Fig 2: DMW effects on mitochondrial metabolic markers of xenograft model of colorectal cancer. (A–C), LDHA, PPAR-α, and PPAR-γ protein levels detected on control and DMW-treated mice. Protein expression was determined after normalisation with α-tubulin or actin as internal control, with ImageJ software and values expressed as arbitrary units (AU). Lane 1 = molecular marker; lanes 2, 3, and 4 = control mice; lanes 5, 6, and 7 = DMW-treated mice. (D), Pyruvate content in tissue from control and DMW-treated mice was expressed as nmol/µg of protein. ** p < 0.01 vs. control mice.

Fig 3: LDHA interaction with NADH propagates ROS.a Primary chondrocytes were treated with IL-1β (10 ng/mL) in the presence or absence of IKK2i (10 μM) or FX11 (40 μM) for 24 h. DCFDA assay was performed and fluorescence was measured by microplate reader to determine intracellular ROS levels, displaying reduction in ROS levels. Results are representative of one experiment out of three, with bars representing mean ± S.D. for n = 8 wells. b, c Primary chondrocytes were treated with IL-1β (10 ng/mL) for 24 h. qPCR was performed to measure gene expression of NOX2 and NOX4. Bars represent mean ± S.E.M. for n = 5 independent experiments. d Primary chondrocytes were treated with IL-1β (10 ng/mL) in the presence or absence of 6-ANA (100 μM) or DPI (1 μM) for 24 h. DCFDA assay was performed and fluorescence was measured by microplate reader to determine intracellular ROS levels, displaying reduction in ROS levels. Results are representative of one of three independent experiments, with bars representing mean ± S.D. for n = 8 wells. e Chondrocytes were treated with IL-1β (10 ng/mL) for 36 h in the presence of absence of FX11 (40 μM). NAD+ and NADH levels were measured in chondrocytes to calculate the NADH:NAD+ ratio in biological replicates. Data are pooled from three experiments for n = 6 biological replicates, with error bars representing mean ± S.D. a, d, e One-way ANOVA was performed followed by Tukey’s multiple comparison’s test. b, c Two-tailed student’s t-test was performed. p-values displayed in figure.

Fig 4: Lactate conversion to pyruvate in AF cells. 13C-lactate tracing to pyruvate in rabbit AF cells in cell culture containing 4mM 3-13C-lactate (a) and in AF tissue extract derived from the rabbit functional spine unit (FSU) culture 3days post injection of 5l of 40mM 13C-lactate into the NP region of the disc (b). Intracellular enrichment of 13C lactate or 13C pyruvate AF cells from AF cell culture or AF tissue of the ex vivo disc organ culture is reported as atomic percent excess (APE) of the total amount of lactate or pyruvate, e.g., 10% APE of pyruvate indicates 10% of total pyruvate contains 13C. Percent (%) APE shown (M+1) indicates that one 13C carbon is present. c, Schematic of gene expression and assembly of lactate dehydrogenase (LDH), the primary enzyme that catalyzes the interconversion of lactate and pyruvate. LDH exists in five isozymes composed of a tetramer of M and H protein subunits encoded by the LDHA and LDHB genes, respectively. LDH5, composed of four M subunits, preferentially converts pyruvate to lactate while LDH1, consisting of four H subunits, preferentially converts lactate to pyruvate. Western blot of LDHM (d) and LDHH (e) in rabbit AF and NP tissue protein extract and their protein levels were quantified by normalizing to -actin as loading control (graphs). Data are means SD of four independent experiments (four rabbits). * p<0.05

Fig 5: Inflammation promotes aerobic glycolysis in chondrocytes.RNA sequencing was performed on primary chondrocytes treated with IL-1β for 24 h and compared to untreated chondrocytes. Volcano plots were generated representing catabolic and anabolic genes (a) and metabolic genes (b). (c, d) Primary sternal chondrocytes were treated with IL-1β (10 ng/mL) in the presence or absence of IKK2i (10 μM) for 72 h prior to performing seahorse assay. All values were normalized to cell viability of treatments relative to untreated cells as measured by MTT assay. c ECAR measurement in glycolysis stress test (Injection 1: No treatment, Injection 2: Glucose, Injection 3: Oligomycin, Injection 4: 2-DG) or d OCR measurement in MitoStress test (Injection 1: No treatment, Injection 2: Oligomycin, Injection 3: FCCP, Injection 4: Antimycin A/Rotenone) was performed on Seahorse Instrument. Measurements were performed every 5 min with per timepoint for each condition. Graphs shown in c, d are mean ± S.D. for 8 wells, representative of one of three independent experiments. (Untreated = Blue, IL-1β = Red, IKK2i = Green, IKK2i + IL-1β = Purple). e Quantification of glycolysis from one timepoint in Glycolysis Stress Test or f–g quantification of basal respiration and maximal respiration for one timepoint each from MitoStress test (mean ± S.D. for n = 8 wells). e–g One-way ANOVA was performed followed by Tukey’s Multiple Comparison’s test, with p-values indicated in figure. h–j IKK2f/f chondrocytes were infected with adenoviral-GFP, or adenoviral-cre (labeled IKK2−/−), then treated with IL-1β (10 ng/mL) for 24 h. qPCR as performed for LDHA, GLUT1, and G6PD2. Data shown is representative of one experiment out of three, with bars representing mean of duplicates. k G6PD activity was measured in primary chondrocytes treated with IL-1β (10 ng/mL) for 24 h compared to untreated cells. Bars are mean ± S.D. for n = 4 biological replicates. Two-tailed unpaired Student’s t-test was utilized for statistical analysis. l Deletion of IKK2 under the conditions described for panels h–j was confirmed by performing qPCR for IKK2, with bars representing mean of duplicates.