fig3
Figure 3. Protective effects of JOSD2 knockdown against OGD-induced apoptosis and injury-associated responses in AC16 cells. (A) RT-qPCR validation of JOSD2 mRNA levels in AC16 cells transduced with sh-Scr or three independent shRNA constructs targeting JOSD2 (n = 3 per group); (B) Western blots showing JOSD2 protein levels in sh-Scr and sh-JOSD2 (sh-3) stable AC16 cells; (C) Densitometric quantification of JOSD2 protein levels (n = 3); (D) RT-qPCR analysis of cardiac stress marker genes NPPA and NPPB in sh-Scr and sh-JOSD2 AC16 cells under basal conditions and after OGD (n = 4 per group); (E) Western blots of apoptosis-associated proteins (BAX, BCL-2, c-Cas-3, c-Cas-9) in sh-Scr and sh-JOSD2 AC16 cells under basal conditions and after OGD; (F) Densitometric quantification of apoptosis-associated proteins (n = 3); (G) Representative flow cytometry plots assessing apoptosis in sh-Scr and sh-JOSD2 AC16 cells under control conditions and after OGD for 6 h or 9 h; (H) Quantification of apoptotic cells from flow cytometry analyses (n = 3 independent biological replicate experiments); (I) Representative TUNEL staining images in sh-Scr and sh-JOSD2 AC16 cells under control conditions and after OGD. Scale bar, 50 μm; (J) Quantification of TUNEL+ cells (n = 5); (K) Heatmap showing the expression patterns of anti-apoptotic and pro-apoptotic genes derived from RNA-seq comparing sh-Scr+OGD versus sh-JOSD2+OGD. All results are representative of at least three independent experiments. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test for (A), an unpaired two-tailed Student’s t-test for (C), and two-way ANOVA with Tukey’s post hoc test for (D), (F), (H), and (J). 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; NPPB: natriuretic peptide B; TUNEL: transferase dUTP nick-end labelling; ANOVA: analysis of variance; ACTB: beta actin; NPPA: natriuretic peptide A; PI-PE: propidium iodide-P-phycoerythrin; Annexin V-FITC: Annexin V-fluorescein isothiocyanate.
