Figure 4. LPS affects C5aR1 expression and PVL susceptibility in FBXO11-deficient macrophages.

(A) Schematic illustration of THP1 PMA–induced differentiation and LPS treatment. (B) Western blot analysis of C5aR1 in WT and FBXO11^−/− macrophages, treated with or without LPS for 24 h. Protein abundance was normalised to β-actin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05; by two-way ANOVA with Sidak’s multiple comparisons test. (C) qRT-PCR analysis of relative C5aR1 mRNA level, comparing the LPS-treated and -untreated macrophages. The mRNA levels were normalised to the control values of GAPDH, and fold change was LPS-treated cells that were compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Flow cytometric analysis and MFI of C5aR1 in LPS-treated or -untreated WT and FBXO11^−/− macrophages. Grey line represents isotype, black line represents untreated, and red line represents LPS-treated macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated or -untreated WT and FBXO11^−/− macrophages treated with PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 for + PVL versus + PVL + LPS at 14 h post toxin treatment; by unpaired t test.

Source data are available for this figure.