell-cell contacts in representative images. Mean and 95% confidence intervals of measured values at indicated positions are shown. Monoculture: N = 16, co-culture: N = 16. P values were calculated using multiple unpaired t-tests.