Fig. 3: RAC1B function is essential for the BCSC maintenance in response to doxorubicin treatment in MCF7 cells.
A Cell growth curve of parental MCF7, RAC1B-proficient Clone 22 (WT) and RAC1B-null single-cell clones in post-treatment recovery period after 2.5 uM doxorubicin treatment for 24 h. Cell numbers are presented as percentage of the pre-treatment cell number for each individual clone. Each data point represents the mean of 3 independent experiments. Significant differences were observed for all 3 RAC1b-null clones compared to parental MCF7 at 96- and 120-h post-treatment recovery time (*p < 0.05; two-tailed unpaired t-test). B Cell growth curve of stable-transgenic MCF7 clone with doxycycline-inducible RAC1B overexpression continuously treated with (RAC1B-ovex) or without (MCF7) 2.5 uM doxycycline in post-recovery period after 2.5 uM doxorubicin treatment for 24 h. Percentage cell number calculations are as described in (A). Each data point represents the mean of 3 independent experiments. Significant differences were observed at 48-, 72-, 96- and 120-h post-treatment recovery time (*p < 0.05; **p < 0.01; two-tailed unpaired t-test). C Mammosphere-forming efficiency (%MFE) of parental MCF7 and single-cell clones when plated in mammosphere culture 24 h after the end of 2.5 uM doxorubicin treatment. Bar graphs represent the mean ± SEM of 3 independent experiments with individual values shown. (**p < 0.01; ***p < 0.005; one-way ANOVA). D Mammosphere-forming efficiency (%MFE) of parental MCF7 and RAC1B-ovex MCF7 clone when plated in mammosphere culture 0 or 24 h after the end of 2.5 uM doxorubicin treatment. Bar graphs represent the mean ± SEM of 3 independent experiments with individual values shown. (**p < 0.01; one-way ANOVA). E Fold change in the size of Aldefluorbright cell subpopulation in parental MCF7, RAC1B-proficient, RAC1B-null or RAC1B-overexpressing clones 24 h after the end of 2.5 uM doxorubicin treatment compared with their own untreated control groups. Fold change differences are shown in Log2 scale and represent the mean ± SEM of 3 independent experiments. (*p < 0.05; one-way ANOVA).
