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. 2020 Aug 16;1:62–68. doi: 10.1016/j.crmicr.2020.08.001

Fig. 3.

Fig 3

Efficiency and specificity of chemical inhibitors blocking different endocytic pathways. Confocal micrographs showing inhibition of internalization of transferrin, FITC-CTxB and FITC-dextran following treatment with (A) CPZ (15 μM), (C) Mβ-CD (5 mM) and (E) EIPA (50 μM), respectively. Dynasore (100 μM) treatment blocked entry of both (G) transferrin and (I) CtxB. hBMECs were allowed to internalize transferrin (100 ng/ml, for 2 min), FITC-CTxB (500 ng/ml, 15 min) or FITC-Dextran (1 mg/ml, 15 min) following treatment with inhibitors. Fluorescence images were captured following fixation and incubation of cells with necessary primary and secondary Ab and representative images are shown. Scale bar, 5 μm. Total fluorescence intensity of internalized transferrin (B, H), FITC-CTxB (D, J) and FITC-dextran (F) in presence or absence of non-cytotoxic concentrations of CPZ (B), Mβ-CD (D), EIPA (F) and Dynasore (H, J) were calculated from fluorescence images (n ≥ 200 cells per coverslip in triplicate) of cells following fixation at indicated time points and incubation with necessary primary and secondary Ab. Data are presented as mean ± SEM of triplicate experiments. Statistical tests were performed using unpaired Student's t-test, ***p < 0.001. (K) Confocal micrographs depicting specificity of the endocytic inhibitors. hBMECs were treated with combinations of inhibitors to block multiple endocytosis pathways leaving a single port of entry open. The following inhibitor combinations were used: Dynasore (Blocked pathways: clathrin and caveolae; operating pathway: dynamin independent pathways); CPZ + EIPA (Blocked pathways: clathrin and dymanin independent pathways; operating pathway: caveolae) and MβCD + EIPA (Blocked pathways: caveolae and dynamin independent pathways; operating pathway: clathrin). Cells were treated with designated endocytic inhibitors at the earlier determined concentration for the desired time period as described above. Fluorescence images (n ≥ 50) were captured following fixation and incubation of cells with necessary primary and secondary Ab and representative images are shown. Scale bar, 5 μm.