Journal: Nature Communications
Article Title: Homoharringtonine exhibits senotherapeutic activity that mitigates diet- and age-associated obesity and insulin resistance and extends lifespan in mice
doi: 10.1038/s41467-026-70475-3
Figure Lengend Snippet: A Schematic of the drug affinity responsive target stability (DARTS) approach. B Western blotting of candidate proteins in replicatively senescent human dermal fibroblasts. Replicatively senescent human visceral preadipocytes (RS HPAs) were transfected with siRNA, incubated for 4 days, then treated with homoharringtonine (HHT) or phosphate buffered saline (PBS) for 4 days ( C–E ). C Cell number by CCK-8 assay ( n = 7 per group). D Immunoblots of poly (ADP-ribose) polymerase (PARP), procaspase-3 (Procasp-3) and cleaved casp-3 (Casp-3). E Quantification of Casp-3 ( n = 3 per group). F Cell number by CCK-8 assay in HSPA5-overexpressing HPAs ( n = 4 per group). G IHC images and quantification of HSPA5 (brown) and Casp-3 (red) in NS and RS HPAs after 2-day HHT treatment ( n = 6 fields from 2 independent experiments). Boxed region enlarged and split into HSPA5 (brown) and Casp-3 (red) channels. H Quantification of Casp-3-positive cells in HHT-treated RS HPAs with high or low HSPA5 ( n = 6 fields from 2 experiments). I Levels of HSPA5, p53, p21, and p16 proteins in HPAs at different passages. J HHT dose-response curves in HPAs at different passages (Low, P2–P3; Mid, P6–P7; High, P12–P13) (n = 5 per group). K Surface plasmon resonance affinity assay. L Predicted HHT–HSPA5 binding; H-bonds (magenta) and pi–cation (blue). M Inhibition of ATPase activity of HSPA5 by HHT and by HA15 ( n = 3 per group). N HSPA5 levels in adipose tissues of chow diet (Ch)- and high-fat diet (HF)-fed mice administered PBS or HHT ( n = 3 in Ch; n = 5 in HF). O IHC images and quantification of HSPA5 in adipose tissue of Ch- and HF-fed mice administered PBS or HHT ( n = 3 per group). P HSPA5 in adipose tissues of aged mice administered PBS or HHT ( n = 4 per group). Data are expressed as means ± SEM and analyzed via one-way ANOVA with a post-hoc test or two-tailed Student’s t test. DAB, 3,3′-diaminobenzidine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSPA5, heat shock protein family A member 5; HSPA8, heat shock protein family A member 8; PKM2, pyruvate kinase M2; RPL7, ribosomal protein L7; siSC, scrambled siRNA; TXNRD1, thioredoxin reductase 1. Illustration created in part (A) with BioRender ( https://BioRender.com/02ykozw ).
Article Snippet: Following final TBS-T washes, color development was performed using enzyme-specific substrate systems: HRP activity was visualized with 3,3′-diaminobenzidine (DAB) substrate (Cell Signaling Technology, #7076, or Dako, K5007) to yield brown staining, and AP activity was visualized with Fast Red TR/Naphthol AS-MX (Sigma-Aldrich, F4648) to yield red staining.
Techniques: Western Blot, Transfection, Incubation, Saline, CCK-8 Assay, SPR Assay, Binding Assay, Inhibition, Activity Assay, Two Tailed Test
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