fig4
Figure 4. Exacerbation of OGD-induced cardiomyocyte injury and apoptosis by JOSD2 overexpression. (A) RT-qPCR validation of JOSD2 mRNA levels in AC16 cells transduced with JOSD2-overexpressing lentiviral vectors (OE-JOSD2) or control vector (OE-Vector) (n = 4 per group); (B) Western blots showing JOSD2 protein levels in OE-JOSD2 and OE-Vector stable AC16 cells; (C) Densitometric quantification of JOSD2 protein levels (n = 3); (D) RT-qPCR analysis of cardiac stress marker NPPA and NPPB in OE-Vector, OE-JOSD2, OE-Vector+OGD, and OE-JOSD2+OGD cells (n = 4 per group); (E) Western blots of apoptosis-associated proteins (BAX, BCL-2, c-Cas-3, c-Cas-9) in OE-Vector and OE-JOSD2 cells with and without OGD; (F) Densitometric quantification of apoptosis-associated proteins in (E) (n = 3); (G) Representative flow cytometry plots assessing apoptosis in OE-Vector and OE-JOSD2 cells under control conditions and after OGD; (H) Quantification of apoptotic cells from flow cytometry analyses (n = 3 independent biological replicates); (I) Representative TUNEL staining images in OE-Vector and OE-JOSD2 AC16 cells under control conditions and after OGD. Scale bar, 50 μm; (J) Quantification of TUNEL+ cells (n = 5); (K) CCK-8 assay assessing cell viability in OE-Vector and OE-JOSD2 cells under control conditions and after OGD (n = 4). All results are representative of at least three independent experiments. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test for (A) and (C), and two-way ANOVA with Tukey’s post hoc test for (D), (F), (H), (J), and (K). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. JOSD2: Josephin domain-containing protein 2; RT-qPCR: reverse transcription quantitative polymerase chain reaction; MI: myocardial infarction; OGD: oxygen-glucose deprivation; TUNEL: transferase dUTP nick-end labelling; SD: standard deviation; NPPB: natriuretic peptide B; ANOVA: analysis of variance; NPPA: natriuretic peptide A; PI-PE: propidium iodide-P-phycoerythrin; Annexin V-FITC: annexin V-fluorescein isothiocyanate; ACTB: beta actin.
