Figure 5. HCT116 cells expressing the Δ133p53β isoform display amoeboid-like movements.
(A) Still time-lapse images of the accompanying video (Supplementary data, video1) of HCT116 cells transfected with the GFP-tagged Δ133p53β isoform. Cells were observed 48 hr after transfection. For the video, images were captured every 4 min during 12 hr. The panel represents 1/10 images i.e. one image every 40 min. Δ133p53β-transfected cells can be distinguished from non-transfected cells through expression of GFP (green). The Δ133p53β-transfected cells are rounded and exhibit blebbing movements on their surface. The arrow shows a cell that detaches from the others and from the dish during the time-lapse. The arrowhead shows a cell that still adheres to the other epithelial cells and to the substratum at the beginning of the experiment and then becomes progressively rounded. (B) Quantitative analysis of blebbing versus adherent cell number in the Myc positive cells. FACS analysis of the percentage of non-apoptotic blebbing Myc-positive HCT116 cells compared to total Myc-positive transfected with cells upon transfection of Myc-Δ133p53β, or Myc-empty expression vector (vector). Results were normalized to Myc-empty vector transfected cells. (C) Western blot analysis of the expression of E-Cadherin and β1-integrin in HCT116 cells expressing the GFP-tagged Δ133p53β isoform; Control: GFP-tag vector. Adherent: cells still adherent to the substratum; Blebbing: cells detached from the substratum and showing blebbing movements. Loading normalization was performed using an anti-α-tubulin antibody. (D) Δ133p53β-transfected HCT116 cells were quantified for their migration ability after 2 hr of migration through the Boyden chamber or for their invasiveness after 24 hr of the invasion through Matrigel, as indicated. The values are plotted as means ± SEMs of at least 3 independent experiments. (E) 3-D LoVo cell scattering. The numbers of scattered cells were quantified using Metamorph software (left). Cells were judged as « scattered » when individual cells or clusters of cells had lost contact with the main colony, as visualized (right). Values (means ± SEMs) were calculated from 4 independent experiments (n = 48) ***p<0.001. (F) Wound healing assay in LoVo cells infected with shRNA non relevant (shNS: shLuciferase) or sh∆133p53. Cells were observed 25 hr after infection. Still time-lapse images of the accompanying videos (Supplementary data, Videos 2 and 3) For the videos, images were captured every 1 hr during 25 hr. The arrows show cells detaching from the others and migrating as individual cells. The arrowhead shows a cluster of cells which leaves the cohesive epithelium and which collectively migrate and enter into the gap. (G) Schematic representation of the role for ∆133p53β in reprogramming cells toward the invasive process. For WT or mutant TP53 cells devoid of ∆133p53b expression, introduction of ∆133p53β promotes EMT and invasion. Reciprocally WT or mutant TP53 cells expressing ∆133p53β have enhanced invasive activity. Depletion of ∆133p53β reverts EMT and inhibits invasion.
Figure 5—figure supplement 1. Control experiments of Δ133p53β expression and effects in colon cancer cells.
