Extended Data Fig. 6: Normal cells eliminate RasV12 cells from pile-up colony via the interaction between LILRB3 and MHC class I.
From: Epithelial cells remove precancerous cells by cell competition via MHC class I–LILRB3 interaction
a, LILRB3 knockout in normal cells retained RasV12 cell in the epithelial monolayer, and the remaining RasV12 cells formed tumor like cell mass in vitro. Schematic of in vitro pile-up colony formation assay (top) and representative XY images of fluorescence staining (bottom) are shown. HaCaT-RasV12 cells were mixed with HaCaT-LILRB3 KO cells to retain RasV12 cells in an epithelial monolayer. After 48 h incubation, pile-up colonies were cultured for the indicated time period, Day 2 to Day 6. The cells were subjected to immunohistochemistry using phalloidin (red) and Hoechst (blue). b-d, The rec.MHC-α3 treatment suppressed pile-up colony formation and outcompeted RasV12 cells undergo apoptosis. Schematic of in vitro pile-up colony formation assay (b,c top). Normal HaCaT and HaCaT-RasV12 cells were mixed. The cells were treated with doxycycline and rec.AltR-D1/D2 protein to retain RasV12 cells in an epithelial monolayer (Day 0). After 48 h incubation (Day 2), pile-up colonies were cultured with or without rec.MHC-α3 protein for the indicated period from Day 2 to Day 6. The samples were subjected to immunohistochemistry using phalloidin (red), Hoechst (blue) (b-d), and an anti-cleaved Caspase-3 (white and white arrowhead) (d). Representative XY images of fluorescence staining (b,c, bottom and d, left) and quantification data of the number of active Caspase-3 positive cell in outmost RasV12 cells on Day 4 (d, right) are shown; broken line, the position of pile-up colony on Day 4. Data are presented as mean ± s.d; n = 6 [α3 (-)] or 15 [α3 (+)] cells examined over three biologically independent experiments; ***P < 0.001 by unpaired two-sided Student’s t-test. a-d, Scale bars, 20 μm.
