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. 2018 Feb 14;9(17):13859–13869. doi: 10.18632/oncotarget.24492

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Copyright: © 2018 Ito et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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(A) Immunohistochemical staining for AR and SV40 T antigen in the prostate lobes of TRAP rats. Representative photographs for each staining of LP (left). Hematoxylin was used as a nuclear counterstain. The percentages of positive cells for each staining (right). Data are presented as the mean ± SD, n = 12 per group, ^**P < 0.01, ^***P < 0.001 vs. the control group. (B) A Western blotting analysis for AR, SV40 T antigen, and β-actin in VP and LP of TRAP. The intensity of each band was measured and normalized by β-actin. Western blotting was repeated at least three times, and each lane represents an individual rat. Data are presented the relative value for a Control (Ventral: lane 3, Lateral: lane 2) as the mean ± SD, n = 4 per group. (C, D) A quantitative RT-PCR analysis for AR (C), AT1R, and AT2R (D) in LP of TRAP rats. The average results were calculated from five rats per group. Each PCR was repeated 4 times. The data was normalized with Gapdh as an endogenous control gene. (E) RT-PCR array analysis of the effect of C21 on the expression of 84 androgen-regulated genes in LP of TRAP rats. RNA samples from all rats in the Control (n = 12) and the 2 mg/kg/day C21 (n = 12) groups were analyzed. The data was normalized to the Actb gene. Top left, scatter plot: Green dots represent genes that were down-regulated. The center line indicates no changed in gene expression, while the boundary dotted lines represent a 2-fold change in expression. Top right, clustergram: non-supervised hierarchical clustering of the entire data set to display a heat map indicating gene alteration across groups (83 out of every 84 genes). Red shows relatively high expression level (n = 7), green shows relatively low expression level (n = 76) as compared with the other group. Bottom, List of genes down-regulated more than double by C21 treatment.

(A) Immunohistochemical staining for AR and SV40 T antigen in the prostate lobes of TRAP rats. Representative photographs for each staining of LP (left). Hematoxylin was used as a nuclear counterstain. The percentages of positive cells for each staining (right). Data are presented as the mean ± SD, n = 12 per group, ^**P < 0.01, ^***P < 0.001 vs. the control group. (B) A Western blotting analysis for AR, SV40 T antigen, and β-actin in VP and LP of TRAP. The intensity of each band was measured and normalized by β-actin. Western blotting was repeated at least three times, and each lane represents an individual rat. Data are presented the relative value for a Control (Ventral: lane 3, Lateral: lane 2) as the mean ± SD, n = 4 per group. (C, D) A quantitative RT-PCR analysis for AR (C), AT1R, and AT2R (D) in LP of TRAP rats. The average results were calculated from five rats per group. Each PCR was repeated 4 times. The data was normalized with Gapdh as an endogenous control gene. (E) RT-PCR array analysis of the effect of C21 on the expression of 84 androgen-regulated genes in LP of TRAP rats. RNA samples from all rats in the Control (n = 12) and the 2 mg/kg/day C21 (n = 12) groups were analyzed. The data was normalized to the Actb gene. Top left, scatter plot: Green dots represent genes that were down-regulated. The center line indicates no changed in gene expression, while the boundary dotted lines represent a 2-fold change in expression. Top right, clustergram: non-supervised hierarchical clustering of the entire data set to display a heat map indicating gene alteration across groups (83 out of every 84 genes). Red shows relatively high expression level (n = 7), green shows relatively low expression level (n = 76) as compared with the other group. Bottom, List of genes down-regulated more than double by C21 treatment.