Fig. 4
Lateral inhibition maintains a sparse spatial organization of altruists. A Immunoblotting to detect indicated proteins extracted from Mi/EGFPHigh and Mi/EGFPLow fractions from indicated cancer cell lines. B,C Mi/EGFPLow and Mi/EGFPHigh cells from indicated cancer cell lines were exposed to conditioned media harvested from separate batches of Mi/EGFPLow and Mi/EGFPHigh cells. After four days, the formers’ fluorescence levels were analyzed by FACS (B) and extracted protein of exposed Mi/EGFPLow cell studied by immunoblotting (C). D, E Mi/EGFPLow cells from indicated cell lines were treated with indicated recombinant proteins in combination with neutralizing antibodies and the fluorescence level analyzed by FACS after four days. F-H Mi/EGFPLow cell fractions from indicated cancer cell lines, transfected with control or GAB1 siRNA, were exposed to recombinant IGFBP2 or CCL28. After four days, their fluorescence levels were analyzed by FACS (F, IGFBP2; G, CCL28) and their extracted protein by immunoblotting (H). CM-R: Mi/EGFPLow recipient cells exposed to recombinant protein. I Box plots of fold change in enrichment of GAB1 mRNA pulled-down using biotinylated miR-125b mimics. J Relative luciferase activities of HEK293T cells following transfection of wild-type or mutant reporter construct for GAB1 and indicated mimics. WT: wild type. K Immunoblotting to detect expression of indicated proteins extracted from miR-125bm or Controlm-transfected MCF7 or MDA-MB-468 cells with or without docetaxel treatment (Upper), or miR-125b LNA-inhibitor- or control-LNA-transfected MDA-MB-231 or MDA-MB-415 cells (Lower). L,M Schema of lateral inhibition model mediated by diffusible IGFBP2 and CCL28 secreted by miR-125bHigh altruists. Upon exposure to IGFBP2 and CCL28, heightened PI3K-AKT signaling is induced in the recipient cells, resulting in reduction in miR-125b expression and adoption of the non-altruistic social fate (L). Hypothesized level of the diffusible proteins, extent of PI3K activation and probability of altruist arising as the distance from altruist increases is depicted, in association with the altruist’s ability to influence social fates beyond the immediate neighboring cells (M). N Simulation of lateral inhibition dynamics showing pattern generation when diffusion coefficient d = 1 (as in the case of Notch-Delta signaling) and d>1 (mediated by diffusible proteins such as IGFBP2 and CCL28) (Left column). Spatial patterns of Mi/EGFPHigh and Mi/EGFPLow cells in indicated cell lines, with or without IGFBP2 & CCL28 antibodies treatment, are shown (Center and Right columns). Experiments repeated two times, representative data are shown for (A-H, K, N). Mean percentage ± s.d. cells for technical triplicates of representative set are shown in same colour as corresponding histograms (B,D-G). Data are mean ± s.d. from three independent biological sets of triplicates (I, J). Statistical analysis was performed using two-tailed one sample t-test against 1 (I) or 100 (J). NT: no treatment; DTX: docetaxel treatment. Exact P values are shown