2.

Assessment of the ability of MA-diyne to metabolically label proteins. (A) Mouse primary chondrocytes were incubated with the indicated concentration of MA-diyne for 6 h. The cell lysates were then clicked with IR680-azide followed by in-gel fluorescence analysis. (B) Mouse primary chondrocytes were incubated with 100 μM MA-diyne for the indicated time points. Cell lysates were then clicked with IR680-azide and in-gel fluorescence analyses. β actin was used as a loading control. (C) Confocal microscopic fluorescence image depicting the ready uptake of the MA-diyne into the cells and subcellular localization of malonylated proteins in the primary chondrocytes. The primary chondrocytes grown on coverslips were labeled with 100 μM MA-diyne for 6 h and then subjected to click chemistry with Carboxyrhodamine 110 Azide (fluorescein isothiocyanate-FITC tag) after fixing and permeabilization. Scale bar: 50 μM. (D) Box plot showing the concentration of intracellular malonyl-CoA in the cells after incubation with different concentrations of MA-diyne for 4 h. n = 4. Data are presented as mean ± SEM. Three group comparisons were evaluated using two-way ANOVA. Significance is noted as ns p > 0.05, *p < 0.05, and **p < 0.01.