(A) LPS induces an increase of about 10-fold in the number of Dipt-lacZ cells that stain positively for β-galactosidase. Ecdysone sensitizes the cells and promotes the response.
(B) Dipt-lacZ induction by LPS requires known Imd signaling components, but not Tl pathway members. The fraction of β-galactosidase-positive cells was normalized to the induced control (normalized %), and influence of RNAi of Tl pathway members (dif, spz, and tub) or Imd pathway members (PGRP-LC, Imd, Ird5, and Dredd) is shown.
(C–H) Activity stain (X-Gal) for β-galactosidase.
(C) Untreated cells.
(D) Cells treated with ecdysone alone.
(E) Cells treated with ecdysone and LPS. About 10% of cells express detectable β-galactosidase.
(F) RNAi against the DDRi sick reduces Dipt-lacZ expression in response to LPS.
(G) RNAi of a representative EDRi, the Ras signaling pathway component Cnk, enhances Dipt-lacZ induction by LPS.
(H) RNAi of a representative CDRi, the actin regulator SCAR induces Dipt-lacZ in the absence of LPS.
(I–J) Immunofluorescence of S2 cells with actin in red, tubulin in green, and DNA in blue. Scale bars in (I) and (J) indicate 10 μm.
(I) Wild-type cells have a characteristic rounded morphology.
(J) RNAi against many CDRi genes disrupts morphological features of wild-type S2 cells. S2 cells are shown treated with MESR4 dsRNA. Cells are significantly larger in appearance and less round, with irregular tubulin and actin networks.