Fig. 2

Cell surface molecules regulated by mutant p53 are involved in CIC formation. a 3D images of a CIC structure in H1299 cells. The top right insert is showing the horizontal plane by confocal imaging. Scale bar = 5 μm. b Fluorescent image of live A431 cells that have been labeled with pHrodo. Scale bars = 50 μm. c Confocal image of A431 cells that have been stained with Calcein AM. Scale bars = 20 μm. d Quantification of CIC for H1299 co-cultures seeded on plastics coated with different matrices. Each bar represents +/−SEM of triplicate experiments **p = 0.0072. e Immunofluorescence image of a CIC structure formed between H1299 cells (green and red as indicated). Dapi is in blue and CD49e (integrin α5) is in yellow. Scale bar = 50 μm. f Number of CIC events in mutant p53 (green)/KO p53 (red) A431 co-cultures in which engulfment by red cells was quantified in the presence of mAb16. Each bar represents +/−SEM of triplicate experiments **p = 0.003. g Quantification of CIC in H1299 EV/H1299 175H co-cultures in the presence or absence of Y27632 or mAb16. Each bar represents +/−SEM of triplicate experiments *p = 0.01–0.04. h Quantification of total CIC structures per 20 hpf observed in A431 Ctr 273H/mCherry and KO 273H/GFP co-cultures treated with and without EGF for 24 h. Either red or green engulfing cells were quantified for KO 273H or ctr 273H respectively. Each bar represents +/−SEM of triplicate experiments Each bar represents +/−SEM of triplicate experiments *p = 0.03 or **p = 0.004. i Quantification of total CIC structures per 20 hpf observed in A431 Ctr 273H/mCherry and KO 273H/GFP co-cultured cells treated with and without Gefitinib (24 h). Either red or green engulfing cells were quantified for KO 273H or ctr 273H respectively Each bar represents +/−SEM of triplicate experiments **p = 0.0019. j Western blot showing pEGFR and EGFR following treatment of A431 control and p53 CRISPR KO cells, with and without Gefitinib and EGF. Actin was used as loading control