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. 2014 Aug 17;5(18):8665–8680. doi: 10.18632/oncotarget.2356

Figure 5. Withaferin A suppresses the CSC properties of iCSCL-10A cells.

Figure 5

(A) Flow cytometric analysis of CD44 and CD24 expression in iCSCL-10A cells treated for 48 hrs with DMSO (control) or WA (1 μM). The numbers indicate the percentage of each subpopulation according to the CD44/CD24 expression profile. (B) Semi-quantitative PCR for ALDH1A1 and Nanog in iCSCL-10A cells treated for 48 hrs with DMSO or 1μM WA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was analyzed as a control. (C) Immunoblotting of stem cell and EMT marker proteins in iCSCL-10A cells treated for 48 hrs with DMSO or 1μM WA. Vinculin was used as a loading control. (D) Cell invasion assays were performed using chemotaxis chambers in transwell tissue culture dishes as described in the Materials and methods. After 48 hrs of treatment with DMSO or 1μM WA, iCSCL-10A cells were seeded in transwells. Representative microscopic fields are shown (upper). Invasive cells were counted and transwells were scored in triplicates. The mean values ± SD were calculated from three independent experiments (lower). (E) Effects of WA on wound healing. Confluent monolayers of iCSCL-10A cells were treated for 48 hrs with DMSO or 1 μM WA and a wounded was made using a pipette tip. After 6 hrs, the cells were fixed, images were captured and wound closure was scored using ImageJ software. Phase contrast microscopy images of the cells are shown (upper). Values represent the mean ± SEM (n = 3, lower). Scale bar, 1 mm.