Research Paper Volume 12, Issue 5 pp 4322—4336

Figure 3. lncRNA sirt1 antisense (AS) upregulates sirt1 expression by increasing the stability of sirt1 mRNA. (A) Real-time qPCR analysis of sirt1 mRNA levels in RLE-6TN cells treated with 10 ng/ml TGF-β1 treatment for various time, as indicated. (B) QPCR of sirt1 mRNA in RLE-6TN cells transfected with ad-sirt1 AS or ad-NC. (C) Western blot analysis and quantitative analyses of sirt1 protein levels in RLE-6TN cells transfected with ad-sirt1 AS or ad-NC. (D) QPCR was used to validate the knockdown efficiency of lentivirus-mediated sh-sirt1 AS. (E) QPCR of sirt1 mRNA in RLE-6TN cells transfected with shRNA against sirt1 AS (sh-sirt1 AS) or sh-NC. (F) Western blot assay and quantitative analyses of sirt1 protein levels in RLE-6TN cells transfected with sh-NC or sh-sirt1 AS. (G) Nucleocytoplasmic separation results confirmed that sirt1 AS was almost all expressed in the cytoplasm of RLE-6TN cells by using qPCR analysis. (H) RNA FISH was used to determine the location of endogenous sirt1 AS (red) expression in RLE-6TN cells. (I) Nonoverlapping (P1-P2) and overlapping (P3-P4) primer positions for ribonuclease protection assay (RPA). (J) RPA was performed on RNA samples from RLE-6TN cells. (K) RNA stability assay of sirt1 mRNA expression in RLE-6TN cells transfected with ad-sirt1 AS or ad-NC. * P<0.05 vs. negative control group.