Cytoplasmic Actin Is an Extracellular Insect Immune Factor which Is Secreted upon Immune Challenge and Mediates Phagocytosis and Direct Killing of Bacteria, and Is a Plasmodium Antagonist
(A) Immune-challenged (LPS-Pa, LPS-EcK12, LPS-Ec; 10 μg/mL Lys-PGN; 20 μg/mL, or DAP-PGN; 1 μg/mL) Sua5B (An. gambiae), and S2 (D. melongaster) insect cell supernatants and soluble lysate fractions examined for the presence of actin (upper panel) or tubulin (lower panel). (B) Viability of Sua5B and S2 cell lines after treatment with LPS-Pa (10 μg/mL), Lys-PGN (20 μg/mL) or DAP-PGN (1 μg/mL) for 24 hr was determined using the Cell Titer Fluor Cell Viability Assay. (C) Cell viability of Sua5B and S2 cells after saponin (1–10−6%)-induced cell lysis and (D) the amount of actin released into the supernatant was determined for Sua5B (upper panel) and S2 cells (lower panel) at varying saponin concentrations (10−5, 10−4, 10−3 and 1%). (E) Supernatant fractions of Sua5B and Moss55 cells challenged with LPS (10 μg/mL) or Lys-PGN (20 μg/mL) for 24 hr in the presence of 5 μM GW4869 or DMSO (control) and probed for actin. (F) Exosomes (EXO) and lysate fractions isolated from LPS and Lys-PGN stimulated Sua5B or Moss55 cells analyzed for actin. (G) Supernatant fraction of caspar and cactus silenced Sua5B cells probed for the presence of actin.