Rho123

The glucose uptake ability of CDCs was evaluated by using the fluorescent glucose 2-NBDG (Cayman). After 3 days of cultivation of CDCs on different substrates, all the culture medium was removed and replaced with glucose-free DMEM containing 50 µM 2-NBDG for 30 min. Consequently, the fluorescence intensity of the cells was measured by flow cytometry (BD FACSCanto). Following a 3-day cultivation, the culture medium of each group was collected to test the extracellular lactate contents, and the reagent kit was the Lactate Colorimetric Assay Kit (Nanjing JianCheng).
For the intracellular ATP content assay, CDCs were cultured on different substrates for 3 days and formed CSps, and single cells were isolated from the CSps of each group and seeded in a 96-well plate at a density of 1 × 10⁵. The intracellular ATP content was determined by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). For the mitochondrial membrane potential measurement, digested cells were washed twice with PBS and incubated with 2 µM Rho123 (Beyotime) for 20 min in a dark environment at 37 °C. Fluorescence images were taken under a fluorescence microscope (Olympus, FV3000: Olympus FV31S-SW software displayed), and the fluorescence intensity of the cells was analyzed with ImageJ software V1.8.0.112.

Anti-PARP-1 (ab74290), Anti-Cleaved-PARP-1 (ab32064), Anti-Caspase 3 (ab13847), Anti-Cleaved-Caspase 3 (ab2302), Anti-β-actin, Anti-Caspase 6 (ab185645), Anti-Caspase 7 (ab255818), Anti-SNRK (ab96762), Anti-P62 (ab109012), Anti-LC3 (ab192890), Anti-Trib3 (ab73547), Anti-PPARα (ab215270), Anti-p-p65 (ab76302), Anti-p65 (ab16502), Anti-UCP3 (ab193470), Anti-IgG (ab133470), Anti-His (ab18184) are purchased from Abcam (Britain). TRIzol reagent was acquired from Invitrogen (USA); an SYBR RT-PCR Kit and DNA PCR kit were from Takara Bio Inc. (Japan); RNase R was from Epicenter (USA). RNA-Binding Protein Immunoprecipitation Kit was purchased from Millipore (USA). T7 RNA polymerase and RNase-free DNase I were bought from Promega (USA).
Primers, mimic, and siRNAs were designed and synthesized by Sangon Biotech (China). Rho123, Caspase 3 activity Kit, CCK-8 Kit and ATP Assay Kit were purchased from Beyotime (China). A Dual-luciferase reporter assay system was purchased from Promega (USA). Z-vad, JSH-23, SP600125, 2-MeOE2 were purchased from Selleck (USA).

Rho123 (Beyotime, China), which can bind specifically to mitochondria, has been used in numerous investigations to estimate MMP with some modifications [28 (link)]. The low MMP increases the fluorescence intensity of Rho123 in cells and vice versa. In this study, treated cells were harvested, washed, and then incubated with Rho123 (5 mg/ml) in PBS for 60 min in the dark at 37 °C. The fluorescence was measured by flow cytometry.

Rho123 (Beyotime, Nantong, China) was used to detect MMP. Rho123 dye was added to the cell suspension at a final concentration of 2 μg/mL, and the cells were further incubated at 30 °C for 20 min. Then the cells were washed twice with PBS and finally resuspended in PBS. The fluorescence was detected by Flow Cytometry (CytoFLEX, Beckman, China) (FITC channel).