Fig 1.
Phosphoproteomic analysis of XCT-790 and tamoxifen treated MDA-MB-231 cells.
(A) Steps of the work flow chart of samples treated for the phosphroproteomic analysis. (B) Outcome of number of identified unique phosphorylated proteins in each group.
Table 1.
Significant phosphoproteomic changes inXCT-790 as well as tamoxifen treated samples.
A table of statistically significant hits from phosphoproteomic analysis of cells treated with XCT-790 as well as tamoxifen.
Fig 2.
XCT-790 and tamoxifen treatments regulate signaling of MAPK pathway.
(A) MDA-MB-231 cells were grown in starvation media for 24 hrs and treated with either 10 μM XCT-790 or 100 nM tamoxifen for 5, 15, 30 or 60 minutes. Cells were lysed as described in “Materials and Methods” and indicated proteins were detected by immunoblot. (B) MDA-MB-231 cells were grown in starvation media for 24 hrs and treated with 10 μM XCT-790 and 100 nM tamoxifen for 5, 15, 30 or 60 minutes. Cells were lysed as described in “Materials and Methods” and indicated proteins were detected by immunoblot. * indicates phosphorylation changes in [PXpTP] motif as compared to control. ‘C’ is untreated control.
Fig 3.
XCT-790 and tamoxifen treatments upregulate phosphorylation of p-ERK on Y204/187.
(A) MDA-MB-231 cells were grown in starvation media for 24 hrs and treated with either 10 μM XCT-790 or 100 nM tamoxifen for 5, 15, 30 or 60 minutes. Cells were lysed as described in “Materials and Methods” and indicated proteins were detected by immunoblot. (B) Quantification of p-ERK Y204/187 protein levels normalized to total ERK signal from ‘A’. (C) Quantification of p-RSK1 S380 protein levels normalized to actin signal from ‘A’. (D) MDA-MB-231 cells were grown in starvation media for 24 hrs and treated with 10 μM XCT-790 and 100nM tamoxifen for 5, 15, 30 or 60 minutes. Cells were lysed as described in “Materials and Methods” and indicated proteins were detected by immunoblot. (E) Quantification of p-ERK Y204/187 protein levels normalized to actin signal from ‘D’. (F) Quantification of p-RSK1 S380 protein levels normalized to actin signal from ‘D’. * represents P<0.05, ** represents P<0.01 and *** represents P<0.001.
Fig 4.
Tamoxifen potentiates phosphorylation of ERK on Y204/187 in the absence of ERRα.
(A) sh Control and sh ERRα cells were treated with 100 nM of tamoxifen for 5, 15, 30, or 60 minutes. Cells were lysed as described in “Materials and Methods” and the indicated proteins were detected by immunoblot. (B) Relative levels of p-ERK Y204/187 normalized to actin, ** represents P<0.01. (C) Relative levels of p-ERK Y204/187 and ERRα, normalized to actin from ‘A’. (D) MDA-MB 231 cells were treated with 10 μM XCT-790 and/or 100 nM tamoxifen for 30 minutes followed by lysis with NE-PER Nuclear Cytoplasmic extraction kit as described in ‘Materials and Methods’ and lysates from nuclear and cytoplasmic extractions were immunoblotted with the indicated antibodies.
Fig 5.
Inhibition of ERRα potentiates apoptotic effects of U0126 in TNBC cells.
(A) MDA-MB-231 cells were serum starved for 24 hrs and treated with 10 μM XCT-790, 100 nM tamoxifen, or 10 μM U0126, alone or in combination for 48 hrs. Cells were imaged under 20x magnification using BZX-800 microscope from Keyence. Scale bar represents 100 μm. (B) MDA-MB-231 cells were seeded in 96-well plate in complete DMEM media, supplemented with 10% FBS and treated with 10 μM XCT-790, 100 nM tamoxifen, or 10 μM U0126, alone or in combination for 6 days and cell viability was measured using Neutral Red cytotoxicity assay as described in “Materials and Methods”. (C) MDA-MB-231 cells were serum starved for 24 hrs and treated with 10 μM XCT-790, 100 nM tamoxifen, or 10 μM U0126, alone or in combination for 24 hrs as indicated. Cells were lysed as described in “Materials and Methods” and immunoblotted for indicated proteins. Arrow indicates cleaved PARP fragment. (D) Quantification of cleaved PARP fragment from “B” normalized to actin. * represents P<0.05, ** represents P<0.01 and *** represents P<0.001.
Fig 6.
Inhibition of ERRα together with U0126 prevents cell migration and invasion in TNBC cells.
(A) MDA-MB-231 cells were seeded in a 6-well plate in complete DMEM media and allowed to attach overnight. The following day cells were placed in reduced (1%) serum media and following 8–10 hrs of pretreatment with 10rμM XCT-790, 100nM tamoxifen, or 10rμM U0126, alone or in combination and wound/scratch was generated. Wound closure was measured 17 hrs post generation. (B) Cell migration/Boyden chamber assay was performed as described in “Materials and Methods”. Representative images of MDA-MB-231 cells stained with 4′,6-diamidino-2-phenylindole (DAPI) following 15 hrs migration assay are shown. (C) Quantification of the Wound Healing assay from (A) was performed and graphed using paired Student’s t-test. * represents P<0.05. ** represents P<0.01. (D) Histogram representing the number of cells migrated relative to untreated control. * represents P<0.05, ** represents P<0.01 and *** represents P<0.001.
Fig 7.
(A) The represented TNBC cell lines were lysed as described in “Materials and Methods” and immunoblotted for the indicated proteins. (B) MDA-MB 231 cells were lysed and immunoprecipitated as described in “Materials and Methods” and proteins were detected by immunoblot. (C) MDA-MB 436 cells were lysed and immunoprecipitated as described in “Materials and Methods” and proteins were detected by immunoblot.