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. 2019 Apr 29;20(6):e48101. doi: 10.15252/embr.201948101

Figure 4. Accumulated shorter LSP transcripts in TEFM‐depleted mitochondria do not contribute to mtDNA replication.

Figure 4

  1. In organello transcription analyses using heart mitochondria isolated from 8‐week‐old control and Tefm knockout mice (n = 5 mice for each group). The steady‐state levels of 12S rRNA and mt‐tRNAV were used as endogenous controls, due to their unchanged levels. VDAC was used as loading control for the equal input of mitochondrial extracts. This experiment has performed five times with either two or three replicates.
  2. Northern blot analyses of 7S RNA level in 4‐ and 8‐week‐old control and Tefm knockout mouse hearts (n = 12 mice at each time‐point for each group). 18S rRNA was used as loading control. RNA from tissue‐specific Mterf4 knockout mouse hearts and tissue‐specific Polrmt knockout mouse hearts was loaded as positive and negative control, respectively.
  3. Southern blot analyses of mtDNA to obtain 7S DNA in 8‐week‐old control and Tefm knockout mice. Heart mitochondria from three control or knockout mice were pooled for preparation of mtDNA. Equal amounts of mtDNA shown on the top of the gel were loaded as the endogenous control. This experiment has performed three times.
  4. In organello replication assays were performed on heart mitochondria isolated from 8‐week‐old control and Tefm knockout mice (n = 5 mice for each group). VDAC was used as loading control.
  5. Northern blot analyses of the 7S RNA levels in 4‐ and 8‐week‐old control and Tefm knockout mouse hearts (n = 12 mice for each group). 5S rRNA was used as loading control. RNA from tissue‐specific Mterf4 knockout mouse hearts and tissue‐specific Polrmt knockout mouse hearts was loaded as positive and negative control, respectively.
  6. Mapping 3′ termini of mitochondrial 7S RNA/early LSP transcripts in control (L/L) and Tefm knockout (L/L, cre) mouse hearts at the age of 8 weeks. A 3′ RACE was performed using linker‐ligated RNA and a forward primer corresponding to the first 45 bp from the transcription initiation site at LSP. CSB, conserved sequence block.