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. 2023 Aug 8;12:e83831. doi: 10.7554/eLife.83831

Figure 3. Pharmacological inhibition of EPAC1, but not EPAC2, protects cardiomyocytes from doxorubicin (Dox)-induced DNA damage.

Figure 3.

(a) Neonatal rat ventricular myocyte (NRVM) were untreated or treated with Dox (1 µM) ± the specific EPAC1 inhibitor CE3F4 (10 µM) for 16 hr and the level of the DNA damage marker H2AX-pS139 was analyzed by western blot. Actin was used as a loading control. Protein levels relative to Ctl are presented in bar graphs (mean ± SEM, n=3–5). **p<0.01 vs. control, ##p<0.01 vs. Dox alone. (b) NRVM were transfected with control (shCtl) or EPAC1 (shEPAC1) shRNA for 12 hr before treatment with Dox for 16 hr. The relative levels of EPAC1 and H2AX-pS139 were measured by immunoblotting with actin as a loading control (mean ± SEM, n=3–5). ***p<0.001 vs. shCtl, ##p<0.01 vs. shCtl + Dox. (c, d) NRVM were untreated or treated with Dox (1 µM) and either the EPAC activator 8-CPT (10 µM) or the EPAC1 inhibitor ESI-09 (1 µM) or the EPAC2 inhibitor ESI-05 (1 µM) for 24 hr. (c) The level of the DNA damage marker H2AX-pS139 was analyzed by western blot. Actin was used as a loading control. Protein levels relative to Ctl are presented in bar graphs (mean ± SEM, n=3). *p<0.05, **p<0.01, ***p<0.001 vs. control, #p<0.05 vs. Dox alone. (d) The level of free topoisomerase IIβ (TopIIβ) was quantified by band depletion assay. Actin was used as a loading control. Protein levels relative to Ctl are presented in bar graphs (mean ± SEM, n=3–5). ***p<0.001 vs. control, ###p<0.001 vs. Dox alone.

Figure 3—source data 1. Raw data for Figure 3.
Figure 3—source data 2. Uncropped WB for Figure 3.
Figure 3—source data 3. Raw WB imaging for Figure 3.