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. 2018 Oct 23;7(21):e009700. doi: 10.1161/JAHA.118.009700

Figure 6.

Figure 6

Sirt1 antisense (AS) long noncoding RNA (lncRNA) increases stability of Sirt1 mRNA. A, Real‐time quantitative PCR (qPCR) analysis of Sirt1 mRNA levels in embryonic day 16.5 (E16.5), postnatal day 1 (P1), and postnatal day 28 (P28) mouse hearts (n=6 mice per group). B, Real‐time qPCR analysis of Sirt1 mRNA levels in isolated cardiomyocytes (CMs) transfected with locked nucleic acid–control (LNA‐NC) or LNA–Sirt1 AS lncRNA (SAS) (n=3 mice per group). C, Western blotting analysis and quantitative analyses of Sirt1 protein levels in isolated CMs transfected with LNA‐NC or LNA‐SAS. β‐Actin was used as a loading control (n=3 mice per group). D, Real‐time qPCR analysis of Sirt1 mRNA levels in isolated CMs transfected with various multiplicities of infection (MOIs) of adenovirus (Ad)–SAS (n=3 mice per group). E, Western blotting analysis and quantitative analyses of Sirt1 protein levels in isolated CMs transfected with various MOIs of Ad‐SAS. β‐Actin was used as a loading control (n=3 mice per group). F, Western blotting analysis and quantitative analyses of Sirt1 protein levels in adeno‐associated virus 9 (AAV9)–NC– or AAV9‐SAS–injected adult mice hearts 28 days after injection. β‐Actin was used as a loading control (n=6 mice per group). G, Real‐time qPCR of cytoplasmic and nuclear fractions to detect Sirt1 AS lncRNA expression in isolated CMs (n=3 mice per group). H, RNA fluorescence in situ hybridization assay of Sirt1 AS lncRNA immunofluorescence detection in isolated CMs. Bar=20 μm. I, Nonoverlapping and overlapping primer positions for ribonuclease protection assay (RPA). J, RPA performed on RNA samples from isolated CMs (n=3 mice per group). K, RNA stability assay of Sirt1 mRNA expression in isolated CMs transfected with Ad‐NC or Ad‐SAS (n=3 mice per group). Statistical significance was calculated using 1‐way ANOVA followed by least significant difference post hoc test in A, D, E, J, and K, and 2‐tailed unpaired Student t test in B, C, F, and G. Data represent mean±SEM. DAPI indicates 4’,6‐diamidino‐2‐phenylindole. *P<0.05.