(A and B) LGR6 knockdown in THP-1 cells. (A) Gating strategy: Cells were gated on FSC-SSC dot plots (left), GFP⁺ populations were selected on the histograms (middle), then LGR6 expression was determined within the GFP⁺ population (right). (B) LGR6 expression (MFI) in GFP⁺ THP-1 cells. Results are mean ± SEM (n = 4). ****P < 0.001 versus control shRNA (Ren 713) and mock vector. (C) [³H]-MaR1 binding. THP-1 cells were incubated with 2-nM [³H]-MaR1-ME in the presence or absence of unlabeled 1-μM MaR1-ME for 60 minutes at 4°C. Results are specific binding (CPM), calculated as total CPM ([³H]-MaR1 plus vehicle) – nonspecific CPM ([³H]-MaR1 plus unlabeled MaR1-ME). Results are mean ± SEM (n = 3). *P < 0.05, versus shRNA LGR6. Two-tailed paired Student’s t test. (D) cAMP. THP-1 cells were incubated with MaR1 (1–100 nM) for 15 minutes and cAMP levels determined. Results are mean ± SEM (n = 3). **P < 0.01, versus shRNA LGR6; ^##P < 0.01, versus 1 nM (mock vector). (E–H) Phagocytosis. THP-1 cells were incubated with 10-nM MaR1 or vehicle (0.01% ethanol) for 15 minutes prior to addition of BacLight Red-labeled (PE-Texas Red) E. coli (1:50 THP-1:E.coli) for 45 minutes at 37°C. Flow cytometry was carried out. (E) Representative histograms for GFP (top panels), GFP⁺ BacLight Red E. coli+ (middle panels) and GFP- BacLight Red E. coli+ (bottom panels). (F) Quantification of BacLight Red E. coli (MFI) in GFP⁺ cells (top) and GFP- cells (bottom). Results are mean ± SEM (n = 3). *P < 0.05; **P < 0.01, MaR1 versus vehicle. (G) Structures of MaR1 and 12E-MaR1 (left) and representative histograms (right) of BacLight Red E. coli in GFP⁺ cells. (H) Percent increase of phagocytosis. ****P < 0.0001, versus MaR1-treated mock vector transfected cells. (B and H) One-way ANOVA or (D and F) 2-way ANOVA with Tukey’s multiple comparisons test.