Figure 3

Manipulation of mTERF expression affects relative levels of antisense transcripts of the 16S rRNA and ND1 genes. (A) Schematic diagram of 16S rDNA-ND1 region of human mtDNA. For full details of primer sequences and location, see Additional File 1, Table S1. (B) Relative steady-state levels of anti-16S and anti-ND1 transcripts, determined by Q-RT-PCR using proximity probe hybridization (probe sets R1, N1 and C1 for normalization), after various manipulations of mTERF expression, i.e. two mTERF overexpressor clones (OE) compared with vector-transfected cells, doxycyclin-induced versus uninduced Flp-In™ T-Rex™-293 cells transfected with mTERF expression construct, and HEK293T cells transfected with mTERF-targeted shRNA versus mock-transfected cells. In each case, data were normalized to the corresponding control cells. * denotes statistically significant differences from control cells (t test, p < 0.02). (C, D) Relative changes in anti-16S and anti-ND1 transcripts, based on replotting of data from the experiment of panel B for each transcript individually, (C) following induced expression of mTERF in Flp-In™ T-Rex™-293 cells and (D) in HEK293T cells transfected with mTERF-targeted shRNA versus mock-transfected cells. Data were normalized to values for corresponding untreated control cells, using 18S as internal normalization standard. * denotes statistically significant differences from corresponding control cells (t test, p < 0.01). (E, F) Relative steady-state levels of 12S, 16S and 18S sense-strand transcripts, as determined by Q-RT-PCR using of proximity probe hybridization (primer sets T1, R2, C1 respectively, as described in Additional File 1, Table S1), and of mTERF mRNA relative to 18S rRNA (probes sets M1 and C1, see Additional File 1, Table S1), (E) following induced expression of mTERF in Flp-In™ T-Rex™-293 cells and (F) in HEK293T cells transfected with mTERF-targeted shRNA versus mock-transfected cells. * denotes statistically significant differences (t test, p < 0.02). See also Additional File 1, Fig. S3.