Reactive oxygen species assay kit

The Seahorse XF Glycolytic Rate Assay Kit (103344‐100) and Seahorse XF Cellular Mitochondrial Stress Test Kit (103015‐100) were obtained from Agilent Technologies, and β‐glycerol disodium (G9422), ascorbic acid (A92902) and dexamethasone (D4902) were obtained from Sigma‒Aldrich. Recombinant mouse RANKL (CJ94) was obtained from NovoProtein Technology Co., Ltd. The PFK activity detection kit was obtained from Soleibao Technology Co., Ltd. DyLight 488 goat anti‐mouse IgG and DyLight 596 goat anti‐rabbit IgG were obtained from Abbkine Scientific. MK‐2206 dihydrochloride (GC16304) and SC79 (GC11645) were obtained from GLPBIO, and AICAR (Acadesine, S1802), Dorsomorphin (Compound C, S7840) and PFK15 (S7289) were obtained from Selleck. The Reactive Oxygen Species Assay Kit (S0033S), GSH and GSSG Assay Kit (S0053) and NADP+/NADPH Assay Kit (S0179) were obtained from Beyotime Biotechnology Ltd. CARM1 (NM_199141) Human Recombinant Protein and PPP1CA (NM_002708) Human Recombinant Protein were obtained from OriGene.
The information on the primary antibodies applied in this research are listed in Table S2.

Intracellular ATP and ROS levels were measured using the ATP Assay and Reactive Oxygen Species Assay Kits according to the manufacturer's instructions (Beyotime, Shanghai, China).

Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) was obtained from Hyclone (South Logan, UT, USA). Fetal bovine serum (FBS) was purchased from ZETA Life (Menlo Park, CA, USA). ZEA (Z2125) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Nec-1 (HY-15760) and GSK’872 (HY-101872) were purchased from MedChem Express (Monmouth Junction, NJ, USA). NSA (T7129), NAC (T0875), BAPTA-AM (T6245), and cell counting kit-8 were purchased from TargetMol (Boston, MA, USA). Calcein/PI Cell Viability/Cytotoxicity assay kits, enhanced ATP assay kits, reactive oxygen species assay kits, and Fluo-4 AM were purchased from Beyotime Biotechnology (Beijing, China). JC-1 staining assay kits were purchased from Goyoo Biotechnology (Nanjing, China). Cytotoxicity LDH assay kits were obtained from Promega Biotechnology (Madison, WI, USA). The anti-P-RIPK3 (ser227, ab209384, 1:1000) antibody was purchased from Abcam (Cambridge, UK). The anti-P-MLKL (ser358, bsm-33331M, 1:1000) antibody was obtained from Bioss Biotechnology (Wuhan, China).

The fruiting bodies of M. conica were obtained from Changchun in Jilin Province (China). DEAE-52 cellulose, Sephadex G100, DPPH (1, 1-diphenyl-2-picrylhydrazyl), standard monosaccharides, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich Co., Ltd. (St. Louis, MO, USA). The Reactive Oxygen Species Assay kits were produced by Beyotime Inc. (Beyotime, Shanghai, China). TRIzol-A+ reagent, FastQuant RT Kits (With gDNase), and Quant qRT-PCR kits were obtained from Tiangen Inc. (Tiangen, Beijing, China). All the antibodies for Western blot analysis were provided by Cell Signaling Technology Inc. (Beverly, MA, USA). Other chemicals and reagents were of analytical grade.

Intracellular were measured using the Reactive Oxygen Species Assay Kits as directed by the manufacturer (Beyotime, Shanghai, China). Briefly, cells were incubated with fresh DMEM medium containing 10 μM DCFH-DA at 37°C for 30 min. Then the cells were washed twice with PBS and resuspended in cold PBS for flow cytometry analysis. Mitochondrial ROS levels in appropriately treated BEAS-2B cells were determined by staining with MitoSox according to the manufacturer’s instructions (Thermo Fisher Scientific). A fluorescence microscope was used to examine stained cells (Olympus Life Science; Tokyo, Japan).