Skip to main content
. 2021 Dec 28;14(1):136. doi: 10.3390/cancers14010136

Figure 1.

Figure 1

Comparison of colon cancer HCT116 WT versus HCT116 p21-/- cells. (A) Phase contrast and immunofluorescence confocal microscopy images of HCT116 WT and p21-/- cells. Top panels: phase contrast magnification = 100×; panels below: confocal magnification = 630×, blue: DAPI, green: F-Actin (scale bar of Phase contrast = 200 µm, confocal scale bar = 20 µm; n ≥ 4). (B) Spheroid formation ability of HCT116 WT and p21-/- cells in a hanging drop assay after 24 and 96 h (scale bar = 500 µm). (C) Western blots of HCT116 WT, HCT116 p21-/-, (D) DLD1 WT, and DLD1 p21-/- cells grown in 2D cultures, used to analyze the expression of EMT markers (Vimentin, ZEB1) and p21 and the epithelial marker E-cadherin (CDH1); n ≥ 2. Fold expressions are presented relative to GAPDH loading control. * Markers were blotted on the same gel. Fold changes of VIM expression in DLD1 p21-/- cells when compared to WT cells as determined by qPCR (** p-value < 0.01, n = 3; biological triplicate and technical triplicate). (E) Immunohistochemical staining for E-cadherin in fixed cells grown in 2D cultures and 3D cultures with hanging drops (scale bar = 50 µm). (F) Western blots of HCT116 WT, HCT116 p21-/-, DLD1 WT, and DLD1 p21-/- cells to investigate the stemness markers CD133, CD44, ALDH1/2, OCT4A, and ABCG2 (n ≥ 2). Fold expressions presented relative to GAPDH loading control. * Markers were blotted on the same gel (G) Evaluation of the wound healing assay after 24 and 40 h for cells treated with 10 nM of mitomycin C (average of 7 wells with 3 single measurements per well) (Image-Pro Plus). (* p-value < 0.05, *** p-value < 0.001). (H) Representative light microscopy images of fixed and extracted chorioallantoic membrane (CAM) tumors derived from HCT116 WT and p21-/- cells (magnification = 100×; scaling segments = 1 mm; n ≥ 8) and representative images of formalin-fixed and paraffin-embedded (FFPE) CAM sections stained with HE for histological analysis (magnification = 200×; scale bar = 50 µm, n ≥ 8). Arrows show blood vessels and the yellow dotted line marks the pushing invasion front.