Figure 3. GLE reduces ALDH1 activity, CD44⁺/CD24^– population, and deforms mammospheres in TNBC cells.

(A) FACS images and quantified results of ALDH1 activity in MDA-MB-231 cells at 24 h reveal that GLE significantly decreases ALDH1 activity, compared to vehicle. (B) Representative flow cytometry dot plots of CD44 and CD24 expression in SUM-149 cells treated with GLE at 24 h show a significant increase in double-negative stained cells and a decrease in the CD44⁺/CD24^– (stem cell) population. (C) The statistical analyses performed in the MDA-MB-231 cells display that GLE significantly decreases the CD44⁺/CD24^– population at 24 h in comparison to vehicle. (D) TNBC cell lines were seeded to form mammospheres in non-serum non-adherent culture conditions in the absence (Vehicle) or presence of GLE, with results showing a decrease in size and deformation of the mammospheres at 24 h. (E) Quantification of the circularity for SUM-149 mammospheres shows that GLE significantly decreases the circularity at 24 h compared to vehicle, (F) while in the MDA-MB-231 mammospheres, GLE shows a tendency (P < 0.07) to decrease circularity compared to vehicle. (G) mRNA expression of STAT3 was measured by qRT-PCR analysis after mammospheres were treated with vehicle and GLE, with results showing that GLE significantly decreases the gene expression of STAT3 in both TNBC cell lines compared to vehicle. Results were normalized to β-actin. Columns represent mean ± SEM from 2 or 3 independent experiments. Data for the circularity analysis are from n = 20 replicates from 3 independent experiments. ^*P ≤ 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001, was considered statistically significant compared to vehicle.