cellular senescence detection kit spider βgal  (Dojindo Labs)

Journal: Advanced Science

Article Title: Farnesyltransferase Deficiency in Cardiomyocytes Initiates Senescence and Contributes to Cardiac Fibrosis

doi: 10.1002/advs.202511530

Figure Lengend Snippet: Fntb Knockout Triggers DNA Damage Response (DDR) and Cellular Senescence in Cardiomyocytes. (a) Transcriptomic profiling of adult mouse primary cardiomyocytes (AMCMs) isolated from Fntb ‐cKO mice 2 weeks post‐tamoxifen induction. Weighted gene co‐expression network analysis (WGCNA) identified 13 gene modules hierarchically clustered by size. Module‐trait correlations are quantified by Pearson coefficients (top values) and corresponding p‐values (bottom values); (b) Top 10 enriched pathways in Module 6 ranked by fold enrichment. Circle size indicates gene count per pathway; color gradient reflects Adjusted p‐values. DDR pathways highlighted in purple; (c) Western blot analysis of Atm and phospho‐Atm (Ser1981) expression in Fntb ‐cKO AMCMs ( n = 5/group); (d) Immunofluorescence detection of γH 2 AX foci (white arrows) in AMCMs with quantitative analysis of γH 2 AX‐positive cells ( n = 5/group), scale bar: 50 µm; (e) Western blot analysis of phospho‐Chk1 (Ser317) and phospho‐Chk2 (Thr68) levels ( n = 5/group); (f) WT and Fntb ‐cKO mice received intraperitoneal injections of the DDR inhibitor KU55933 (5 mg/kg) or vehicle (DMSO/saline) every 48 h for 12 weeks. (g) Interstitial fibrosis assessed by PSR staining and quantitative analysis in KU55933‐treated cohorts ( n = 4–6/group), scale bar: 50 µm; (h,i) Representative Western blot (h) and quantitative analysis (i) of anti‐apoptotic proteins Hsp70 and Bcl‐2, along with pro‐apoptotic markers Bax and cleaved caspase‐3 ( n = 4–6/group); (j) RT‐qPCR analysis of cyclin‐dependent kinase inhibitors in Fntb‐cKO AMCMs ( n = 4/group); (k,l) Representative images (l) and quantification (k) of senescence‐associated β‐galactosidase (SA‐β‐Gal) activity in AMCMs at 24 weeks post‐induction ( n = 4/group). White arrows denote SA‐β‐Gal‐positive cells. scale bar: 100 µm. All data represent mean ± SEM. ns: not significant; Multi‐group comparisons were analyzed using two‐way ANOVA with Tukey‐Kramer post hoc test. Two‐group comparisons were performed with an unpaired two‐tailed Student's t ‐test.

Article Snippet: SA‐β‐Gal activity was detected using the Cellular Senescence Detection Kit (Dojindo Molecular Technologies, #SG03).

Techniques: Knock-Out, Isolation, Expressing, Western Blot, Immunofluorescence, Saline, Staining, Quantitative RT-PCR, Activity Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Farnesyltransferase Deficiency in Cardiomyocytes Initiates Senescence and Contributes to Cardiac Fibrosis

doi: 10.1002/advs.202511530

Figure Lengend Snippet: FNTB Depletion Induces Senescence‐Associated Secretory Phenotype (SASP) in Cardiomyocytes to Promote Fibroblast Activation. (a) Conditioned media transfer paradigm. Primary AMCMs from tamoxifen‐induced WT and Fntb ‐cKO mice were cultured for 24 h. Conditioned media were collected and applied to WT cardiac fibroblasts. (b,c) Fibroblast activation was evaluated by quantifying α‐SMA‐positive cells (b, n = 4/group) and imaging α‐SMA expression (c), scale bar: 100 µm; (d) mRNA levels of Col1a1 , Col3a1 , Mmp2 , and Timp2 in fibroblasts treated with conditioned media were measured by RT‐qPCR ( n = 4/group); (e) Fibroblast proliferative capacity was assessed using CCK‐8 assay under conditioned media treatment ( n = 4/group); (f) Gata4 protein expression in Fntb ‐cKO AMCMs was analyzed by Western blot ( n = 5/group); (g) SASP‐related transcript levels were validated in AMCMs using RT‐qPCR ( n = 4–5/group, upper panel), Corresponding RNA‐Sequencing data from prior analyses were displayed as a heatmap (lower panel), with color gradients reflecting expression differences between genotypes (red: upregulation in KO; blue: downregulation). (h) Protein level elevation of Gdf15, Tgf‐β2, and Timp2 in Fntb ‐cKO AMCMs;(i) Quantitative analysis of Gdf15, Tgf‐β2, and Timp2 protein level ( n = 5/group); (j) Secreted Gdf15 and Tgf‐β2 in cardiomyocyte conditioned media were quantified by ELISA ( n = 6/group); (k,l) Representative immunofluorescence images (k) and quantitative analysis (l) of α‐SMA‐positive fibroblasts treated with conditioned media from Ad‐shFntb‐infected NRCMs transfected with negative control siRNA (si‐NC), si‐Tgfb2, or si‐Gdf15 ( n = 5/group), scale bar: 100 µm; (m) mRNA levels of ECM‐related genes, including Col1a1 , Col3a1 , Timp1 , and Mmp2 , in fibroblasts under the indicated treatments were measured by RT‐qPCR ( n = 6/group); (n) Fibroblast proliferative capacity was assessed using CCK‐8 assay ( n = 6/group). All data represent mean ± SEM. Multi‐group comparisons were analyzed using two‐way ANOVA with Tukey‐Kramer post hoc test. Two‐group comparisons were performed with an unpaired two‐tailed Student's t ‐test.

Article Snippet: SA‐β‐Gal activity was detected using the Cellular Senescence Detection Kit (Dojindo Molecular Technologies, #SG03).

Techniques: Activation Assay, Cell Culture, Imaging, Expressing, Quantitative RT-PCR, CCK-8 Assay, Western Blot, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Infection, Transfection, Negative Control, Two Tailed Test