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. 2021 Nov 29;12:714163. doi: 10.3389/fphar.2021.714163

FIGURE 4.

FIGURE 4

Cortisol elevated by CUMS enhances the stemness of breast cancer cells via GRP78. (A) The relative levels of cortisol, corticosterone, and cortisone in the plasma of the mice exposed to CUMS were measured by ELISAs. (B) GRP78 expression in 4T1 cells was detected by western blots after treatment with cortisol at a gradient concentration for 24 h. (C) The 4T1 cells were treated with cortisol at a gradient concentration for 24 h, and the relative mRNA expression of GRP78 was quantified by qPCR. (D) The ALDH+ cell population induced by cortisol was analyzed in the 4T1 cells after treatment with HA15 (10 μM), an inhibitor of GRP78, for 24 h. (E) The effect of HA15 on the size and number of the mammospheres induced by cortisol was quantified (the scale bars indicate 100 μm). (F) The ALDH+ cell population induced by cortisol was analyzed by flow cytometry following the GRP78 knockdown with shRNA (verification by western blot in the upper right panel). (G) The effects of GRP78 knockdown by shRNA on the size and number of the mammospheres induced by cortisol were examined (the scale bars indicate 100 μm). Data are represented as the mean value ±SD. One representative experiment of three independent experiments is displayed. Unpaired two-sided Student’s t-tests (A, D, E, F, G), one-way ANOVA, and Dunnett t-tests as post hoc tests (C) were applied.