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. 2023 Feb 2;51(6):2655–2670. doi: 10.1093/nar/gkad043

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Re-expression of AR partly rescues the AR target expression and growth of SMAD3-KD PCa cells. (A) Western blot showing re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells. Optimal amounts of lentivirus were used to restore the expression of AR and AR-V7 in the SMAD3-KD cells to the levels seen in control cells. (B) Real-time RT-PCR results showing that re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued the expression of example AR targets. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (C and D) Re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued colony formation in cell-culture plates. The indicated cells were seeded at low density and maintained for 2 weeks. Colony numbers were scored in the 12 high-power fields. The example image represents 4 high-power fields. Colony number per field was presented as mean ± SD (n = 12), and ANOVA was used for statistical analysis (***P < 0.001). (E and F) Re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued the colony formation in soft agar. The indicated cells were grown in soft agar for 3 weeks and colony numbers were scored in 12 high-power fields. The example image represents 1 high-power field. Colony number per field was presented as mean ± SD (n = 12), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (G) Western blot showing re-expression of AR in the SMAD3-KD C4-2 cells. (H) Real-time RT-PCR results showing that re-expression of AR in the SMAD3-KD C4-2 cells partly rescued the expression of example AR targets. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (I) Re-expression of AR in the SMAD3-KD C4-2 cells partly rescued the colony formation in cell-culture plates. The procedure is as described in C and D. Colony numbers were scored in 8 high-power fields. Colony number per field was presented as mean ± SD (n = 8), and ANOVA was used for statistical analysis (***P < 0.001). (J) Re-expression of AR in the SMAD3-KD C4-2 cells partly rescued the colony formation in soft agar. The procedure is as described in E and F. Colony number per field was presented as mean ± SD (n = 12), and ANOVA was used for statistical analysis (***P < 0.001). (K and L) Re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued the xenograft tumor formation. The indicated cells (1 × 10⁶) were subcutaneously injected into athymic nude mice (n = 10 per group). Xenograft tumors were collected at 5 weeks post injection. The image (K) and weight (L) of xenograft tumors are shown. Tumor weight was presented as mean ± SD (n = 10), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001).