Fig. 3: OPA1 deletion impairs macrophage polarization.
From: OPA1 drives macrophage metabolism and functional commitment via p65 signaling
OPA1f/f and OPA1M/M BMDMs were differentiated with MCSF (40 ng/mL) for 7 days, then BMDMs were stimulated with: M0: MCSF (10 ng/mL); M1: LPS (500 ng/mL) and IFNγ (25 ng/mL); M2: IL-4 (20 ng/mL) for 24 h. Relative gene expression for M1 polarization markers (A) Il6, (B) Tnf and C Nos2. Rplp0 was used as housekeeping in all the experiments (n = 5). D IL6 and E TNFα protein levels were measured by ELISA in cell culture supernatants upon M1 polarization (n = 4). F Nitric Oxide (NO) level quantification in M1 macrophages measured by DAR-4M AM staining. Relative gene expression for M2 markers (G) Mrc1 (H) Arg1 and I Retnla by real-time RT-PCR. Rplp0 was used as housekeeping in all the experiments (n = 5). J Representative western blot and K quantification for NOS2 and ARG1 of total proteins (n = 5). GAPDH was used as a loading control. Inflammasome activation was performed in M0 macrophages that were primed by 100 ng/ml LPS for 4 h and activated by ATP (5 mM, for the last 15 min) (L) ELISA of IL1β in the supernatants (n = 4), no stimulated (NS) cells were used as a control. Data are presented as mean ± SEM. Statistical analysis was performed by Unpaired nonparametric t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
