NRCMs were treated for 24 h with 1 µM TMG to induce protein O-GlcNAcylation. During XF analysis NRCMs were permeabilized and provided with succinate (to support Complex II-dependent respiration), as well as Rot (to inhibit Complex I activity). (A) XF assay of Complex II-dependent respiration: following three baseline OCR measurements in MAS buffer, the permeabilization agent, saponin, and succinate + Rot were injected. After two measurements, oligomycin (Oligo) and AA + Rot (AA–Rot) were injected sequentially, with two measurements recorded after each injection. (B) State 3 OCR: the AA + Rot rate was subtracted from the succinate-stimulated rate to determine the State 3 rates. (C) State 4o OCR: the AA + Rot rate was subtracted from the oligomycin rate to obtain State 4o rates. (D) RCR: State 3/State 4o. As indicated in bars, n = 7 independent experiments; *P < 0.05.