Extended Data Fig. 8: Extended data for Fig. 6.
From: Epithelial cells remove precancerous cells by cell competition via MHC class I–LILRB3 interaction
a, Mouse Pirb is the homologous gene of Canis ALTR. PirB has 6 immunoglobulin-like domains (D1 − D6) in the extracellular region shown as a circle. D1/D2 and D3/D4 domains of PirB have high homology with D1/D2 domain of AltR shown as blue-filled circles. In the intracellular region, ITIM domains are shown as blue squares. b, Rec.AltR-D1/D2 protein interacted with mouse MHC class I (mMHC-I). Cell lysates of mouse Raw264 cells were mixed with GST or rec.GST–AltR-D1/D2–HA protein (rec.AltR-D1/D2) and subjected to pulldown assay with glutathione-conjugated beads. Interacted mouse MHC class I was detected with an anti-MHC-I (clone: OX-18). White arrowhead: rec.AltR-D1/D2 protein. Black arrowhead: GST. *: mMHC-I. c, The interaction analysis of rec.GST–MHC-α3–Flag with PirB-D1-8mer using Bio-Layer Interferometry (BLI). Kd = 2.88 ± 0.20 nM. d, PirB was upregulated in surrounding normal cells. NMuMG and/or NMuMG-pTRE3G-GFP–RasV12 (NMuMG-RasV12) cells were single/mixed-cultured treated with doxycycline for 12 h. The cells were stained with an anti-PirB (red), phalloidin (white) and Hoechst (blue). e, Images of air-evacuated lungs. Collected lungs were fixed and then air-evacuated under the low-pressure condition. Arrowhead: tumorigenic nodule. f, Images of H&E staining (top) and immunofluorescence staining (bottom) of a nodule; E-cadherin, red; CK19, white; Hoechst, blue. g, Urethane induced Kras-mutated nodules in lungs. The genomic DNAs of nodules microdissected from the lungs were extracted and subjected to PCR analysis. The amplicons around the Kras exon 2 (codon 12, G12V) or exon 3 (codon 61, Q61L/R) were used for the direct-sequencing analysis. The observed Kras mutation and mutation rate are shown in a graph; ND., not detected. h, HaCaT-RasQ61L cells were extruded from the epithelial monolayer. HaCaT cells were mixed with HaCaT-RasV12 or -pTRE3G-Myc-RasQ61L cells and cultured with doxycycline for 16 h. The cells were stained with an anti-Myc (green), phalloidin (red), and Hoechst (blue) (Method 1). Representative XZ images of fluorescence staining (left) and quantification data of apical extrusion (right). Broken lines indicate the apical lines of the cell monolayers. i, Knockdown efficiency of Pirb by AAV6-shPirb infection. Mouse Raw 264 cells were infected with AAV6-shRNA for control (shCtrl) or Pirb (shPirb) and cultured for 48 h. The cells were subjected to qPCR analysis. The knockdown efficiency is shown as relative expression to AAV6-shCtrl-infected cells. d-f,h, Scale bars, 10 μm (d,h), 2 mm (e), or 100 μm (f). h,i, Data are presented as mean ± s.d.; n = 3 biologically independent experiments; **P < 0.01 by unpaired two-sided Student’s t-test.
