Anti nrf2

To measure protein levels, immunoblot analysis was conducted based on a previously published protocol [6 (link)]. The samples were lysed using RIPA buffer (#P0013B, Beyotime Biotechnology, Shanghai, China). An equal amount of protein (20 mg) was loaded onto an SDS-polyacrylamide gel and transferred to the polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 4% bovine serum albumin (BSA, #ST023, Beyotime Biotechnology), followed by overnight incubation with primary antibodies at 4 °C. Afterwards, the membranes were incubated with appropriate secondary antibodies. After washing, the membranes were visualized using ECL (#P0018FS, Beyotime Biotechnology). The antibodies contained anti-NRF2 (1:1000, #16396-1-AP, Proteintech, Wuhan, China), anti-GAPDH (1:10000, #10494-1-AP, Proteintech, Chicago, IL, USA), anti-KEAP1 (1:1000, #80744-1-RR, Proteintech), and HRP-conjugated anti-rabbit IgG antibody (#7074, Cell Signaling Technology, Danvers, MA, USA).

LPF peptide was synthesized by Genscript Biotech Corporation (Nanjing, China). D-glucose (G8270) and MTT (M2003) were purchased from Sigma. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH, A101386) was from Aladdin Biochemical Technology (Shanghai, China). Sodium fluorescein and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were from Wako Pure Chemical (Osaka, Japan). Anti-O-GlcNAc antibody (CTD110.6) (sc-59623) was from Santa Cruz Biotechnology. Lipid Peroxidation Assay Kit (S0131S), Glutathione (GSH) Assay Kit (S0052), Hematoxylin and Eosin Staining Kit (C0105S), DAPI (C1002), and H₂DCFDA (S0033S) were purchased from Beyotime Biotechnology (Shanghai, China). Anti-Pax3 antibody was from DSHB. Primary antibodies, anti-NRF2 and anti-Keap1, were purchased from Proteintech. Anti-β-Actin and HRP-conjugated secondary anti-rabbit and anti-mouse antibodies were purchased from Fude Biological Technology (Hangzhou, China). Alexa Fluor 488 goat anti-rabbit IgG was purchased from Invitrogen (Carlsbad, CA, USA).

For immunoblotting, antibodies used in this study were purchased from EMD Millipore (Oakville, Canada), Sigma Aldrich (Oakville, Canada), Proteintech (Illinois, USA), and Cell Signaling Technology (Whitby, Ontario). The following primary antibody was used from EMD Millipore (Oakville, Canada): anti-androgen receptor (Millipore Cat# 06-680, 1:1,000). The following primary antibodies were used from Sigma Aldrich (Oakville, Canada): anti-fast skeletal myosin (M4267, 1:15,000), anti-slow skeletal myosin (Sigma-Aldrich Cat# M8421, 1:10,000), and anti-beta actin (Sigma-Aldrich Cat# A2066, 1:10,000). The following primary antibodies were used from Proteintech (Illinois, USA): anti-PGC1α (Proteintech Cat# 66369-1-Ig, 1:5,000), anti-NRF-2 (Proteintech Cat# 16396-1-AP, 1:500), and anti-TFAM (Proteintech Cat# 22586-1-AP, 1:1,000). The following HRP-conjugated secondary antibodies were used from Cell Signaling Technology (Whitby, Ontario): goat anti-rabbit IgG, HRP-linked (Cell Signaling Technology Cat# 7074, 1:5,000) and horse anti-mouse IgG, HRP-linked (Cell Signaling Technology Cat# 7076, 1:5,000).

Specific details regarding protein extraction were described in our published paper [21 (link)]. The primary detection antibodies used in our experiment were anti-beta-actin (1:1000, Proteintech, Rosemont, IL, USA), anti-Sirt7 (1:1000, Santa, Dallas, TX, USA), anti-alkaline phosphatase (ALP) (1:1000, ABclonal, Wuhan, China), anti-MCP-1 (1:1000, ABclonal, Wuhan, China), anti-Nrf2 (1:1000, Proteintech, Rosemont, IL, USA), anti-NQO1 (1:1000, Proteintech, USA), anti-Hmox1 (1:1000, Proteintech, Rosemont, IL, USA), anti-ICAM-1 (1:1000, Abcam, Waltham, MA, USA), anti-BAX (1:1000, CST, Boston, MA, USA), anti-runt-related transcription factor 2 (Runx2) (1:1000, Proteintech, Rosemont, IL USA) and anti-cleaved caspase3 (1:1000, CST, Boston, MA, USA). The secondary antibodies used were goat anti-rabbit and anti-mouse IgG-HRP (1:5000, Fudebio, Hangzhou, China). The quantitation of Immunoblotting images was analyzed using ImageJ software (NIH, Bethesda, MA, USA).

hBM-MSCs were differentiated in osteogenic differentiation medium adding AU (0, 0.01, 0.1, 1 μM) for 3 days. With regard to inhibitor studies, cells were treated with AU (1 μM) and noggin (100 ng/mL) /LDN193189 (100 nM) for 3 days. Pertaining to AU antioxidant injury studies, cells were treated with both 1 μM AU and 300 μM H₂O₂ for 3 days. The ODM adding AU and H₂O₂ was changed after 2 days of osteogenesis. Thereafter, cells were treated with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS for three times (3 min each), the cells were incubated for 5 min with 0.1% TritonX-100 in PBS and blocked in 5% bovine serum albumin for 30 min. Treated cells were washed for three times and incubated with primary antibodies (anti-COL1A1 (1:200; Abcam), anti-RUNX2 (1:200; Abcam) and anti-Nrf2 (1:200; Proteintech)) at 4 °C for at least 12 h. Then the cells were incubated with fluorescence-conjugated secondary antibody for 1 h and stained with DAPI staining solution (Nanjing KeyGen Biotech, Nanjing, China). A fluorescent microscope (Leica Camera) was used to capture the images, and the fields were randomly selected.

The IHC protocol was performed as described previously [15 (link)]. Briefly, tumour sections of patients and nude mouse xenografts were analysed by IHC using the EnVision™ System (Dako, Carpinteria, CA). Primary antibodies used for IHC were anti-BDH2 (1:100, Proteintech, 27,207), anti-Ki67 (1:100, Abcam, ab15580), anti-cleaved caspase-3 (1:200, CST, #9661), anti-LC3B (1:200, Abcam, ab48394), anti-Nrf2 (1:50, Proteintech, 16,396), anti-p-Akt (1:100, Proteintech, 66,444), and anti-p-mTOR (1:100, Abcam, ab109268).

Total protein separation and western blotting were performed as described previously [16 (link)]. Western blot analysis was performed using anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9661), anti-p-AMPK (Thr172) (#50081), anti-p-ERK1/2 (#4370), and anti-HA-Tag (#3724) antibodies purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3 (14600–1-AP), anti-P62/SQSTM1 (18420–1-AP), anti-phospho-Akt (Ser-473) (66444–1-Ig), anti-Akt (10176–2-AP), anti-mTOR (20657–1-AP), anti-P38 MAPK (14064–1-AP), anti-JNK (51151–1-AP), anti-ERK1/2 (16443–1-AP), anti-AMPK (10929–2-AP), anti-Nrf2 (16396–1-AP), anti-Keap1 (10503–2-AP), anti-FLAG-tag (66008–2-AP), anti-MYC-tag (60003–2-AP), and anti-β-actin (66009–1-Ig) antibodies were obtained from Proteintech Group Co., Ltd. (Wuhan, China). An anti-phospho-mTOR (Ser-2448) (ab109268) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-p-p38 (sc-7973), anti-p-JNK (sc-6254), and anti-Ub (sc-8017) antibodies were obtained from Santa Cruz Biotechnology., Inc. (Santa Cruz, CA, USA).