Figure 7.
Inhibiting PR Methylation Affects Breast Cancer Cell Proliferation and PR Stability
(A) Immunofluorescence of T47D PRKO, T47DWT, and T47DR637K cells, before and after stimulation with 10 nM of R5020, stained with anti-PR antibody. The nuclei were counterstained with DAPI (blue) (Obj: X40).
(B) WCE of T47DWT or T47DR637K, stimulated with R5020 (10 nM) for 1 h, were used for IP with anti-PR antibody or control IgG and immunoblotted with pan-meth-R and PR antibodies.
(C) WCE from T47DWT and T47DR637K cells stimulated with R5020 for the indicated times were immunoblotted.
(D) Half-life of the endogenous PR in T47DWT and T47DR637K cells. Left panel: WCE from T47DWT and T47DR637K cells treated with cycloheximide before the stimulation with R5020 were immunoblotted with the indicated antibodies. The amount of PR was quantified by densitometry; two different expositions are shown to well quantify the PR band intensity for each time. Right panel: The half-life curves for each cell line is graphically represented. This experiment is representative of three independent experiments.
(E) Analysis of T47DWT and T47DR637K cell proliferation by Incucyte technology, performed as described in Figure 6B.
(F) T47DWT and T47DR637K cells were stimulated with 10 nM of R5020, and colony growth was measured at 10 days after staining with crystal violet. Both (E) and (F) graphs show the mean ± SD of one experiment representative of three. The p value was determined using the Student's t test: ∗∗ indicates p ≤ 0.01 and ∗∗∗p ≤ 0.001.
(G and H) T47DWT and T47DR637K cells were stimulated with R5020 (10 nM). EGFR and EGR1 expression were analyzed (G) by IB and (H) by RT-qPCR (after 6 h of R5020 induction). The mean ± SEM of, at least, three independent experiments is shown. The p value was calculated using a paired t test: ∗ indicates p ≤ 0.05.