GPR84 knockdown suppresses β-catenin expression and in vitro colony formation of stem cell–derived MLL pre-LSCs. (A) Western blot analysis of GPR84 knockdown efficiency and β-catenin expression in scrambled control (Scr) or 2 independent GPR84 shRNAs (KD#1 and KD#2) transduced KLSMLL pre-LSCs. (B) Colony-forming assays of Scr or GPR84 KD KLSMLL pre-LSCs. Numbers of colonies per dish at the third round of replating are shown. (C) Total number of cells per dish after the third round of replating. (D) Flow cytometric analysis of cell-cycle status in Scr or GPR84 KD#1 KLSMLL pre-LSCs. Error bars represent triplicates obtained in independent experiments. (E) Western blots of GPR84 and β-catenin in Scr or GPR84 KD#1 KLSMLL pre-LSCs expressing empty vector (EV) or KD#1-resistant GPR84 cDNA (mutG). (F) Colony-forming assays of Scr or GPR84 KD#1 KLSMLL pre-LSCs expressing EV or mutG. *P < .05, **P < .01, ***P < .0005, NS, not significant (P > .05).