Phosphofructokinase

ADSCs were lysed using RIPA lysis buffer, and the total protein concentration was measured with a BCA protein assay kit (all from ThermoFisher Scientific). The transferred protein on the PVDF membrane (Millipore) was blocked with 5% non-fat milk for 1 h, followed by incubation with primary antibodies overnight at 4 °C. After washing with TBST, the PVDF membrane was incubated with the corresponding HRP-conjugated second antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. The primary antibodies used were: β-Actin (1:1,000), Tom20 (1:1000), Hexokinase 1 (HK1, 1:1000); Hexokinase 2 (HK2, 1:1000); Phosphofructokinase (PFK, 1:1000); Lactate dehydrogenase A (LDHA, 1:1000); Citrate Synthase (CS, 1:1000); Dihydrolipoamide succinyltransferase (DLST, 1:1000), Succinate dehydrogenase A (SDHA, 1:1000), and Fumarase (FH, 1:1000) (all obtained from Abcam). Mitochondrial fission factor (MFF, 1:1000); Mitofusin 2 (MFN2, 1:1000); Opticatrophy protein 1 (OPA1, 1:1000) (all obtained from Proteintech).

Commercially available colorimetric assay kits of enolase (Abcam), pyruvate dehydrogenase (Abcam), phosphofructokinase (Abcam), and lactate dehydrogenase (Abcam) were used to measure corresponding enzymatic activities. In brief, S. aureus cell cultures with or without treatment of AgNO₃ were harvested, washed, suspended, and sonicated in lysis buffers. The protein concentration of supernatant after centrifugation was measured by BCA assay. Enzyme activities were measured according to the standard procedures provided by the manufacture. The enzymatic activity was normalized to protein concentration. The activity of alkyl hydroperoxide reductase⁷⁰ , Pgl^{71 (link)}, and 6PGDH^{72 (link)} were performed according to previous reports.

UMSCs were lysed in RIPA lysis buffer (Thermo Fisher Scientific), and total protein concentration was measured with BCA protein assay kit (Thermo Fisher Scientific). The PVDF membrane (Invitrogen) transferred protein was blocked with 5% non-fat milk and then incubated with primary antibodies overnight at 4 ℃. After washing with TBST for three times, the PVDF membrane was incubated with corresponding HRP-conjugated second antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. The primary antibody information is as follows, Bcl-2 (1:1000, abclonal), cleaved Caspase-3 (1:1000, abclonal), glutathione peroxidase 4 (GPX4, 1:1000); hexokinase 1 (HK1, 1:1000); hexokinase 2 (HK2, 1:1000); phosphofructokinase (PFK, 1:1000); lactate dehydrogenase A (LDHA, 1:1000); citrate synthase (CS, 1:1000); aconitase 2 (ACO2, 1:1000); fumarase (FH, 1:1000); succinate dehydrogenase A (SDHA, 1:1000); all were obtained from Abcam.