Alexa fluor 647 annexin 5 and propidium iodide

The conditionally immortalized human podocytes (HPC) was a gift from Dr. Moin Saleem at Bristol University, UK. The cells were cultured in RPMI 1640 containing 10% fetal bovine serum and pennicilin/streptomycin (100 U/ml of each) (Gibco, USA) and 1% insulin-transferrin-selenium (ITS, Invitrogen). Other reagents used in the studies were as follows: podocyte transfection reagent (LipofecTAMINE 2000, Invitrogen); plasmid DNA extraction kit (Purelink HiPure Plasmid DNA Purification Kit, Invitrogen), antibodies against total p65, phospho-p65, total p38 and phospho-p38 (Cell Signaling Technologies), TLR9 and CD2AP (Abcam), podocin (Sigma), GAPDH (Kangchen), anti-rabbit IgG (Bioworld); puromycin aminonucleosides (Sigma); Alexa Fluor® 647 Annexin V and Propidium Iodide (Biolegend); Reverse transcription kit, DRR037A, and quantitative PCR kit, DRR820A (Takara); RIPA cell lysis buffer; BCA protein quantification kit (Bi-yun-tian, Shanghai); psiRNA-hTLR9 (Invivogen); RNA extraction kit (mirVana miRNA extraction kit, Ambion); E. coli DH5a competent cells (TransGen Biotech, Beijing).

The antibodies against MCC, CD2AP, total β-catenin, p16, p21, p27, p57, CDK1, Cyclin E1, Cyclin D2, PCNA, and GAPDH (Proteintech, USA); antibodies against active β-catenin, total ERK1/2 and phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies, USA); WT1 antibody (Abcam, USA); puromycin aminonucleosides (Sigma, USA); Alexa Fluor® 647 Annexin V and Propidium Iodide (Biolegend); Reverse transcription kit, DRR037A (Takara); quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara).