Fig. 4

Two distinct subpopulations expand and show proliferative properties in response to RT. A Very small subpopulations (<1%), represented by barcodes b and f, expanded significantly following RT (fold change in % of cells relative to the control of each time point). B–G 4T1 cells were irradiated with 15Gy. 6 days post-RT, cells were incubated with antibodies against Ki67, cMet, and Her2 and nuclei were stained with DAPI (fluorogel II). B, E 40× lens; scale bar represents 50 μm. C, F Sum intensities of Ki67 (C, left panel); Her2 (C, right panel); Ki67 (F, left panel); and cMet (F, right panel) were calculated from 8 to 10 fields using the NIS-Elements software (Nikon). D, G Correlation plots between D Ki67 and Her2 and G Ki67 and cMet were generated for each indicated condition to test co-activation represented in C, F. R values indicating the degree of correlation between Ki67 and Her2 (D) and Ki67 and cMet (G) were calculated before and after RT. H Survival rates of 4T1 cells in response to Trastuzumab (T), Crizotinib (C), RT, RT+C, RT+T, and RT+T+C as detected by MB survival assays 6 days post RT (upper panel), and cell viability as measured by the MTT assay (lower panel). Drugs were added from 3 days prior to RT until the end of the experiment. I Key downstream to Her2 and cMet signaling proteins are shown following different treatments. The predicted combination induced high levels of cleaved caspase-3 compared to radiation alone, irradiation+T, and irradiation+C. Downregulation of pAKT, pERK, and p-S6 was detected when T+C was applied prior to RT. For A: quantification of subpopulations was performed using at least ~30,000 cells from each condition, which were obtained from at least three flasks and from at least three independent experiments for each time point. For C and F, statistically significant differences between all presented conditions and the cells treated with RT+T+C were determined using a two-tailed Student’s t test (*P < 0.05); for H, upper and lower panels *P < 0.01