Fig. 5: Long-term BIK expression promotes aggressive cell phenotypes.

a Experimental scheme depicting the generation of “LTC” cells. b Left: Western blot analysis performed for MCF-7 Tet-on cells after 10 passages in Dox showing the persistence of BIK expression and DNA damage. Right: Western blot analysis showing BIK expression turned off and DNA damage resolved after Dox withdrawal. Cell lysates made from cells expressing BIK were used as a positive control for anti-BIK and -γH2AX antibodies. c Left: Representative images depicting the anchorage-independent growth of MCF-7 LTC cell lines. Right: Quantitation of the fold changes in the number of soft-agar colonies relative to control. Three independent experiments were performed. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. d Left: Representative images from mammosphere formation assay performed with MCF-7 LTC cell lines. Mammosphere-forming efficiency (MFE) was calculated after 12 days in culture. Scale bar 250 µm. Right: Bar graph depicting quantitation of the MFE from three independent experiments. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. e Top: Representative images from colony-formation assay performed for MDA-MB-231 LTC cells. Scale bar 5 mm. The satellite images show a magnified view of the colonies. Bottom Left: Colony area was calculated for at least 350 colonies from each group from three different experiments. Error bars represent SEM. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. Bottom Right: Colony density was calculated for at least 350 colonies from each group from three different experiments. Error bars represent SEM. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. f Left: Representative images at the indicated time-points from the collective cell migration assay performed for MDA-MB-231 LTC cells. Right: Quantitation of the movement of the cell-front over 15 h. A total of nine positions from three independent experiments were analyzed for each group. Error bars represent SD. Linear regression analysis was performed to calculate differences between groups. Scale bar 100 µm. g Rose plots depicting the spread of cell movements of the MDA-MB-231 LTC cells. h Speed (left) and persistence (right) for MDA-MB-231 LTC cells were calculated by taking the average speed of cells over 24 h. At least 57 tracks were analyzed from three independent experiments. Error bars represent SEM. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups.