A3672

For animal experiments, 28 female Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and maintained according to the protocol approved by the Ethics Committee of the Hospital of Stomatology at Wuhan University (S07921060J). Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. In total, 28 SD rats were randomly divided into four groups (i.e. A, B, C, D). The submucosal injection method on the right cheek was used in this experiment. Arecoline (S2614, Selleck, USA), LSKL (HY-P0299, MCE, USA) and SLLK (HY-P0301, MCE, USA) were dissolved in DMSO (A3672, Applichem, GER) to 10 mg/mL for storage and were diluted to 2 mg/mL in 0.9% normal saline (NS) immediately before injection. To establish the OSF lesion (n = 7), the rats in group A were injected with 20% DMSO and the rats in group B were injected with Arecoline. As for drug administration assay (n = 7), the rats in group C were injected with SLLK and the rats in group D were injected with LSKL. All groups were injected twice a week and with 100 μL each time. The mouth opening of animals were measured once a week. After 12 weeks, the rats were humanely euthanized, and the buccal tissue was attained for further research.

Temozolomide (TMZ, T2577) and neocarzinostatin (NCS, N9162) were both purchased from Sigma-Aldrich. Calpains were inhibited using 50 µM PD150606 (Calbiochem, 513,022). 20 µM Q-VD-OPh (Sigma-Aldrich, SML0063) were used to inhibit caspases; 100 µM Nec-1 (Sigma-Aldrich, N9037) to inhibit necroptosis. We used 100 nM of the BH3 mimetic ABT-737 (Selleckchem) and 1 µM of the Mcl-1 inhibitor S63845 (Selleckchem) to induce apoptosis. Except for NCS, all chemicals were dissolved in DMSO (A3672, AppliChem). NCS is delivered in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 5.5 by the manufacturer.

The following ETC inhibitors, reagents and small molecules were used for cell treatment throughout the study (final concentrations are provided in the respective figures or figure legends): Antimycin A dissolved in DMSO (A8674, Sigma Aldrich (different lots); sc-202467, Santa Cruz; ab141904, Abcam), Rotenone dissolved in DMSO (R8875, Sigma Aldrich), Oligomycin dissolved in DMSO (O4876, Sigma Aldrich), trans-ISRIB dissolved in DMSO (16258, Cayman Chemical), Salubrinal dissolved in DMSO (SML0951, Sigma Aldrich), Celastrol dissolved in DMSO (C0869, Sigma Aldrich), Geranylgeranylacetone dissolved in DMSO (G5048, Sigma Aldrich), CDDO Methyl Ester dissolved in DMSO (SMB00376, Sigma Aldrich), N-Acetyl-L-cysteine dissolved in H₂O (A7250, Sigma Aldrich), Sodium pyruvate dissolved in H₂O (P5280, Sigma Aldrich), L-Aspartic acid dissolved in 1 M HCl (A7219, Sigma Aldrich), H₂O₂ diluted in PBS (9681, Carl Roth), DMSO (A3672, AppliChem).