Fig. 6.

Cholesterol depletion hyper-activates LTβR-dependent pro-inflammatory response

a, b The concentrations of secreted CXCL8 were measured with ELISA in media collected from cells preincubated for 1 h with MβCD and then stimulated or not for 4 h (a) or 8 h (b) with Ago or LTα1β2 in the presence or absence of MβCD. Data represent the means ± SEM, n = 4. *P ≤ 0.05; **P ≤ 0.01 by Mann-Whitney or Student’s t-test. c Lysates of A549 cells pretreated for 1 h with MβCD and then stimulated or not for 8 h with LTα1β2 or Ago in the presence or absence of MβCD were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control. Graph shows densitometric analysis for ICAM1 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 = 4; ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01 by one sample t-test (in grey) or by Mann-Whitney (in black). d, f Adhesion of Jurkat, NK cells, neutrophils and T lymphocytes to A549 cells (d) and HUVECs (f) treated as in a or e, respectively. Graphs represent quantification of immune cell adhesion to A549 and HUVECs relative to control (untreated) cells. Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n = 3 (d), n ≥ 3 (f); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01 by one sample t-test. e Lysates of HUVECs preincubated for 1 h with MβCD and then stimulated or not for 6 h with LTα1β2 in the presence or absence of MβCD were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control.