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. 2021 Oct 19;41(7):943–959. doi: 10.1038/s41388-021-02060-5

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© The Author(s) 2021, corrected publication 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — LNCaP cells were transfected with nontargeting siRNA (siControl) as negative control or siSAT1 targeting the additional exon in lncRNASAT1. After 24 h of transfection, cells were treated with SAL or 0.1% DMSO as solvent control for 48 h followed by RNA extraction. A The knockdown of lncRNASAT1 in LNCaP cells was confirmed with qRT-PCR (n = 3). B The level of cellular senescence was analyzed by quantification of SA-β-Gal- positive stained LNCaP cells (n = 3). C The levels of CDKN2B mRNA and p15^INK4B protein were analyzed with and without lncRNASAT1 knockdown (n = 3). D The level of lncRNASAT1 was analyzed in the CDKN2B-knockdown LNCaP cells using qRT-PCR (n = 3). E Changes of panAKT, p-AKT, panS6, and p-S6 levels after knockdown of lncRNASAT1 (n = 3). F Growth curves of LNCaP cells with and without SAL treatment comparing with and without knockdown of lncRNASAT1 analyzed by crystal violet staining (n = 2). G LncRNASAT1 suppression mediates apoptosis in LNCaP cells. Western blot analysis of cleaved PARP in response to lncRNASAT1 knockdown after 48 h of treatment with DMSO as solvent control or 1 nM R1881 (SAL). The protein expression was normalized to the loading control β-Actin using LabImage 1D software (n = 2). H, I qRT-PCR was used to analyze the expression of AR target genes through knockdown of lncRNASAT1 in LNCAP cells treated with and without SAL. Relative expression was calculated compared with solvent control. The mean ± SEM values were calculated from three independent experiments (n = 3). Indicated are (H) the positively androgen-regulated KLK3 and FKBP5 genes and (I) the negatively androgen-repressed hTERT gene. J LNCaP cells were incubated for 24 h with DMSO or SAL followed by cytosolic and nuclear extraction. Immunoprecipitation was performed using an AR antibody or a matched immunoglobulin G (IgG) as control. RNA-immunoprecipitated lncRNASAT1 was analyzed by qRT-PCR (n = 2). K RNA-ChIP was performed with cross-linked chromatin from LNCaP cells treated with DMSO or SAL for 24 h followed by immunoprecipitation of AR and qRT-PCR detection of the lncRNASAT1 (n = 4). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, n.s. not significant.

Fig. 7 — LNCaP cells were transfected with nontargeting siRNA (siControl) as negative control or siSAT1 targeting the additional exon in lncRNASAT1. After 24 h of transfection, cells were treated with SAL or 0.1% DMSO as solvent control for 48 h followed by RNA extraction. A The knockdown of lncRNASAT1 in LNCaP cells was confirmed with qRT-PCR (n = 3). B The level of cellular senescence was analyzed by quantification of SA-β-Gal- positive stained LNCaP cells (n = 3). C The levels of CDKN2B mRNA and p15^INK4B protein were analyzed with and without lncRNASAT1 knockdown (n = 3). D The level of lncRNASAT1 was analyzed in the CDKN2B-knockdown LNCaP cells using qRT-PCR (n = 3). E Changes of panAKT, p-AKT, panS6, and p-S6 levels after knockdown of lncRNASAT1 (n = 3). F Growth curves of LNCaP cells with and without SAL treatment comparing with and without knockdown of lncRNASAT1 analyzed by crystal violet staining (n = 2). G LncRNASAT1 suppression mediates apoptosis in LNCaP cells. Western blot analysis of cleaved PARP in response to lncRNASAT1 knockdown after 48 h of treatment with DMSO as solvent control or 1 nM R1881 (SAL). The protein expression was normalized to the loading control β-Actin using LabImage 1D software (n = 2). H, I qRT-PCR was used to analyze the expression of AR target genes through knockdown of lncRNASAT1 in LNCAP cells treated with and without SAL. Relative expression was calculated compared with solvent control. The mean ± SEM values were calculated from three independent experiments (n = 3). Indicated are (H) the positively androgen-regulated KLK3 and FKBP5 genes and (I) the negatively androgen-repressed hTERT gene. J LNCaP cells were incubated for 24 h with DMSO or SAL followed by cytosolic and nuclear extraction. Immunoprecipitation was performed using an AR antibody or a matched immunoglobulin G (IgG) as control. RNA-immunoprecipitated lncRNASAT1 was analyzed by qRT-PCR (n = 2). K RNA-ChIP was performed with cross-linked chromatin from LNCaP cells treated with DMSO or SAL for 24 h followed by immunoprecipitation of AR and qRT-PCR detection of the lncRNASAT1 (n = 4). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, n.s. not significant.