Fig. 5.

Treatment with curcumin enhances the suppression of cancer stemness by miR‐181a inhibitor. (A) Confocal microscopy of acridine orange staining in MDA‐MB‐231/A cells treated with curcumin following miR‐181a transfection. (Magnification: ×40, zoom: 2.0). The scale bar for magnified images equals 10 μm. Nuclei were counterstained with Hoechst 33342. Scale bar, 20 μm. The percentage of MDA‐MB‐231/A cells containing acridine orange puncta is shown. The number of nuclei was used to normalize the values. Data were presented as mean ± SD. Statistical analyses were performed with one‐tailed Student’s t‐test (*P < 0.05, **P < 0.01). (B) Confocal microscopy of autophagic MDA‐MB‐231/A cells stained with Cyto‐ID dye following miR‐181a transfection and curcumin treatment (magnification: ×40, zoom: 2.0). The scale bar for magnified images equals 10 μm. Nuclei were counterstained with Hoechst 33342. Scale bar, 20 μm. The number of Cyto‐ID puncta was normalized with the number of nuclei. Data were presented as mean ± SD. Statistical analyses were performed with one‐tailed Student’s t‐test (*P < 0.05; ns, nonsignificant difference). (C) qRT‐PCR analysis showing the miR‐181a expression levels in MDA‐MB‐231/A stable KO cells. Data were presented as mean ± SD of three independent experiments. Statistical analyses were performed with one‐tailed Student’s t‐test (***P < 0.001). (D) Limiting dilution assays performed on curcumin‐treated MDA‐MB‐231/A stable miR‐181a‐KO cells. Stem cell frequency was calculated using extreme limiting dilution assay analysis.