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. 2020 Oct 30;3:626. doi: 10.1038/s42003-020-01322-4

Fig. 6. Depletion of mito-ncR-805 does not affect steady-state mitochondrial replication, transcription, or translation but a subset of neMITO.

Fig. 6

a Antisense inhibitor (AI) of mito-ncR-805 (AI805) was transfected into MLE12 cells to decrease mito-ncR-805 levels. The sequence of the AI805 is shown. Northern blot and b FISH analysis of mito-ncR-805. Scale bar 10 μm. c, d Cells were transfected with AI805 or non-targeting RNA. Total, mitochondrial, and nuclear extracts were isolated. mito-ncR-805 levels were analyzed by RT-qPCR. e–g MLE12 cells were transfected with AI805 or non-targeting RNA. e RNA was extracted and the expression of six mitochondrial genes was tested: two encoded by the light strand and four by the heavy strand (see Fig. 2d for their location in mitochondrial genome). f Representative phospho-images of mitochondrial translation products labeled with [35S]-methionine and [35S]-cysteine for 60 min following the addition of emetine to inhibit non-mitochondrial translation or the combination of emetine and chloramphenicol to inhibit all translation. g DNA was extracted and mitochondrial copy number was determined (n = 3 biological experiments for each condition; p = 0.05). h MLE12 cells were transfected with AI805 or with non-targeting RNA. Levels of mito-ncR-805 in cells transfected with non-targeting RNA were 1.18 +/− 0.2 and in cells transfected with AI805 0.28 +/− 0.04 in 3 independent biological experiments. RNA was analyzed for changes in the expression of neMITO using Qiagen Mitochondrial Energy Metabolism RT2 Profiler PCR Arrays. All gene expression was normalized to GAPDH. Genes with expressed fold changes ≥1.7-fold were grouped based on known function. i Cells were transfected with AI805, ncR805, or with non-targeting RNA and analyzed by RT-qPCR for the expression levels of four representative genes found to be downregulated in h. Significant P values are marked by asterisks, and P values were calculated from at least 3 representative biological experiments as Control versus AI805 or Control versus ncR805 (pNdufb7C/AI = 7.01036E−05, pNdufb7C/ncR805 = 0.004, pNdufs8C/AI = 0.005, pNdufs8C/ncR805 = 3.03509E–05, pAtp6vC/AI = 0.004, pAtp6vC/ncR805 = 0.0004, pOxaL1C/AI = 0.017, pOxaL1C/ncR805 = 0.029).