Monoclonal anti caspase 3

Human kidney proximal tubular epithelial cells (HK-2) were a cell line purchased from the American Type Culture Collection (ATCC, USA). Antibodies were from the following sources: polyclonal anti-TLR4 from Abcam (USA). Monoclonal anti-PGC-1α, monoclonal anti-caspase-3, and NF-κB p65 were from Cell Signaling Technology (Boston, USA). Polyclonal phospho-NF-κB p65 (ser536) antibody was from Bioworld (USA), and polyclonal anti-cytochrome C was from Proteintech (Wuhan, China). Beta-actin and all secondary antibodies for Western blot and immunofluorescence were from Proteintech (Wuhan, China). TAK242 was from MedChem Express (USA), and parthenolide was from Sigma (USA). Plasmids containing pcDNA4 myc PGC-1α (pcDNA4/PGC-1α) was bought from Addgene (USA). Lipofectamine 2000, MitoTracker Red CMXRos, MitoSOX, and TRIzol were purchased from Invitrogen (USA). PrimeScript™ RT reagent Kit with gDNA Eraser and SYBR® Premix Ex Taq™ (Tli RNase H Plus) were from TaKaRa (Japan). Annexin V-FITC Apoptosis Detection Kit was from Beyotime (Shanghai, China). TMRM Detection Kit was from Genmed Scientifics Inc. (USA). Other reagents, including DMEM/F12 medium, bovine serum albumin (FBS), and trypsin, were obtained from Gibco (USA).

The cells were fixed in 4% paraformaldehyde (PFA) in PBS for 30 min at 37 °C, and were permeabilized using 0.1% Triton-X-100 in PBS for 1 h at room temperature. After blocking with PBS containing 5% bovine serum albumin (BSA; minimum 98% electrophoresis grade, Sigma-Aldrich), cells were immunostained with monoclonal mouse anti-class III β-tubulin (TuJ1; 1 : 1,000; Covance, Madison, WI, USA; Cat# MMS-435 P-250, RRID: AB_10063408) and monoclonal anti-caspase 3 (1; 200; Cell Signaling, Danvers, MA, USA; Cat# 9668, RRID: AB_2069870) in PBS containing 5% BSA overnight at 4 °C. The samples were subsequently probed with mouse directed secondary antibodies for 2 h at room temperature and mounted onto coverslips using a fluorescent mounting medium (Dako, Carpinteria, CA, USA). The images were taken with an upright microscope (Olympus, Tokyo, Japan) and analyzed using a DP-controller image system (Olympus).
For the assessment of neurite length, we measured the longest neurite for each TuJ1–positive neuron and calculated the average of neurite length using ImageJ software (NIH). The effect of PHD inhibitors on cell survival was quantified by caspase 3 immunostaining. We estimated the ratio of caspase 3-positive neurons to total neurons.