Figure 3. Autophagy of S. Typhimurium requires phosphatidic acid phosphatase and phospholipase D. (a).

Pathways for the production of DAG. PC (phosphatidylcholine), PLD (phopholipase D), PA (phosphatidic acid), PAP (phosphatidic acid phosphatase), propr. (propranolol hydrochloride), PLC (phospholipase C), SMS (sphingomyelin synthase), SM (sphingomyelin). (b) HeLa cells were transfected with GFP-LC3 and infected with RFP-Sal. Cells were treated with growth medium (GM), U73122(10 μM), DMSO, propranolol hydrochloride (propr., 250 μM) or H₂O at 10 min p.i. for the remainder of the infection, and fixed at 1 h p.i. (c) HeLa cells were transfected with PKCδ-C1-GFP and infected with RFP-Sal. Cells were treated with GM, propranolol hydrochloride (propr., 250 μM) or D609 at the indicated concentrations at 10 min p.i. for the remainder of the infection, and fixed at 45 min p.i.. (d) HeLa cells were co-transfected with GFP-LC3 and either control siRNA (si-CTRL) or siRNA specifically targeting Atg12 (si-ATG12) or PAP2B (si-PAP). Cells were infected with RFP-Sal and fixed at 1 h p.i. (e) HeLa cells were transfected with RFP-LC3 and either GFP, HA-PLD1 DN (dominant negative) or HA-PLD2 DN. Cells were infected with S. Typhimurium, fixed at 1 h p.i. and immunostained for S. Typhimurium and HA tag. The percentage of DAG⁺ or LC3⁺ S. Typhimurium was determined by fluorescence microscopy. Data represent the mean ± standard error (s.e.m.) for at least three independent experiments.