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. 2020 Jun 18;10(6):925. doi: 10.3390/biom10060925

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Caudatin inhibits the glucocorticoid receptor (GR) signal through the ubiquitin (Ub)-dependent degradation of GR. (A) The expression level of GR in the total protein of MDA-MB-231-derived mammospheres was measured after treatment of the mammospheres with caudatin for 48 h using western blot analyses. (B) After treatment with caudatin, the transcriptional expression of the GR gene was detected in mammospheres by real-time reverse transcription- quantitative polymerase chain reaction (RT-qPCR) using specific primers. β-actin was used as an internal control. (C) Mammospheres incubated with caudatin were lysed for immunoprecipitation (IP) with an anti-GR antibody, and western blot analysis was performed using anti-ubiquitin and anti-GR antibodies. (D) Mammospheres were incubated with caudatin and MG-132 (0.5 μM) for 24 h and lysed for western blot analysis. (E) The expression level of GR in the cytosolic and nuclear protein fractions of MDA-MB-231-derived mammospheres was measured after treatment of the mammospheres with caudatin using western blot analysis. (F) Immunofluorescence (IF) analysis of GR (green) expression and localization in MDA-MB-231 cells under caudatin treatment was performed. (G) The effect of knocking down GR expression using GR-specific siRNA on mammosphere formation was evaluated. (H) The effect of RU-486, an antagonist of GR, on mammosphere formation was evaluated. The data are presented as the mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 versus the DMSO-treated control group.

Caudatin inhibits the glucocorticoid receptor (GR) signal through the ubiquitin (Ub)-dependent degradation of GR. (A) The expression level of GR in the total protein of MDA-MB-231-derived mammospheres was measured after treatment of the mammospheres with caudatin for 48 h using western blot analyses. (B) After treatment with caudatin, the transcriptional expression of the GR gene was detected in mammospheres by real-time reverse transcription- quantitative polymerase chain reaction (RT-qPCR) using specific primers. β-actin was used as an internal control. (C) Mammospheres incubated with caudatin were lysed for immunoprecipitation (IP) with an anti-GR antibody, and western blot analysis was performed using anti-ubiquitin and anti-GR antibodies. (D) Mammospheres were incubated with caudatin and MG-132 (0.5 μM) for 24 h and lysed for western blot analysis. (E) The expression level of GR in the cytosolic and nuclear protein fractions of MDA-MB-231-derived mammospheres was measured after treatment of the mammospheres with caudatin using western blot analysis. (F) Immunofluorescence (IF) analysis of GR (green) expression and localization in MDA-MB-231 cells under caudatin treatment was performed. (G) The effect of knocking down GR expression using GR-specific siRNA on mammosphere formation was evaluated. (H) The effect of RU-486, an antagonist of GR, on mammosphere formation was evaluated. The data are presented as the mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 versus the DMSO-treated control group.