Fig. 2

Cholesterol depletion by MβCD enhances LTβR-triggered activity of the NF-κB pathway and reduces internalization of the ligand-bound receptor. a, b Lysates of A549 cells preincubated for 1 h with MβCD or vehicle and stimulated for 0.5, 1 or 4 h with LTβR agonist (Ago) were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control. Graphs show densitometric analysis for the indicated proteins from Western blotting (protein levels normalized to vinculin). Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n = 6 (a), n = 4 (b); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01 by one sample t-test (in grey) and Student’s t-test (in black). c Immunofluorescence staining of ligand-bound LTβR and EEA1 in A549 cells upon 0.5 h stimulation with LTβR agonist in cells preincubated with vehicle (Veh.) or MβCD. Insets: magnified views of boxed regions in the main images. Scale bars, 20 μm. d Analysis of integral intensity and the number of LTβR- and EEA1-positive vesicles in cells treated as in B. Values are presented as fold change versus control - vehicle-treated cells marked as a black line, set as 1. Data represent the means ± SEM, n = 3. ns - P > 0.05; *P ≤ 0.05 by one sample t-test