Extended Data Fig. 7: Extended data for Fig. 5.
From: Epithelial cells remove precancerous cells by cell competition via MHC class I–LILRB3 interaction
a, The mix proportion of normal and HaCaT-RasV12 cells used in tumorigenesis assay in Fig. 5b,c,e are described. ‘Figure#’ indicates the figure number in Fig. 5. ‘Genotype’ indicates genotypes of HaCaT or HaCaT-RasV12 cells. ‘HaCaT:RasV12’ means a ratio of the actual cell number used for the experiments as a unit of 105 cells. ‘Mix rate’ indicates ratio of HaCaT cell number over the RasV12 cell number. ‘Rec.α3’ indicates whether rec.MHC-α3 protein was coinjected (+) or not (–). b, High magnification fluorescence images of Fig. 5d. Tumors collected from mice at 2 week were fixed, and the frozen sections of the collected tumors were subjected to immunohistochemistry with phalloidin (white) and Hoechst (blue). c, LILRB3 was induced in the normal cells contacting with RasV12-HLA TKO cells. HaCaT and/or HaCaT-RasV12-HLA TKO cells were single/mixed-cultured and treated with doxycycline for 12 h. Immunohistochemistry were conducted under the non-permeabilized condition using an anti-LILRB3 (red) and phalloidin (white). Representative XY images of fluorescence staining are shown. d, Rec.MHC-α3 protein promoted apical extrusion of HaCaT-RasV12-HLA TKO cells under the mix condition. Normal/RasV12-HaCaT cells were mixed and cultured with or without rec.MHC-α3 protein upon doxycycline treatment for 16 h. The extrusion efficiency was quantified and shown as a graph. Data are presented as mean ± s.d; n = 3 biologically independent experiments; **P < 0.01 by unpaired two-sided Student’s t-test. e, Tumor formed by coinjection of normal and RasV12 cells recruited macrophage at the peripheral of tumors. HaCaT WT or -LILRB3 KO cells were mixed with HaCaT-RasV12 cells at a ratio of 10:1 and coinjected with rec.MHC-α3 protein into nude mice. After 2 weeks, tumors were collected and fixed, followed by frozen sectioning. Frozen sections of tumors were subjected to immunohistochemistry with an anti-F4/80 (Macrophage marker, red), an anti-CK19 (Epithelial cell marker, white) and Hoechst (blue). Representative XY images of fluorescence staining are shown. b,c,e, Scale bars, 200 μm (b) or 10 μm (c,e).
