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

Journal: EMBO Molecular Medicine

Article Title: A versatile drug delivery system targeting senescent cells

doi: 10.15252/emmm.201809355

Figure Lengend Snippet: A SK‐MEL‐103 melanoma cells were treated with palbociclib (1 μM) for 1 or 14 days, and senescence induction was assessed by SAβgal staining. Next, cultures were exposed to free doxorubicin (50 μM) or GalNP(dox) (1 mg/ml, filtered) for 30 min. Pictures show representative images illustrating doxorubicin fluorescence by confocal microscopy. Scale bar: 50 μm. B SK‐MEL‐103 melanoma cells were treated with palbociclib (5 μM) for 14 days, cultures were exposed to GalNP(dox) (0.06 mg/ml, filtered), and annexin V signal was quantified over time. Representative pictures are shown in Appendix Fig S3F . C Athymic nude female mice carrying subcutaneous SK‐MEL‐103 xenografts were treated daily with palbociclib (oral gavage, 50 mg/kg) and/or GalNP(dox) (tail vein injection, 200 μl of a solution with 4 mg/ml of GalNP containing a total of 1 mg/kg of deliverable doxorubicin), alone or in combination, as indicated. For each tumor, the relative tumor volume change was calculated relative to its baseline prior to treatment. Values are expressed as mean ± SEM. Individual tumor size measurements are shown in Appendix Fig S3G . D Similar to (B) but using GalNP(nav) (tail vein injection, 200 μl of a solution with 4 mg/ml of GalNP containing a total of 1 mg/kg of deliverable navitoclax), as indicated. Individual tumor size measurements are shown in Appendix Fig S3F . E Left, fold change of tumor size of SK‐MEL‐103 xenografts, after the indicated daily treatments. Data for palbociclib, and for palbociclib plus GalNP(dox), correspond to the same data in panel (C), at day 17. Data for free doxorubicin (daily tail vein injection, 1 mg/kg, for 17 days) were obtained in parallel. Right, mRNA levels of cardiotoxicity markers in hearts from the same mice. Actb and Gapdh were used for input normalization. Values are relative to control mice and are expressed as mean ± SD, and statistical significance was assessed by one‐way ANOVA and Dunnett's multiple comparisons test (versus palbociclib‐alone treated group). F Left, fold change of tumor size, as in (C), after the indicated daily treatments. Data for palbociclib, and for palbociclib plus GalNP(nav), correspond to the same data in panel (D), at day 13. Data for free navitoclax (daily oral gavage, 25 mg/kg, for 13 days) were obtained in parallel. Right, platelet levels in the blood of the same mice. Values are expressed as mean ± SEM in the case of tumor size, and as mean ± SD in the case of platelet counting, and statistical significance was assessed by one‐way ANOVA and Dunnett's multiple comparisons test (versus palbociclib‐alone treated group).

Article Snippet: Loading of the NP scaffolds with the different compounds was performed as follows: For NP(rho), 200 mg of NPs were suspended in a solution of 16 ml of EtOH together with 76.65 mg of rhodamine B (Sigma, #R6226); for NP(icg), 200 mg of mesoporous silica nanoparticles (MSNs) were suspended in a solution of 5 ml of water together with 5 mg of indocyanine green (Sigma, #I2633); for NP(dox), 200 mg of MSNs were suspended in a solution of 12.5 ml of water together with 110 mg of doxorubicin (Carbosynth, #AD15377); for NP(nav), 250 mg of NPs were suspended in 27 ml EtOH together with 91.3 mg of navitoclax (Active BioChem, #A‐1001).

Techniques: Staining, Fluorescence, Confocal Microscopy, Injection

Journal: Kidney360

Article Title: Targeting Senescent Cells as Therapy for CKD

doi: 10.34067/KID.0000000000000316

Figure Lengend Snippet: Completed, ongoing, and planned clinical trials of senolytics in human disease

Article Snippet: ABT-263 (navitoclax) has been encapsulated in a nanocarrier and a senolytic nanoparticle, Gal-NP(nav), generated.

Techniques: Clinical Proteomics