Journal: International Journal of Immunopathology and Pharmacology

Article Title: Targeted chelation therapy decreases NLRP3 expression by vascular cells and acts as senomorphic in chronic kidney disorder induced vascular calcification

doi: 10.1177/03946320251391142

Figure Lengend Snippet: (a) EDTA is Senomorphic and not Senolytic—Immortalized human vascular smooth muscle cells cultured under high phosphate conditions for 3 days and were treated with either EDTA or ABT 263 (senolytic agent) for 24 h. IHC-based Expression level analysis of Caspase 3 was used as a marker for senolytic (inducing selective apoptosis of senescent cells) activity. We observed that in comparison to ABT 263, EDTA treatment significantly brought down Caspase 3 activity, indicating that EDTA does not induce apoptosis. Experiment performed in triplicate, repeated four times, results represented as Mean ± S.D and (b) EDTA but not ABT263 Decreases NLRP3 expression—Immortalized human vascular smooth muscle cells cultured under high phosphate conditions for 3 days and were treated with either EDTA or ABT 263 (senolytic agent) for 24 h. IHC-based Expression level analysis of NLRP3 reveals that EDTA treatment and not ABT 263 decreased NLRP3 activity. Justifying the anti-apoptotic and senomorphic nature of EDTA treatment. Experiment performed in triplicate, repeated four times, results represented as Mean ± S.D.

Article Snippet: 130127, Madison, WI), EDTA (Sigma, E6511), vitamin D3 (VitD3, Cholecalciferol, Sigma, #C9756-5G), NaCl (sigma, S3014), KCl (Sigma P9541), NaHCO3 (Sigma, S5761), glucose (Sigma, G7021), NaH2PO4 (Sigma, S0876), dPBS (Gibco, 10010-031), DMEM (Gibco-1885-076), FBS, Pen-Sterp, Human Aortic Smooth Muscle Cells (SMCs) (Promocell, C12533 ), SMC Medium II (Promocell 22062), ABT263, HSA (Evonik Birmingham Laboratory, 777HSA097), HEPES (Sigma, H3375), absolute ethanol (Fisher BP 2818500), mPEGNHS, MW 2000 (BOC Sciences), Traut’s reagent (G-Biosciences, Saint Louis, MO, BC95), 5-Dodecanoylaminofluorescein di-β-D-Galactopyranoside (C12FDG, MedChem Express HY-126839), Cell Staining buffer (BioLegend 00-4222-26), anti-CD16/32 mouse monoclonal antibody (BioLegend Cat#156604), anti-CD45-APC (eBiosciences Cat#17-0461-82) DAPI (BioLegend Cat#422801), Rabbit anti-Rat Caspase-3 (Cell Signaling Technology, C8487), NLRP3 (Novus Biologicals, NBP2-12446), and PiT-1 (Santa Cruz Biotechnology, SC98824), anti-rabbit IgG secondary antibody Cy5 (Invitrogen, A10523) cDNA synthesis Kit (Bio-Rad, Cat# 1725034), iTaq—Universal SYBR Green Super mix 12 reagent (Bio-Rad, Cat# 172512), SA-βGal Staining Kit (Cell Signaling, 9860), Rat IL-6 ELISA Kit (RnD SYSTEMS DY-506), Rat IL-1β ELISA Kit (RnD SYSTEMS SRLB00), T-Per protein extraction buffer (ThermoFischer, 78510), BCA assay kit (Thermo Fisher Scientific, A55865).

Techniques: Cell Culture, Expressing, Marker, Activity Assay, Comparison