Androgen deprivation induces senescence markers in LNCaP cells.

<p>(<b>A</b>) Senescence associated beta-galactosidase (SA-beta-gal) staining under indicated conditions and timepoints. FBS, fetal bovine serum indicating androgen-replete culture; CSS, charcoal-stripped serum indicating androgen- deprived culture. 9D FBS post-SEN denote LNCaP cells that were subjected to 14 days CSS culture and then switched to FBS media for 9 days. Quantitation of positively stained cells is presented to the right of the representative images. (<b>B</b>) Ki67 pan-proliferation marker staining is shown at indicated conditions and time points. DAPI staining is used to mark cell nuclei for purposes of counting Ki67-positive cells. Percentage of stained cells is graphed on the right. (<b>C</b>) Propidium iodide cell cycle analysis, at indicated timepoints and culture conditions. Note the S-phase fraction drops from 15.6% in FBS to 0.4% at 10d CSS and to 0.8% after restoration to FBS media for 9 days (9D FBS post-SEN). (<b>D</b>) Proliferation curve for LNCaP cells cultured in CSS for 3, 8 or 14 days before being switched back to FBS media. (<b>E</b>) Measurement of total ROS levels via flow cytometry in LNCaP cells at day 1, day 4 and day 7 of indicated culture conditions. Note the right-shifted peak in red corresponding to increasing ROS levels in the CSS-cultured LNCaP cells relative to FBS-cultured cells. (<b>F</b>) DNA double-strand break (DSB) foci detected via H2AX/53BP1 co-staining (green = H2AX, red = 53BP1) for LNCaP cells cultured under the indicated conditions. Representative images for cells with the different numbers of counted foci are shown. Note that the percentage of cells with 5+ foci increases with the duration of CSS culture.