Fig. 4: Low-dose TM and 2-DG preconditioning increases the number of CD44^high/CD24^low CSC-like cells.

A, B FACS analysis of CD44^high/CD24^low CSC-like cells in TM-preconditioned cancer cells. (A) Low-HA Has2^+Neo cells were treated with different doses of TM for 8 days and analyzed for CD24 and CD44 expression by flow cytometry. Data are the mean ± SD from n = 4. Two-tailed Student’s t-test. **p < 0.01. (B) Low-HA Has2^+Neo cells were treated with 0.1 or 0.2 μg/ml TM for 8 days and further cultured in the absence of TM for an additional 8 days. The numbers of CD44^high/CD24^low CSC-like cells were counted at different time points. Data are the mean ± SD from n = 3. Two-tailed Student’s t-test. *p < 0.05, **p < 0.01. C Effects of GlcN supplementation on the number of CD44^high/CD24^low cells. Low-HA Has2^+Neo cells were treated with 0.1 µg/ml TM and/or 500 µM GlcN for 8 days and then analyzed for CD24 and CD44 expression by flow cytometry. Data are the mean ± SD from n = 3. Two-tailed Student’s t-test. *p < 0.05. D Mammosphere formation. Has2^+Neo cells were treated with low-dose TM for 8 days and cultured for an additional 7 days in ultralow attachment surface 24-well plates with spheroid-forming medium containing the same concentrations of TM. Representative images of mammospheres were taken, and the mammosphere number was counted under a phase-contrast microscope. Scale bar: 100 µm. Data are the mean ± SD from n = 3. Two-tailed Student’s t-test. *^{, #}p < 0.05, **^{, ##}p < 0.01. E The effects of 2-DG preconditioning on the number of CD44^high/CD24^low CSC-like cells. Low-HA Has2^+Neo cells were treated with different doses of 2-DG for 8 days and analyzed for CD24 and CD44 expression by flow cytometry. Data are the mean ± SD from n = 4. Two-tailed Student’s t-test. *p < 0.05, **p < 0.01.