Fig 1: Clinicopathological relevance of CXXC5 expressions in breast cancer patients. (a) CXXC5 expression in breast cancer (BC) as compared to normal breast tissue from METABRIC. (b) CXXC5 expression in ER-negative versus ER-positive breast cancer patients from METABRIC. (c) CXXC5 expression in different molecular subtypes of tumors of breast cancer patients from METABRIC. (d) Pearson correlation analysis between mRNA expressions of CXXC5 and ESR1 (ERa) in patients from METABRIC. (e) Pearson correlation analysis between mRNA expression of CXXC5 and ERa protein level in breast cancer patients from TCGA. (f) Kaplan Meier survival analysis of basal breast cancer patients based on CXXC5 expression separated from the median.
Fig 2: Intracellular Localization of CXXC5.MCF7 cells grown on coverslips in 12-well culture plates with medium containing FBS for 48 h were un-transfected (UT) or transfected with pcDNA3.1(-) bearing the Flag-CXXC5 (F-C5) cDNA. Thirty six hour after, cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.4% Triton-X100 in PBS. For the detection of the endogenous CXXC5 protein in un-transfected cells, cells were blocked with 10% normal goat serum (NGS) followed by an incubation with ab106533 in PBS containing 2% NGS. Cells were then incubated with an Alexa Fluor®-488 (green channel) conjugated goat anti-rabbit secondary antibody in PBS containing 2% NGS to detect endogenous CXXC5. For the detection of Flag-CXXC5 protein in transfected cells, following a block with 10% bovine serum albumin (BSA) in PBS, cells were incubated with the Flag-M2 antibody in PBS containing 3% BSA. Cells were then incubated with an Alexa Fluor®-488 (green channel) conjugated goat anti-mouse secondary antibody in PBS containing 3% BSA. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). (blue channel). Merge images are indicated. A representative image from two independent experiments is shown. Scale bar is 5 µm.
Fig 3: Chromatin Immunoprecipitation assay (ChIP).MCF7 cells grown in medium containing CD-FBS for 72 h treated without (EtOH, 0.01%) with 10-8 M E2 for 1 h prior to ChIP. Cells were fixed with 0.75% paraformaldehyde, lysed, sonicated and subjected to ChIP using IgG or an ERa specific HC20x antibody followed by the incubation with Protein A/G conjugated magnetic beads. Shown (a) are PCR reactions subjected to 2% agarose gel electrophoresis from a representative experiment performed three independent times. (b) Samples were also subjected to RT-qPCR for quantitative analysis with primers specific to the estrogen responsive region of CXXC5. (c) RT-qPCR results the estrogen responsive region of TFF1 with the same experimental inputs described in (b) with primers specific to the estrogen responsive region of TFF1. Sizes of the DNA fragments in base pairs are indicated. Asterisk (*) denotes significant change depicted as percent (%) of input.
Fig 4: Transcriptional Responses of CXXC5 and TFF1 to ER ligands.MCF7 cells grown in medium containing charcoal-dextran treated fetal bovine serum (CD-FBS) for 48 h were treated without (ethanol, EtOH, 0.01% as vehicle control) or with 10-9 M E2 for 3 h, 6 h or 24 h. Cells were subsequently subjected to total RNA isolation for the expression of CXXC5 (a) or TFF1 (b). MCF7 cells were also treated without (EtOH) or with 10-9 M E2 and/or 10-7 M ICI for 24 hours for CXXC5 expression (c). RT-qPCR results, which are the mean ± SD of three independent determinations in triplicates normalized to the expression of RPLP0, depict fold changes in mRNA levels in response to ligand compared with those treated with EtOH, which is set to 1 at each time point (a,b) or at 24 h (c). Asterisk (*) indicates significant change.
Fig 5: Transcriptional responses from the estrogen responsive CXXC5 region-driven gene reporter.(a) MCF7 cells grown in medium containing CD-FBS for 48 h were transiently transfected with pGL3 bearing none (Basic-Luc), the estrogen responsive region of CXXC5 (CXXC5-Luc) or OXT (OXT-Luc) driving Firefly Luciferase cDNA expression as the reporter in the absence (EtOH, 0.01%) or presence of 10-9 M E2 for 24 h. The transfection efficiency was monitored by the co-expression of pCMV–RL that drives the expression of Renilla Luciferase cDNA. 24 h later, cellular extracts were subjected to luciferase assays. Shown is the mean ± SD of three independent experiments performed in triplicate. Firefly/Renilla luciferase activities are presented as fold change compared to EtOH control of pGL3-Basic, which is set to 1. *a and *b indicate significant difference from E2 of Basic-Luc and the corresponding EtOH control, respectively. (b) MCF7 cells transfected with CXXC5-Luc treated without (EtOH, 0.01%) or with 10-9 M E2 and/or 10-7 M ICI for 24 h were subjected to luciferase assays. Shown is the mean ± SD of three independent experiments performed in triplicate. Firefly/Renilla luciferase activities are presented as fold changes compared to EtOH, which was set to 1. (c) MDAMB231 cells were transfected as described in (A) with Basic-Luc, CXXC5-Luc, or OXT-Luc reporter together with pCDNA-Flag-ERa vector. Cells were also co-transfected with pCMV-RL for monitoring transfection efficiency. Results are the mean ± SD of three independent experiments performed in triplicate. Firefly/Renilla luciferase activities are presented as fold changes compared to EtOH of pGL3-Basic, which is set to 1. *a indicate significant change from EtOH of Basic-Luc; while *b denotes significant change of E2 compared to EtOH of CXXC5-Luc or OXT-Luc. (d) MDAMB231 cells were transfected with CXXC5-Luc or mutCXXC5-Luc vector, the latter which bears a mutant sequence that changes the ERE sequence in CXXC5 to a non-ERE, together with pcDNA-Flag-ERa vector. Cells were treated without (EtOH, 0.01%) or with 10-9 M E2 for 24 h. Shown is the mean ± SD of three independent experiments performed in triplicate. The normalized Firefly/Renilla luciferase activities are presented as fold change compared to EtOH of CXXC5-Luc, which was set to 1.
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