Impact of CCDC134 loss on TLRs and integrins. (A and B) THP1 DUAL reporter cells stimulated with R848 (5 μg/ml), flagellin (0.1 μg/ml), Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (0.1 μg/ml), Poly(I:C) complexed with lipofectamine (1 μg/ml), cGAMP (3 μg/ml), C12-iE-DAP (5 μg/ml), L18-MDP (10 μg/ml), TNFα (10 ng/ml), or IL-1β (10 ng/ml) for 24 h. Supernatants were analyzed for ISRE and NF-κB reporter activity. (A, left panel, and B) Data show mean ± SD from one representative experiment performed in stimulation triplicates. (A, right panel) Reporter activity relative to sgRen. Data show three independent experiments. Line represents the mean ± SD ISRE reporter activity, R848 sgCCDC134-1 P value <0.0001, sgCCDC134-4 P value <0.0001; Pam3CSK4 sgCCDC134-1 P value = ns, sgCCDC134-4 P value = ns; LPS sgCCDC134-1 P value <0.0027, sgCCDC134-4 P value <0.0030; flagellin sgCCDC134-1 P value = 0.0006, sgCCDC134-4 P value <0.0001; Poly(I:C) complexed with lipofectamine sgCCDC134-1 P value = ns, sgCCDC134-4 P value = ns; cGAMP sgCCDC134-1 P value = ns, sgCCDC134-4 P value = ns. NF-κB reporter activity, R848 sgCCDC134-1 P value <0.0001, sgCCDC134-4 P value <0.0001; Pam3CSK4 sgCCDC134-1 P value = ns, sgCCDC134-4 P value = 0.0315; LPS sgCCDC134-1 P value = 0.0012, sgCCDC134-4 P value = 0.0022; flagellin sgCCDC134-1 P value = 0.0053, sgCCDC134-4 P value = 0.0051; C12-iE-DAP sgCCDC134-1 P value = ns, sgCCDC134-4 P value = ns; L18-MDP sgCCDC134-1 P value = ns, sgCCDC134-4 P value = ns; TNFα sgCCDC134-1 P value = ns, sgCCDC134-4 P value = ns; IL-1β sgCCDC134-1 P value = 0.0329, sgCCDC134-4 P value = 0.0291. Two-tailed one sample t test. Untr.: untreated. (C) TNFα production of indicated knockout THP1 cells stimulated with flagellin (0.1 μg/ml) for 24 h. (D) IL-6 (Top panel) and TNFα (Bottom panel) production of indicated knockout THP1 cells differentiated with 10 nM of PMA for 24 h before stimulation with LPS (0.1 μg/ml), Pam3CSK4 (Pam3.) (0.1 μg/ml) or Pam2CSK4 (Pam2.) (0.1 μg/ml) for 24 h. Untr.: untreated. (E) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated knockout THP1 cells differentiated with 10 nM of PMA for 48 h. (F and G) Immunoblots of indicated knockout Hoxb8-macrophages stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) or LPS (10 ng/ml) for 0–1 h (F) or knockout Raw 264.7 cells (G). Asterisks indicate a non-specific band. (H) TNFα production of indicated knockout Raw 264.7 cells stimulated with CpG-B (ODN1668) (150 nM), LPS (10 ng/ml), or poly(I:C) (500 ng/ml) for 24 h. (I) Representative histograms of surface (upper panel) or surface and intracellular (lower panel) expression of CD11a, CD18, CD49d, or CD44 in sgRen (red) or sgCCDC134 (blue) Hoxb8-macrophages. Gray curves represent unstained controls. (J) Volcano plot of quantified proteins in whole proteome of sgCCDC134 versus sgRen Hoxb8-macrophages (upregulated: red, fold change [FC] >2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01); green: downregulated ISGs signature. In A–D and H, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In E–G, data are representative of two independent experiments. In I, data are representative of three independent experiments. Source data are available for this figure: SourceData FS5.