Figure 6.

Tafazzin knockdown induces cellular hypertrophy and mitochondrial biogenesis. (a) and (b) Confocal images of the ShWT1 and ShTaz1 HeLa cells after staining with 5 μM AO as described in materials and methods. AO stains the nuclei (binds to DNA) and part of the cytoplasm in green (non-aggregated form) and also the acidic vesicles where it accumulates and because of its stacking is subject to a Stokes shift and is re-emitted in red (i.e., mainly the lysosomes and the autophagic vacuoles when present). * p < 0.05 vs. Control HeLa cells (ShWT1). (c) ShTaz1 knockdown cells as well as their revertant ShTAZR1 were treated and assayed for phospho-AMPK (p-AMPK) as described in materials and methods. p-AMPK was corrected for total AMPK and expressed as fold increase vs. ShWT1 control (arbitrarily set at 1). (d) Quantification of red fluorescence from TMRM staining on confocal images where the intensity per cell is taken into account. (e) ShTaz1 cells exhibited increased protein synthesis. HeLa cells were transduced with the shRNA adenovirus and labeled with 3[H]leucine as described in materials and methods. Protein synthesis is expressed as counts per minute (cpm) of 3[H]leucine incorporated. Data represent means ± SE from 4 separate experiments. From (c) to (g) the ± SEM is given on 6 independent preparations (f) Mitochondrial DNA content as assayed by NADH dehydrogenase 1 (ND1) gene content measured by RT-PCR (see materials and methods). (g) Increases expression of the hypertrophic marker brain natriuretic peptide (BNP). RT-PCR is used to detect the BNP mRNA. The BNP promoter was activated in tafazzin knockdown HeLa cells (ShWT1). ShWT1 cells were transfected with a luciferase reporter gene under the control of a BNP promoter and infected with shRNA adenovirus. The BNP promoter activity is detected by luciferase activity, expressed as fold increase vs. SCR control. ns = no significant and * is the p-value ≤ 0.05 and ** is the p-value ≤ 0.01 and *** is the p-value ≤ 0.001.