Fig 2: Pharmacological inhibition or genetic deficiency of Cav-1 do not impair BCG uptake by G-MDSC and M-MDSC. (A) MDSCs were left untreated or pretreated or not with Cytochalasin D for 1 h and then incubated with BCG-GFP at MOI 2 for 6 h. Cells were then analyzed by flow cytometry for % BCG uptake by GFP detection (gated by dotted line) in G-MDSC and M-MDSC subsets by gates shown in Figure 1A. Dotted lines indicate gating for positive detection of uptake by GFP fluorescence. (B) As in A but several pooled experiments are shown and MDSCs were incubated with cytochalasin-D, filipin III, simvastatin or ß-cyclodextrine for 1 h and then stimulated with BCG-GFP at MOI of 2, 5, or 10 for 6 h. (C,D) MDSCs of WT and Cav1-/- mice were incubated with BCG-GFP at MOI 2 for 6 h. Cells were then analyzed by flow cytometry for BCG uptake by GFP detection in G-MDSC and M-MDSC subsets by gating as shown in Figure 1A. Pooled and normalized data of several experiments as performed for (A). (E) MDSCs were stimulated with BCG-GFP at 1 MOI for 16 h. Cytospins were then stained for Cav-1 and DAPI and analyzed by confocal microcopy. G-MDSC and M-MDSCs were defined on the basis of their nuclear shape. Scale bars upper row 7 µm, all other rows 10 µm. (F) Pearson's correlation coefficients for colocalization (“overlap”) of BCG and Cav-1 from n = 5 independent experiments like shown in (E). Data are from ****P < 0.0001; **P < 0.01; ns, not significant unpaired, two-tailed, student's t-test.

Fig 3: Western blot analysis of Tubulin, GNAI2, and CAV1 proteins in cytoplasmic (Cytosol.), non-detergent-resistant membrane (Non-DRMs), and DRM preparations collected from ARPE19 cell homogenates during successive detergent extraction. Five micrograms of proteins from each preparation were loaded on SDS-PAGE, separated, electro-transferred, and immunoblotted with antibodies against the indicated proteins. The micrographs presented were representative of three independent experiments.

Fig 4: Characterization of drug-resistant MCF/DOX and HL-60/HAR cells. (A) ABCB1, p53 and CAV-1 expression levels in both cell lines were detected via western blotting. Accumulations of rhodamine 123 in (B) MCF-7/DOX, MCF-7, (C) HL-60 and HL-60/HAR both cell lines as determined using flow cytometry. One of representative results from three independent experiments is presented. ABCB1, ATP-binding cassette subfamily B member 1; CAV-1, caveolin-1; Rho 123, rhodamine 123; DOX, doxorubicin; HAR, harringtonine.

Fig 5: Expression of mRNA of Cav-1α and Cav-1β in the CAFs, divided into the tumor stages T1 to T4. The expression of the Cav-1α isoform was lower than the expression of Cav-1β trough all tumor stages. With higher tumor stages, there was a slight trend for higher Cav-1α expression, while Cav-1β expression was heterogenous throughout the different tumor stages. Data is shown as box and whisker plots with median and 10 to 90 percentiles, and Kruskal-Wallis test was used for comparison between expression in CAFs of different tumor stages, n = 19. * p < 0.05, ** p < 0.01, *** p ≤ 0.001