Extended Data Fig. 5: Extended data for Fig. 4.
From: Epithelial cells remove precancerous cells by cell competition via MHC class I–LILRB3 interaction
a,b, AltR-V5 expressed in normal cells was phosphorylated under the mixed condition, and the phosphorylation was promoted by MHC class I. MDCK-ALTR KO-AltR-V5-OE cells were cultured alone (a Normal), or mix-cultured with MDCK-RasV12 WT cells (a,b Mix) or -DLA88 KO cells (b) with doxycycline and Na3VO4 for 2 h (b) or 4 h (a,b). The cell lysates were subjected to immunoprecipitation assay with an anti-phospho-tyrosine and followed by detection with an anti-V5. Numeric value below bands is relative intensities of phosphorylated AltR normalized with total AltR. Arrowheads: phosphorylated V5-AltR. *: non-specific band. c,d, An SHP2-specific inhibitor suppressed apical extrusion using MDCK and HaCaT cells. Co-cultured HaCaT or MDCK cells were treated with DMSO or an SHP2-specific inhibitor, SHP099, upon doxycycline treatment for 16 (c, HaCaT cells) or 24 (d, MDCK cells) h. The extrusion efficiency was quantified and shown as graphs (Method 1). e, LILRB3 knockout suppressed the accumulation of filamin in surrounding normal cells. HaCaT WT or -LILRB3 KO cells were mixed with HaCaT-RasV12 cells and cultured with doxycycline for 12 h, followed by immunohistochemistry with an anti-filamin (red) and phalloidin (white). Representative XY images of fluorescence staining (left) and quantification data of filamin accumulation in surrounding HaCaT cells (right) are shown; scale bars, 10 μm. f, The primed normal cells by contact with RasV12 cells induces LILRB3/AltR, which promotes apical extrusion via filamin accumulation. Neighboring normal cells sense physical changes, eg. membrane tension, of RasV12 cells and induce RUNX2, and thereby the induced RUNX2 drives LILRB3 expression (left). The expressed LILRB3 on the cell surface of normal cell further interacts with MHC class I of RasV12 cells. Activated LILRB3 by interaction with MHC class I is phosphorylated and recruits SHP2, leading to activation of SHP2 − ROCK2 signaling pathway, which eventually promotes filamin accumulation (right). c-e, Data are presented as mean ± s.d.; n = 3 biologically independent experiments (c,d); n = 56 (WT) or 61 (LILRB3 KO) cells examined over three biologically independent experiments (e); *P < 0.05, **P < 0.01, ****P < 0.0001 by unpaired two-sided Student’s t-test.
