Extended Data Fig. 3: Extended data for Fig. 2.
From: Epithelial cells remove precancerous cells by cell competition via MHC class I–LILRB3 interaction
a, RasV12 expression induced HLA-B mRNA in MCF10A cells. The raw meta-data reported in9,10 were re-analyzed. In non-malignant mammary epithelial cells, MCF10A, HLA-B mRNA was induced in a RasV12 expression-dependent manner. Data of study #1 and Study #2 were extracted from9,10, respectively. b, RasV12 expression did not affect HLA-A or HLA-C on the cell surface. HaCaT-RasV12 cells were treated with doxycycline for the indicated time period and followed by immunohistochemistry under the non-permeabilized condition. Samples were stained with an anti-HLA-A or anti-HLA-C (white) and phalloidin (blue). Representative XY images of fluorescence staining are shown. c, Schematic of sgRNA targeting HLA-A/B/C exon 1. The guide sequence targeting human HLA-B is indicated in green. PAM is in bold typeface. d, HaCaT-RasV12-HLA TKO cells had no MHC class Ia proteins. HaCaT-RasV12 WT or -HLA TKO cells were single-cultured. The cell lysates were subjected to immunoblotting. Arrowhead: target band. *: non-specific band. e-g, Direct sequencing results of CRISPR/Cas9-mediated gene disruption in MDCK cells. Sequences of MDCK WT cells are shown in top rows, and those of MDCK-DLA12 KO (e), -DLA64 KO (f), or -DLA88 KO (g) cells are shown in lower two rows. PAM is underlined. The red spacing and arrows indicate a deleted nucleotide and the guide sequences, respectively. h, Disruption of TAP1 suppressed the efficiency of apical extrusion. Normal/Rasv12-MDCK cells were mixed and treated with doxycycline for 24 h. Cells were subjected to immunohistochemistry with phalloidin (red) and Hoechst (blue) (Method 1). Representative XZ images of fluorescence staining (top) and quantification data of apical extrusion of RasV12 cells (bottom) are shown. Data are presented as mean ± s.d.; n = 3 biologically independent experiments; **P < 0.01, ***P < 0.001 by unpaired two-sided Student’s t-tests over MDCK-RasV12 WT cells; scale bars, 10 μm. Broken lines indicate the apical lines of the cell monolayers.
