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Fig. 5 | Cancer Cell International

Fig. 5

From: Highly versatile cancer photoimmunotherapy using photosensitizer-conjugated avidin and biotin-conjugated targeting antibodies

Fig. 5

Effect on HuH-7 SP cells by AvIR-mediated PIT. a Clonogenicity of HuH-7 cells was evaluated by tumorsphere-formation assay. HuH-7 cells were sorted to CD133/EpCAM, CD133/EpCAM+, CD133+/EpCAM, or CD133+/EpCAM+ subpopulation. Using each subpopulation of HuH-7 cells, tumorsphere-formation assay was performed in PromoCell Cancer Stem Cell medium. The data are the means ± SEM (n = 3, *p < 0.05, **p < 0.01, one-way ANOVA with Tukey’s test). Representative images of tumorspheres are also shown in the bottom panels. b Identification of SP cells in HuH-7 cell line. HuH-7 cells were stained with Hoechst 33342 and analyzed by flow cytometry. The SP cells, which disappear in the presence of verapamil (50 µg/ml; bottom left panel), are outlined and shown as a percentage of the total cell population. Expression profile of CD133 and EpCAM in HuH-7 cells is shown in the top right panel, in which red dots indicates the SP cells by back-gating. The percentage of each subpopulation is also shown in the bottom right panel (means ± SEM, n = 4; bottom right panel). c Sphere-forming capability of AvIR-PIT-treated HuH-7 cells. By using Bio-CD133 and Bio-EpCAM, CSC-like subpopulation-targeted AvIR-PIT was performed against HuH-7 cells. After removal of the dead cells by using ClioCell magnetic particles, the resulting live cells were examined by tumorsphere-fomation assay. The data are the means ± SEM (n = 3, ND; not detected). d FACS analysis of AvIR-PIT-treated HuH-7 cells. The survived cells collection from CSC-targeted AvIR-PIT was performed as in c. The cells were further cultured by 2 passages and FACS-analyzed on SP and expression of CD133 and EpCAM

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