Extended Data Fig. 2: Organelle-associated functional lncRNAs are identified and validated.
From: Mitochondrial long non-coding RNA GAS5 tunes TCA metabolism in response to nutrient stress
(a-c) The indicated candidates of mitochondria (a), lysosome (b) and ER (c) were verified by organelle purification coupled with RT-qPCR. The positive control of mitochondria (ATP8, MT-RNR1), lysosome (LAMP2) and ER (FGF2, TJP1) was shown as red column; and the negative control (GAPDH, U6, MALAT1, XIST) was shown as white column; the confirmed candidates were shown as blue column; other candidates were shown as grey column. The relative enrichment of each RNAs was calculated by 2^-(CtMito-CtTotal) followed by normalizing all ratio value to the GAPDH in control group (the first GAPDH column). Cut-off line was thereby determined by the relative GAPDH enrichment level. Data are presented as mean values ± S.D., n = 3 biologically independent experiments. (d-e) The knockdown efficiency of siRNA interference for the indicated mitochondria (d) and lysosome (e) candidates was determined by RT-qPCR. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, **P < 0.01, ***P < 0.001. (f) Immunoblotting detection of AMPK activation by measuring the p-AMPK ratio in HEK293T. Cells were interfered by lysosome candidate’s siRNA and treated with 2-hour glucose starvation. (g) Relative cellular ADP/ATP ratio was determined in HEK293T cells by mitochondrial candidate’s siRNA screening. Each candidate had two replicated siRNA, and three biological duplication for each siRNA. Data are presented as mean values ± S.D., n = 6 biologically independent experiments, Two-sided Student’s t-test, **P < 0.01, ***P < 0.001.
