Gapdh

Total RNA was isolated from the cells using TRIzol Reagent (Thermo Scientific, MA). qPCR analysis was performed using standard procedures on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA). PCR reactions were performed in triplicate and the relative amount of cDNA was calculated by the comparative CT method using RPS21 as an endogenous control. RT-PCR was performed in at least three biological replicates. The Taqman probe for NQO1 (Cat: HP101580), PRDX1 (Cat: HP101099), TXN (Cat: HP100418), FTL (Cat: HP104808), and GAPDH (Cat: HP100003) were purchased from Sino Biological Inc. (Beijing, China). The primers for Human GCLC: 5′-ATGTGGACACCCGATGCAGTATT-3′ (forward) and 5′-TGTCTTGCTTGTAGTCAGGATGGTTT-3′ (reverse) and HMOX: 5′-AACAAGCAGAACCCAGTCTATGC-3′ (forward) and 5′-AGGTAGCGGGTATATGCGTGGGCC-3′ (reverse). Gene expression was normalized to housekeeping gene expression (GAPDH).

Total RNA was extracted from cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Aliquots of 1 μg of total RNA were reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen) and oligo-dT (18)-primers (Invitrogen). The real time PCR reaction was performed using SYBR Green Master Mix kit in ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal standard. The Taqman probe for IL-1β (Cat: HP100210) and GAPDH (Cat: HP100003) were purchased from Sino Biological Inc. (Beijing, China). The primers for mouse TRIM59: 5′-ATGCACAATTTTGAGGAGGAG-3′ (forward) and 5′-TCAACGAGAAACTATTTTCC-3′ (reverse). The expression levels of the mRNAs were reported as fold changes vs. control.

Calycosin (cat #B20846), bafilomycin A1 (Baf A1; cat #S17106), rapamycin (Rapa; cat #T54160), and MHY1485 (cat #S31626) were from Yuanye Bio-Technology Co. (Shanghai, China). β-glycerophosphate (cat #G9422) was from Sigma-Aldrich (Shanghai, China). LysoTracker Deep Red was from Beyotime. Self-quenched BODIPY FL conjugate of BSA (DQ-BSA; green; cat #ab286868) was from BioVision, Inc. (Milpitas, CA, USA). Antibodies against RUNX2, BMP2, OPN, Cathepsin B (CTSB), microtubule associated protein 1 light chain 3 beta 2 (LC3B), sequestosome 1 (p62), AMPK, p-AMPK (Thr172), mTOR, p-mTOR, ULK1, p-ULK1 (757), VAMP8, and SNAP29 were from Abcam (Cambridge, UK). Antibodies against SM22α, lysosome-associated membrane protein 2 (LAMP), p-S6K, and ribosomal protein S6 kinase (S6K) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-tubulin, β-actin, GAPDH, and STX17 were from Sino Biological, Inc. (Beijing, China).

Proteins were extracted from C18-4 cells which were treated with various doses of Res for 24 h. Cells were collected and lysed with lysis buffer, and then the protein concentration was detected using the BCA Protein Quantification Kit (Vazyme, Piscataway, NJ, USA). After heat denaturation in 5% SDS–PAGE sample loading buffer, the protein samples were resolved by SDS-PAGE and transferred to a PVDF membrane [55 (link)–56 (link)]. The samples were probed with β-ACTIN (1:1000; Sino Biological Inc., Shanghai, China), GAPDH (1:1000; Sino Biological Inc.), p53 (1:1000; Bioss, Beijing, China), Ac-p53 (1:1000; Cell Signaling Tecnology, Inc., USA), FOXO1 (1:500; Bioworld, Nnjing, China), Ac-FOXO1 (1:500; Cell Signaling Tecnology), BCL2 (1:500; Bioss), P38 (1:500; Sangon Biotech, Shanghai, China), SIRT1 (1:1000; Bioworld) as previously described [24 (link)]. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit/mouse IgG (1:1,000; Boster, Wuhan, China). Protein blots were probed with the indicated primary antibodies and appropriate secondary antibodies and protein bands were visualized using the Thermo Scientific Pierce ECL western blotting substrate, and the results were analyzed using a Tanon-410 automatic gel imaging system.