Fig 7. Tafazzin knockdown and Dox-sensitivity in H9c2 cardiomyocytes.

(A) knockdown of tafazzin was confirmed in H9c2 cells after transfection with 20 nM tafazzin siRNA for 48 h by western blot analysis. (B) Representative western blot showing the level of cleaved caspase-3. After transfection with tafazzin and NTP siRNA for 24 h, cells were treated with 0.5 and 1 μM Dox for 24 h and cleaved caspase-3 levels were quantified using tubulin as a loading control. (C) Quantitative analysis of cleaved caspase-3 from (B) normalized by α-tubulin. (D) Mitochondrial superoxide levels were measured using MitoSOX Red dye in tafazzin and NTP transfected control H9c2 cells at basal levels and (E) after 0.5 and 1μM Dox treatment for 24 h. The changes in mean fluorescence intensity of mitochondrial superoxide sensitive dye MitoSOX Red in healthy and Barth lymphocytes was normalized by the corresponding control sample to compare the Dox-induced effect in both cell types. Mitochondrial complex III inhibitor Antimycin A (20 μM) was used as a positive control. (F) Measurement of mitochondrial membrane potential using JC-1 dye for NTP and Taz siRNA transfected H9c2 after treatment with Dox for 2 h. Y-axis represents the relative fluorescence ratio of 590/540 nm normalized to control without Dox treatment. Basal level of mitochondrial membrane potential in Taz siRNA transfected cells was slightly lower than NTP siRNA transfected cells (1.03 for NTP siRNA transfected cells and 0.9 for Taz siRNA transfected cells). CCCP (10 μM), a commonly used mitochondrial membrane depolarization agent was used as a positive control. To determine the Dox-induced effect between NTP and Taz siRNA transfected H9c2 cells, basal level of ROS and mitochondrial membrane potential of untreated (control) NTP and Taz siRNA transfected H9c2 cells were normalized to 100; and Dox-induced changes in the ROS and mitochondrial membrane potential were normalized against corresponding control. *p<0.05; **p<0.001.