Fig. 5

Disruption of crosstalk between CMSCs and FAs leads to increased velocity of microtubule growth and increased sensitivity to PTX. A Knockdown of αV integrin subunit increases velocity of microtubule growth. Quantification of time-lapse live cell microscopy data of 435S-EB3 cells and 3αV-EB3 cell clone with decreased expression of integrin αV. Violin plot represents measurements of > 450 analyzed microtubules, (n = 3) relative to velocity of MT growth in MDA-MB-435S cells that was set as 1. Data were analyzed by unpaired Student’s t-test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Still images of Additional file 2 : Movie S1 and Additional file 3: S2 represent tracking of one microtubule tip in 104 s through five frames. (B) 435S-EB3 cells transfected with either talin1, talin2, KANK2 or β5-specific siRNA, but not KANK1 siRNA, showed a significant increase in velocity of MT growth compared to cells transfected with control siRNA. Quantification of time-lapse live cell microscopy data. Violin plot represents measurement of > 250 analyzed microtubules, (n ≥ 2) relative to velocity of MT growth in MDA-MB-435S cells transfected with control siRNA that was set as 1. Data were analyzed by one-way ANOVA with Šídák’s multiple comparisons test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. C PTX treatment decreases MT growth velocity much stronger in 3αV-EB3 cells compared to 435S-EB3 cells. Quantification of time-lapse live cell microscopy data of 435S-EB3 cells and 3αV-EB3 clone upon treatment with equitoxic and equimolar concentrations of PTX. Scatter plot with median marked in red represents measurements of > 200 analyzed microtubules, (n = 3) relative to velocity of MT growth in MDA-MB-435S cells that was set as 1. Data were analyzed by one-way ANOVA with Šídák’s multiple comparisons test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. D Cell proliferation decreases upon knockdown of talin1, but not talin2, KANK1 or KANK2. Cell proliferation was measured using ClickIT EdU assay upon transfection with either control, talin1, talin2, combination of talin1 and talin2, KANK1 or KANK2-specific siRNA. Histogram represents measurements of > 30 cells, plotted as mean ± SD (n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. E Talin2 knockdown increases sensitivity to PTX in MDA-MB-435S cells. Twenty-four hours upon transfection, cells were seeded in 96-well plates and 24 h later treated with different concentrations of PTX. Cytotoxicity was measured by MTT assay. Data were analyzed by two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test, with a single pooled variance. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (n = 3). F Talin2 knockdown decreases migration in MDA-MB-435S cells. Serum starved (24 h) cells, transfected previously with either control or talin2-specific siRNA were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 µm. F Histogram data represents averages of five microscope fields of three independently performed experiments, plotted as mean ± SD. Data were analyzed by unpaired Student’s t-test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001