Journal: iScience

Article Title: CPNE5 overexpression inhibits cardiomyocytes apoptosis by promoting the degradation of FAS receptor

doi: 10.1016/j.isci.2025.113302

Figure Lengend Snippet: Overexpression of CPNE5 inhibits the activation of FAS receptor mediated apoptotic pathway in vitro and in vivo (A) Representative image showing immunofluorescence of TUNEL (red, arrows), DAPI (blue, for nuclei), and cardiac troponin (green; bar = 50 μm) in overexpression (Flag- Cpne5 ) or knockdown (si- Cpne5 ) CPNE5 NMCMs under FASL protein treatment. Flag-vector: empty vectors, NC: negative control. (B) Statistical analysis of TUNEL-positive cells in (A). (C and D) After treated with FASL protein, NMCMs overexpression CPNE5 (C) or knockdown CPNE5 group (D) were detected CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein levels by western blot. Bar graph on the right quantification protein level of (C) and (D), normalized to β-actin ( n = 5). (E and G) After TAC (E) or I/R (G) model, AAV9-ctrl and AAV9-OE- Cpne5 mice were detected for CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein levels by Western blot. Bar graph on the right quantification protein level of (E) and (G), normalized to β-actin ( n = 5). (F and H) After TAC (F) or I/R (H) model, WT and KO mice were detected for CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein level by Western blot. Bar graph on the right quantification protein level of (F) and (H), normalized to β-actin ( n = 5). All data are presented as mean ± SD; unpaired two-tailed Student’s t test (two groups) was used to determine statistical significance of experimental data. ∗∗ p < 0.01.

Article Snippet: Following this transfection step, the cells were added with 5μM CHX (MCE, cat. no. HY-12320) for protein stability study, 1 μM Chloroquine (MCE, cat. no. HY-17589A) for lysosome inhibition study, 4 μM MG132 (Selleck Chemicals, cat. no. S2619) for proteasome inhibition study, 500ng/ml FASL protein (MCE, cat. no. HY-P74157) to activate the FAS receptor, 5 μM puromycin 2HCl (Selleck Chemicals, cat. no. S7417) for protein synthesis study.

Techniques: Over Expression, Activation Assay, In Vitro, In Vivo, Immunofluorescence, TUNEL Assay, Knockdown, Plasmid Preparation, Negative Control, Western Blot, Two Tailed Test

Journal: iScience

Article Title: CPNE5 overexpression inhibits cardiomyocytes apoptosis by promoting the degradation of FAS receptor

doi: 10.1016/j.isci.2025.113302

Figure Lengend Snippet: Overexpression of CPNE5 in cardiomyocytes reduces FAS mediated apoptosis of cardiomyocytes (A) Representative image showing immunofluorescence of TUNEL (red, arrows), DAPI (blue, for nuclei), and cardiac troponin (green; bar = 80 μm) in NMCMs, which infected with Flag- Cpne5 and HA- FAS plasm id under FASL protein treatment. Flag-vector: empty vectors. (B) Statistical analysis of TUNEL-positive cells in (A). (C) Representative Western blot in cardiomyocytes infected with Flag- Cpne5 and HA- FAS plasmid under FASL protein treatment. (D–G) Quantification of FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein level of (C), normalized to β-actin ( n = 5). (H) Apoptotic cells were detected using the TUNEL assay after CPNE5 and FAS knockdown under FASL protein stimulation. NC: negative control. bar = 80 μm. (I) Statistical analysis of TUNEL-positive cells in (H). (J) After treated with FASL protein, NMCMs, which treated with si- Cpne5 and si- Fas were detected CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein level by Western blot. (K–N) Quantification FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein levels of (J), normalized to β-actin ( n = 5). All data are presented as mean ± SD; unpaired two-tailed Student’s t test (two groups) and one-way ANOVA with Dunnett’s post hoc test (more than two groups) were used to determine statistical significance of experimental data. ∗∗ p < 0.01.

Article Snippet: Following this transfection step, the cells were added with 5μM CHX (MCE, cat. no. HY-12320) for protein stability study, 1 μM Chloroquine (MCE, cat. no. HY-17589A) for lysosome inhibition study, 4 μM MG132 (Selleck Chemicals, cat. no. S2619) for proteasome inhibition study, 500ng/ml FASL protein (MCE, cat. no. HY-P74157) to activate the FAS receptor, 5 μM puromycin 2HCl (Selleck Chemicals, cat. no. S7417) for protein synthesis study.

Techniques: Over Expression, Immunofluorescence, TUNEL Assay, Infection, Plasmid Preparation, Western Blot, Knockdown, Negative Control, Two Tailed Test