Additional file 1 of Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line
Creators: name: Lončarić, Marija
name: Stojanović, Nikolina
name: Rac-Justament, Anja
name: Coopmans, Kaatje
name: Majhen, Dragomira
name: Humphries, Jonathan D.
name: Humphries, Martin J.
name: Ambriović-Ristov, Andreja
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 1: Fig. S1. KANK1 localization is not affected by talin1 or talin2 knockdown. (A, B) Talin2, but not talin1, knockdown slightly affects KANK1 appearance. Forty-eight hours after transfection with either control, talin1 or talin2-specific siRNA, MDA-MB-435S cells were methanol fixed and stained with anti-talin1 or anti-talin2 antibody followed by Alexa-Fluor 546-conjugated antibody (red) or Alexa-Fluor 488-conjugated antibody (green), respectively. KANK1 was further visualized by anti-KANK1 antibody followed by Alexa-Fluor 555-conjugated antibody or Alexa-Fluor 488-conjugated antibody (shown in magenta). Finally, vinculin was visualized using conjugated anti-vinculin Alexa Fluor 647 antibody (shown in grey) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 µm. (C) KANK1 knockdown does not affect sensitivity to PTX. Sensitivity of cells transfected with either control or KANK1-specific siRNA to PTX was measured by MTT assay. Twenty-four hours upon transfection, cells were seeded in 96-well plates and 24 h later treated with different concentrations of PTX. 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 &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001. Fig. S2. Cell area decreases upon knockdown of talin1, but not talin2. Quantification of data presented in Fig. 2. Violin plot represents measurements of &gt; 30 cells (n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. ns, not significant; *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001. Fig. S3. The CMSC protein liprin-β1 loses its organization upon KANK2, but not upon talin2 knockdown. (A, B) Forty-eight hours after transfection with talin2 or KANK2-specific siRNA, MDA-MB-435S cells were fixed with methanol and stained with anti-liprin-β1 antibody followed by Alexa-Flour 546-conjugated antibody, anti-talin2 antibody followed by Alexa-Fluor IgG2b 488-conjugated antibody (green) and anti-KANK2 antibody followed by Alexa-Flour 647-conjugated antibody and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 µm. Fig. S4. Verification of the MDA-MB-435S and 3αV cell model expressing fluorescently labelled EB3. (A, B) Clone 3αV-EB3 shows decreased expression of talin1, integrin β5 and KANK2 as compared to MDA-MB-435S-EB3 cells. Forty-eight hours after seeding cells were methanol fixed and stained with anti-talin1 followed by Alexa-Fluor 546-conjugated antibody (red), anti-KANK2 antibody or anti-β5 antibody followed by Alexa-Fluor 647-conjugated antibody (magenta) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 µm. (C) Clone 3αV-EB3 demonstrates increased sensitivity to PTX as compared to parental MDA-MB-435S-EB3 cells. Twenty-four hours upon seeding in 96-well plates cells were 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 denotes not significant; * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001 (n = 3). Fig. S5. Knockdown of KANK2 increases talin2-positive FAs size. (A) Forty-eight hours after transfection with either control or KANK2-specific siRNA, MDA-MB-435S cells were methanol fixed and stained with anti-talin2 antibody followed by Alexa-Fluor IgG2b 488-conjugated antibody (green) and anti-KANK2 antibody followed by Alexa-Fluor 647-conjugated antibody (magenta) IRM images were taken. (B) Quantification of data presented in (a). Scatter plot with median marked in red represents measurements of ≥ 30 cells, (n = 2). Data were analyzed by unpaired Student’s t-test. ns, not significant; *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001. Fig. S6. Full images of the blots in Fig. 2A. Images were obtained using Uvitec Alliance Q9 mini, which directly scanned membranes developed with ECL reagents. Fig. S7. Full images of the blots in Fig. 3C. Images were obtained using Uvitec Alliance Q9 mini, which directly scanned membranes developed with ECL reagents.