FIGURE 12.

NOTCH1 is involved in the anti-proliferative effects exerted by ATRA in NOTCH-addicted HCC1599 breast cancer cells. A, HCC1599 cells were treated for 3 and 6 days with increasing concentration of ATRA as indicated and subjected to the CellTiter-Glo assay to determine growth and viability. The graph shows the growth-inhibitory effect exerted by the retinoid. **, significantly different (t test, p < 0.01);*, significantly different (t test, p < 0.05). B, HCC1599 cells were incubated with vehicle (DMSO), DAPT (1 μm), ATRA (1 μm), or a combination for 24 h. Cells were harvested, and total RNA was extracted to measure the expression of NOTCH1 (NICD) by qRT-PCR after reverse transcription. Each value is the mean ± S.D. of six independent replicates. **, significantly different (t test, p < 0.05). C, HCC1599 cells were incubated with vehicle (DMSO), DAPT (1 μm), ATRA (1 μm), or a combination for the indicated amount of time. Western blot analysis was performed to analyze NICD expression levels. Tubulin was used as a loading control. D, cells were treated as in B for the indicated amount of time and subjected to the CellTiter-Glo assay to determine growth and viability. Each value is the mean ± S.D. of three independent replicates. **, significantly different from vehicle-treated cells (t test, p < 0.01). The results are representative of two independent experiments. E, cells were treated as in B, and RNA was extracted to determine the expression levels of the indicated NOTCH1 target genes by qRT-PCR after reverse transcription. Each value is the mean ± S.D. of six independent replicates. **, significantly different from vehicle-treated cells (t test, p < 0.01); *, significantly different (t test, p < 0.05).