Additional file 2 of ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization
Creators: name: Wu, Ambroise
name: Mazurkiewicz, Ewa
name: Donizy, Piotr
name: Kotowski, Krzysztof
name: Pieniazek, Małgorzata
name: Mazur, Antonina J.
name: Czogalla, Aleksander
name: Trombik, Tomasz
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Science Policy
subject: Developmental Biology
subject: Cancer

description: Additional file 2: Figure S1. ABCA1 level is inversely proportional to cholesterol amount in melanoma cell lines. A Representative immunoblots of ABCA1 of WM1341D, SK-Mel-28, A375, WM9 and Hs294T cells. Vinculin was used as a loading control. B Quantification of ABCA1 level from. One-way ANOVA with Sidak’s multiple comparisons test was used for statistical analyses and only the statistical comparisons between Hs294T and the other cells lines are presented. C Total cholesterol quantitative measurement assay of WM1341D, SK-Mel-28, A375, WM9 and Hs294T cells. 5 μg of protein were used for each cell type. Data are mean ± SD. ns: non-significant, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Figure S2. ABCA1 activity is promoting migration and invasion ability in WM1341D and WM9 cell lines. A, B Calculated covered distances of WM1341Dor WM9DMSO- and probucol-treated cells after 72h. C, D Relative invasiveness of WM1341Dor WM9DMSO- and probucol-treated cells expressed as fold change of DMSO-treated cells after 24h. Data are mean ± SD. **p ≤ 0.01 and ****p ≤ 0.0001. Figure S3. ABCA1 activity does not influence MMP-2 and MMP-9 degradation abilities. A, B Representative gelatin gels of zymography assay for MMP-2 and MMP-9 activity assessment of conditioned medium from scrambled and ABCA1 KO cellsor from DMSO- and probucol-treated cells. The same amount of samples was loaded in SDS-PAGE in parallel and gels were stained with Coomassie for total protein visualization and used as a loading control for gelatin gels. Bands on the left of Coomassie-stained gels represent the size protein ladder. C, D Quantification of the MMP-2 and MMP-9 activity expressed as % of active MMPs per Pro-MMPs of conditioned medium from scrambled and ABCA1 KO cellsfromor of conditioned medium from DMSO- and probucol-treated cellsfrom. Data are mean ± SD. ns: non-significant. Figure S4. ABCA1 activity does not influence α-parvin localization and level neither cell spreading. A, B Representative confocal images of scrambled and ABCA1 KO cellsor DMSO- and probucol-treated cellsstained with an antibody recognizing α-parvin followed by a secondary antibody conjugated with Alexa Fluor 488and antibody recognizing active integrin β3 followed by secondary antibody conjugated with Alexa Fluor 647. Scale bars represent 20 µm. C, D Magnified view of scrambledand ABCA1 KOcellsfromor ofDMSO-and probucol-treatedcellsfrom. White arrowheads represent active integrin β3 and α-parvin-rich FAs and magenta arrowheads point at active integrin β3-poor and α-parvin-rich FAs. Scale bars represent 20 µm. E and H Representative immunoblots of α-parvin levels in scrambled and ABCA1 KO cellsor DMSO- and probucol-treated cells. β-actin was used as a loading control. F and I Quantification of α-parvin relative level in scrambled and ABCA1 KO cellsfromor of DMSO- and probucol-treated cellsfrom. Unpaired Student t-test were used for statistical analyses. G and J Quantification of the spreading area of scrambled and ABCA1 KO cellsor DMSO- and probucol-treated cells. Data are mean ± SD. ns: non-significant. Figure S5. ABCA1 activity impacts cells’ PM lateral organization. A, B svFCS diffusion laws of fluorescently labelled sphingomyelinfor scrambledand ABCA1 KOcellsor for DMSO-and probucol-treatedcells. Figure S6. Cholesterol loading disrupts cells’ PM order and pFAK397 stable docking to active integrin β3 clusters. A, B Quantitative analysis of Di-4 mean a lifetime of scrambled, scrambled loaded with cholesteroland ABCA1 KO cellsor of DMSO-, DMSO loaded with cholesterol-and probucol-treated cells. C, D Distribution represented as % of Di-4 intensity lifetime separated into two segments: Di-4 intensity with a lifetime between 3 and 4 ns [3, 4] and between 4 and 5 ns [4, 5] for scrambled, scrambled loaded with cholesteroland ABCA1 KO cellsor for DMSO-, DMSO-treated and loaded with cholesteroland probucol-treated cells. E, F Quantification from confocal imagesof the percentage of cells with active integrin β3 clusters enriched with pFAK397 of scrambled, scrambled loaded with cholesteroland ABCA1 KO cellsor DMSO-, DMSO loaded with cholesterol-and probucol-treated cells. Data are mean ± SD. ns: non-significant, *p ≤ 0.05, ***p ≤ 0.001 and ****p ≤ 0.0001.