Fig. 7: Competitive inhibition of glycolysis by 2-Deoxy-D-Glucose (2-DG) reverses the pathogenic effects of USP32 overexpression in inflammatory chondrocytes.

A Representative western blot and quantitative analysis of USP32, Col2a1, Mmp13, PKM2, Bax, and Cleaved caspase 3 in inflammatory chondrocytes transfected with adUSP32 to overexpress USP32 or adNC for negative control, followed by treatment with 2-DG (5 mM) or vehicle control. B Quantification of protein expression levels (N = 3). For Bax protein expression, which showed non-normal distribution, statistical analysis was performed using the Kruskal-Wallis test followed by Dunn’s post hoc correction for multiple comparisons. C Quantitative RT-qPCR analysis of mRNA expression levels of USP32, PKM2, MMP13, ADAMTs, COL2A1, and BAX across respective groups (N = 3). D Flow cytometric evaluation of apoptosis using Annexin V/PI staining. E TUNEL staining showing apoptotic cells (green) and nuclei (blue, DAPI) with quantitative analysis of TUNEL-positive cells (N = 6) in (F). Scale bar 40 μm. G JC-1 staining to assess mitochondrial membrane potential, with quantitative analysis (N = 6) (H). Scale bar 20 μm. I Representative flow cytometry assessment of JC-1. J Fluorescence intensity changes of ROS levels with quantitative analysis shown in (K). Scale bar 40 μm. L Flow cytometric measurement of intracellular ROS levels. M Measurement of ATP synthesis levels across experimental groups. N Quantification of lactate production in different treatment groups. O Quantification of pyruvate levels in respective groups. Statistical significance is indicated by *P < 0.05, ** P < 0.01, *** P < 0.001.