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. 2022 Jan 26;11:808300. doi: 10.3389/fonc.2021.808300

Figure 1.

Figure 1

Enhanced CD133 levels in sphere cultured HCT116 cells and their involvement in sphere formation. (A) The presence of CD133-positive cell population in monolayer-cultured HCT116 cells was analyzed using allophycocyanin (APC)-fluorescence-based fluorescence-activated cell sorting method. (B, C) Transcript (B) and protein levels (C) of CD133 were assessed in monolayer and sphere cultured HCT116 cells using relative quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis and western blotting. Bar graph represents quantified protein levels from at least three experiments. aP < 0.05 compared with the monolayer HCT116. (D, E) The transcript (D) and protein levels (E) of Kruppel-like factor 4 (KLF4) and ATP-binding cassette subfamily G member 2 (ABCG2) in monolayer and sphere HCT116 cells were assessed. Bar graph represents quantified protein levels from at least three experiments. aP < 0.05 compared with the monolayer HCT116 cells. (F) Transcript levels of CD133 in the non-transfected (NT), non-specific small interfering RNA (siRNA) (siCtrl)- or CD133-specific siRNA (siCD133)-transfected HCT116 cells. aP < 0.05 compared with the siCtrl group. (G) HCT116 cell with either the non-specific siRNA (siCtrl) or CD133-specific siRNA (siCD133) transfection, were grown in sphere culture systems and the protein levels of CD133, KLF4, ABCG2 were examined. Bar graph represents quantified protein levels from at least three experiments. (H) Sphere formation was assessed in the non-transfected (NT), non-specific siRNA (siCtrl)- or CD133-specific siRNA (siCD133)-transfected HCT116 cells. Number of spheres over 70 μm diameter was counted using an image processing ToupView software. aP < 0.05 compared with the siCtrl group. Quantification results of western blotting were relative values to the loading control GAPDH. All values represent the mean ± standard error of the mean (SEM) of more than three experiments.