Impaired TLR3- and TLR4-mediated induction of IFN-responsive genes in LYST-mutant THP-1 cells. (A) Sequence analysis showing indel mutations for all three alleles of LYST in THP1-Blue ISG clones 7 and 5. Red dashes indicate deleted bases, red letters show inserted or substituted bases, and numerals indicate total number of deleted/inserted base pairs. (B) Whole cellular lysates of the indicated THP1-Blue ISG cell clones were immunoblotted (IB) with antibodies against TRIF, actin, and Rab7. Data are representative for three independent experiments. (C) Differentiated WT and LYST-mutant (clones 5 and 7) THP1-Blue–ISG cells that carry an IRF-inducible SEAP reporter construct, were stimulated (stim.) overnight with 1 µg/ml LPS, 20 µg/ml poly(I:C), or 100 ng/ml Pam3CSK4, and the IRF response was assessed by determining the SEAP activity from cell culture supernatants. Supernatants from Pam3CSK4-treated cells were diluted threefold. Data represent mean ± SEM. *, P < 0.05 (Student’s t test). Data are pooled from n = 4 independent experiments. unstim., unstimulated.