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. 2021 Aug 23;11:17077. doi: 10.1038/s41598-021-96449-7

Figure 3.

Figure 3

Translocation of CD24 is associated with chemoresistance and overexpression of Bcl-2. (a) Schematic shows experimental design used to obtain cell populations for MTT (b) and western blot assays (c,d). dfm drug-free medium, Dx doxorubicin, CD24+/DxR CD24+ doxorrubicin-resistant cells. (b) CD24+/DxR cells become tolerant to a second Dx treatment. Parental MDA-MB-231 cells were cultured in the presence of Dx at 0.6 μM for 48 h leading to the obtention of CD24+/DxR cells, which were cultured in a drug-free medium (dfm) for 48 h. CD24+/DxR and naïve MDA-MB-231 cells were submitted to Dx treatment for 24 h and cell viability was measured by MTT. The curves were fitted with non-linear regression as means of triplicates ± SD. ***p < 0.001 (two-way ANOVA with Bonferroni post-test). (c) CD24+/DxR cells are characterized by a reduction of cyclin D1 and Bax expression and increase of Bcl-2 level. Representative image of western blotting are shown and the data have been reproduced two times. (d) CD24+/DxR cells are characterized by a constitutive activation of p38 MAPK. Cells were starved for 2 h and then stimulated with serum (FBS) for 30 min. Cell lysates were immunoblotted with antibodies against the phosphorylated form of p38 (pp38) or ERK (pERK). (e) Downregulation of Bcl-2 and p38 expression in CD24 silenced (SiCD24) cells. SiC (control silenced) and SiCD24 (CD24 silenced) cell lysates were immunoblotted with the depicted antibodies. Representative images of western blotting are shown and the data have been reproduced three times. (f) Correlation between surface CD24 expression and sensitivity to Dx. Cell subpopulations were submitted to Dx treatment for 24 h and then cell viability was measured by MTT assay. Curves were fitted with non-linear regression as means of triplicates ± SD. ***p < 0.001 (two-way ANOVA with Bonferroni post-test). (g) Imiquimod reduces MDA-MB-231 cell proliferation. MDA-MB-231 cells were treated with Imiquimod. Then cells were counted and the curves were fitted as means of triplicates ± SD. ***p < 0.001 (two-way ANOVA with Bonferroni post-test). (h) Imiquimod does not induce chemoresistance to subsequent treatments on MDA-MB-231 cells. MDA-MB-231 cells were pre-treated with Imiquimod (1 μg/ml) for 96 h. After that, cells were incubated with a drug free medium for 48 h and then resubmitted to a second treatment with Imiquimod (1 μg/ml) for 96 h. Cell viability was evaluated by MTT. Data were plotted as means of triplicates ± SD. **p < 0.01 and ***p < 0.001 (T test). (i) Imiquimod does not induce CD24 translocation. MDA-MB-231 cells were treated with Dx or Imiquimod (1 μg/ml and 10 μg/ml) for 48 h. Then, an extracellular staining was performed using anti-CD24/Cy7. The pseudocolor plots are representative of two independent experiments. (j) Imiquimod reduces Bcl-2 expression. MDA-MB-231 cells were treated with Imiquimod (1 μg/ml or 10 μg/ml) in DMEM supplemented with fetal bovine serum (FBS) 10% for 24 h and 48 h. Cell lysates were immunoblotted with the depicted antibodies. The results are representative of two independent experiments.