Fig. 4: Replenishment of NO rescues the defective HIF-1α activation and its related glycolysis in MDM2 knockdown macrophages. | Nature Communications

Fig. 4: Replenishment of NO rescues the defective HIF-1α activation and its related glycolysis in MDM2 knockdown macrophages.

From: MDM2 induces pro-inflammatory and glycolytic responses in M1 macrophages by integrating iNOS-nitric oxide and HIF-1α pathways in mice

Fig. 4

Mouse BMDM were transfected with siMdm2 or siScramble for 24 h, then stimulated with LPS and IFNγ for 20 h, followed by treatment with NO donor S-Nitroso-N-acetylpenicillamine (SNAP) or DMSO for 3 h. a Total nitrate and nitrite in the cell culture medium. n = 4. b Nuclear HIF-1α transcriptional activity in the BMDM. n = 4. c The treated cells were subjected to S-nitrosylation assay, followed by immunoprecipitation (IP) of HIF-1α. Immunoblotting analysis of HIF-1α in the nuclear extract and S-nitrosylated HIF-1α (labeled by iodoTMT Reagent) was performed using an HIF-1α antibody and anti-TMT antibody respectively. Lamin B1 is a nuclear protein and used as loading control. d GlycoPER was measured using Seahorse XF analyzer. The right panel is the area under curve (AUC). n = 5. P values of siScramble-LPS + IFNγ vs siMdm2-LPS + IFNγ are under the curve while siMdm2-LPS + IFNγ vs siMdm2-SNAP + LPS + IFNγ is above and highlighted in yellow. e Lactate in the cell culture supernatant. n = 4. f Relative mRNA levels of glycolytic genes normalized with β-actin and expressed as fold change over siScramble-LPS + IFNγ. n = 4. 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. Representative immunoblot images are shown from at least two independent experiments.

Back to article page