Figure 3.

PCA PKM2-dependently protects cardiomyocytes. Primary neonatal rat ventricular myocytes (NRVMs) were cultured in glucose-free DMEM under 1% O₂ (OGD) for 4 h in the presence of protocatechuic aldehyde (PCA). (A) Cell survival in NRVMs exposed to OGD for 6 h (n = 6). (B) Intracellular ROS production (n = 6). (C) Representative images of mitochondrial permeability transition pore (mPTP) and quantification of relative fluorescence intensity (n = 6, one of three independent experiments). Scale bar: 10 μm. (D) Mitochondrial fission was detected by Mito-Tracker Red (one of 3 independent experiments) and quantification analysis. Scale bar: 10 μm. (E) Caspase 3 activity in NRVMs (n = 6). (F) Representative Western blots of BAX, BCL-2, caspase 3, cleaved caspase 3 (c-caspase 3) and the ratio of BAX/BCL-2 (n = 3). (G) Representative images of TUNEL staining and quantification analysis the percentage of apoptosis cells. Scale bars, 100 μm (n = 3). (H) Data of Annexin V/PI double staining flow cytometry; the percentage for each panel indicates the percentage of apoptotic cells and quantification of the percentage of apoptotic cells (n = 3). Results are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.