Figure 5.

CoCl₂ induces autophagy in H9c2 cardiomyoblasts. Cells were NT (white) or treated with CoCl₂ (300 μM, black) for different lengths of time. Protein (a–c) and mRNAs (d) densitometric quantification of autophagic markers: Beclin-1, LC3 and p62. The densitometric quantification of protein LC3II/LC3I ratio is reported. mRNA expression analysis was performed at 48 h. The t-test was performed on treated versus NT cells. (e) Cell viability was measured by MTT. Cells were NT (white) or treated with CoCl₂ for 48 h (black), CoCl₂+1 mM 3-MA inhibitor of autophagy in the last 12 h (dark grey) and 3-MA alone (light grey). Results are expressed as percentage of MTT reduction, relative to NT control. t-test was calculated comparing CoCl₂ versus CoCl₂+3-MA. (f) Densitometric quantification of protein LC3II/LC3I ratio in cells cultured in the presence or absence of Baf (50 nM). Cells were treated with CoCl₂ for 48 h and Baf was added in the last 6 h. In the graph four conditions of treatment are reported: NT (white), Baf (light grey), CoCl₂ (black) and CoCl₂+Baf (dark grey). t-test was calculated between NT versus Baf and CoCl₂ versus CoCl₂+Baf. Tubulin and β-actin were used as loading control for protein and mRNA, respectively. Representative images are reported below each graph. The values are the mean±S.D. of three independent experiments. In all the experiments, except for (e), values are expressed as fold relative to NT. *P<0.05 and ***P<0.005