Fig. 2: Knockdown of MDM2 expression dampens glycolysis and HIF-1α activation in mouse M1 macrophages.

BMDM were transfected with siMdm2 or siScramble, followed by with LPS and IFNγ for 20 h. a Glycolytic stress test was performed using Seahorse XF analyzer. Rotenone/Antimycin A (Rot/AA) and 2-deoxyglucose (2-DG) were added to inhibit mitochondrial functions and glycolysis respectively. Glycolytic proton efflux rate (GlycoPER) was measured at the basal condition and during the treatments as indicated. n = 4. P values of siScramble-LPS + IFNγ vs siMdm2-LPS + IFNγ are above the curve and highlighted in yellow while siScramble-veh vs siMdm2-veh are below the curve. b Lactate level in the conditioned medium. n = 4 biological replicates. c, d Relative abundance of metabolites in (c) glycolysis and (d) pentose phosphate pathway determined by UPLC-MS/MS. n = 5. e Immunoblotting analysis of HIF-1α, hydroxyl-HIF-1α and β-actin. n = 3. f HIF-1α transcriptional activity in the nuclear fraction of the BMDM. n = 4. g QPCR analysis of the glycolytic genes normalized with β-actin. n = 8 for Glut1, Ldha, Mct4 and Pkm2. n = 4 for Hif1a. Data are displayed as mean ± SEM. Statistical significance was examined using one-way ANOVA with Tukey post-hoc test. N number represents the number of biological replicates.