Pendent nuclear localization of TC-AR (right). Cells were counterstained with DAPI to identify nuclei (left) andModeling Truncated AR in AD BackgroundFigure 3. Cell shape and motility change of LN/TC-AR under different dox treatments. A LN/TC-AR cells were grown in the presence of hormone depleted media and treated with various concentrations of doxycycline or 1 nM DHT. CWR22Rv1 cells were grown in RPMI supplemented with 10 FBS. At 48-hours post-treatment representative images of each sample group were acquired. B LN/TC-AR cells were pre-cultured in serum free media (SFM) for 24 hours then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control. doi:10.1371/journal.pone.0049887.gKnockdown of RHOB affects cell morphology and cell migration of LN/TC-AR cells under doxycycline treatmentsRHOB, a small GTPase, is a member of the Ras-homologous (Rho) gene family, which plays a role in cell motility, apoptosis MK-8931 response and actin organization [22,23]. The aforementioned microarray data showed the overexpression of RHOB is selectively induced by TC-AR. Western blot analysis confirmed the overexpression of RHOB protein in LN/TC-AR treated with Low and High Dox, but not in DHT treated cells without Dox induction (Figure 5A). Furthermore, ChIP to chip analysis revealed that under High Dox conditions, TC-AR is recruited to 3880 bp and 47521 bp downstream of transcription end site (TES) of RHOB (Figure 5B). Given the significant alterations of the cell morphology of LN/TC-AR upon doxycycline induction, we asked whether RHOB contributes to these changes. To this end, shRNA was used to knock down RHOB expression in the LN/TC-AR cell line. Two new cell lines were established: LN/TC-AR/shR-RHOB in which shRNA MedChemExpress 370-86-5 targeting endogenous RHOB is constitutively expressed (TC-AR expression remains doxycycline dependent) and LN/TC-AR/shR-empty in which the shRNA sequence targeting RHOB has been removed. Western blot analysis of these lines revealed efficient knockdown of RHOBexpression even following indirect induction with doxycycline via TC-AR-mediated upregulation (Figure 5C). Images of LN/TC-AR/shR-RHOB cells were taken following treatment with 1 nM DHT, 24272870 Low Dox or High Dox and culture in androgen depleted media for 48 hours. The shape of doxycyclineinduced LN/TC-AR/shR-RHOB cells remained the same as DHT treated or control cells (Figure 5D). We then tested the effect of lower expression of RHOB on the migration of doxycyclineinduced LN/TC-AR cells by performing a migration assay. The result showed that knockdown of RHOB negates the TC-AR overexpression mediated increase in migration of the LN/TC-AR cell line (Figure 5E). In order to test if knockdown of RHOB affects ADI growth of LN/TC-AR cells, an MTT assay was performed. LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle as control and an MTT assay was completed on indicated days. Knockdown of RHOB did not affect the growth of DHT-treated cells, control cells or Low Dox-treated cells (Figure 5F). Thus, RHOB is likely to play a significant role in the morphological changes and migratory properties in LN/TCAR cells, but not significantly involved in the proliferation of the cells.Modeling Truncated AR in AD BackgroundDiscussionIt has been previously reported that simple overexpression of AR is sufficient to circumvent the normal androgen depen.Pendent nuclear localization of TC-AR (right). Cells were counterstained with DAPI to identify nuclei (left) andModeling Truncated AR in AD BackgroundFigure 3. Cell shape and motility change of LN/TC-AR under different dox treatments. A LN/TC-AR cells were grown in the presence of hormone depleted media and treated with various concentrations of doxycycline or 1 nM DHT. CWR22Rv1 cells were grown in RPMI supplemented with 10 FBS. At 48-hours post-treatment representative images of each sample group were acquired. B LN/TC-AR cells were pre-cultured in serum free media (SFM) for 24 hours then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control. doi:10.1371/journal.pone.0049887.gKnockdown of RHOB affects cell morphology and cell migration of LN/TC-AR cells under doxycycline treatmentsRHOB, a small GTPase, is a member of the Ras-homologous (Rho) gene family, which plays a role in cell motility, apoptosis response and actin organization [22,23]. The aforementioned microarray data showed the overexpression of RHOB is selectively induced by TC-AR. Western blot analysis confirmed the overexpression of RHOB protein in LN/TC-AR treated with Low and High Dox, but not in DHT treated cells without Dox induction (Figure 5A). Furthermore, ChIP to chip analysis revealed that under High Dox conditions, TC-AR is recruited to 3880 bp and 47521 bp downstream of transcription end site (TES) of RHOB (Figure 5B). Given the significant alterations of the cell morphology of LN/TC-AR upon doxycycline induction, we asked whether RHOB contributes to these changes. To this end, shRNA was used to knock down RHOB expression in the LN/TC-AR cell line. Two new cell lines were established: LN/TC-AR/shR-RHOB in which shRNA targeting endogenous RHOB is constitutively expressed (TC-AR expression remains doxycycline dependent) and LN/TC-AR/shR-empty in which the shRNA sequence targeting RHOB has been removed. Western blot analysis of these lines revealed efficient knockdown of RHOBexpression even following indirect induction with doxycycline via TC-AR-mediated upregulation (Figure 5C). Images of LN/TC-AR/shR-RHOB cells were taken following treatment with 1 nM DHT, 24272870 Low Dox or High Dox and culture in androgen depleted media for 48 hours. The shape of doxycyclineinduced LN/TC-AR/shR-RHOB cells remained the same as DHT treated or control cells (Figure 5D). We then tested the effect of lower expression of RHOB on the migration of doxycyclineinduced LN/TC-AR cells by performing a migration assay. The result showed that knockdown of RHOB negates the TC-AR overexpression mediated increase in migration of the LN/TC-AR cell line (Figure 5E). In order to test if knockdown of RHOB affects ADI growth of LN/TC-AR cells, an MTT assay was performed. LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle as control and an MTT assay was completed on indicated days. Knockdown of RHOB did not affect the growth of DHT-treated cells, control cells or Low Dox-treated cells (Figure 5F). Thus, RHOB is likely to play a significant role in the morphological changes and migratory properties in LN/TCAR cells, but not significantly involved in the proliferation of the cells.Modeling Truncated AR in AD BackgroundDiscussionIt has been previously reported that simple overexpression of AR is sufficient to circumvent the normal androgen depen.
