Fig. 6

ABCA1 regulates Hs294T PM lateral organization, fluidity and order to promote the formation of fully active FAs. A, B Representative FLIM images of cellular PM, which were obtained after staining of scrambled and ABCA1 KO cells (A) or DMSO- and probucol-treated cells (B) with the Di-4 probe. The code color scale represents the average lifetime of Di-4 with 2.9 ns as a minimum (blue) and 4.5 ns as maximum (red). Scale bars reprensent 20 µm. C, D Quantitative analysis of Di-4 mean lifetime for scrambled and ABCA1 KO cells (C) and for DMSO- and probucol-treated cells (D) (n = 30 from three independent experiments, unpaired Student t-test). E, F 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 and ABCA1 KO cells (E) and for DMSO- and probucol-treated cells (F) (n = 30 from three independent experiments, unpaired Student t-tests). G, H Representative confocal images of scrambled and ABCA1 KO cells (G) or DMSO- and probucol-treated (H) cells loaded or not with cholesterol using a methyl-β-cyclodextrin-cholesterol complex (Chol) for 30 min. Upon fixation, the cells were stained with an antibody recognizing pFAK³⁹⁷ followed by a secondary antibody conjugated with Alexa Fluor 488 (green) and an antibody against active integrin β3 followed by a secondary antibody conjugated with Alexa Fluor 647 (red). The region of interest (blue squares) of the cells was magnified (ROI). White arrowheads point at pFAK³⁹⁷-rich active integrin β3 clusters. Scale bars represent 20 µm. I, J Quantification of clusters number of active integrin β3 of scrambled and ABCA1 KO cells (I) or DMSO- and probucol-treated cells (J) (n = 30 from three independent experiments, one-way ANOVA with Sidak’s multiple comparisons test). K, L Quantification of the size of clusters with active integrin β3 of scrambled and ABCA1 KO cells (K) or DMSO- and probucol-treated cells (L) (n = 30 from three independent experiments, one-way ANOVA with Sidak’s multiple comparisons test). Data are mean ± SD. ns non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001