Fig 2: GRA7-III-induced activation of PLD1 and phagosomal maturation were required for antimicrobial activity in MTB-infected macrophages.(A and B) BMDMs were infected with MTB (MOI = 1) for 4 h and then stimulated with rGRA7 (5 µg/ml) and its mutants for 18 h. Mycobacteria-containing phagosome fractions were subsequently purified by sucrose-step-gradient-ultra-centrifugations, followed by IB to detect aRab5, aRab7, aLAMP1, aLAMP2, aPLD1, aPKCa, and aActin (A) or IP with aPLD1 and IB with aHis, aPLD1, and aActin (B). Quantitative analysis of the PLD1 interacts with His-rGRA7 in MTB-containing phagosomes band normalized to Actin is shown (lower). (C and D) Intracellular survival of MTB was assessed by CFU assay. BMDMs were infected with MTB for 4 h, followed by treatment with rGRA7, and then lysed to determine intracellular bacterial loads. (D, right) IB with aPLD1, a?SC, and aActin in BMDMs. The data are representative of five independent experiments with similar results (A and B). Data shown are the mean ± SD of five experiments (C and D). Significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) compared with PKCa+/+ and PKCa-/- (B) or rVector (C and D). CFU, colony-forming units. ns, not significant.

Fig 3: GRA7 interactions with ASC and PLD1.(A) Identification of PLD1, PKCa, TRAF6, and ASC by mass spectrometry analysis in THP-1 cell lysates incubated with a His-tagged rGRA7 (2 µg). CoBlue, staining of His-rGRA7 with Coomassie blue. Whole cell lysates (WCLs) were used for immuno blotting (IB) with aActin. (B) THP-1 cells were stimulated with rGRA7 (5 µg/ml) for the indicated times, followed by immunoprecipitation (IP) with aHis-agarose bead and IB with aPLD1, aTRAF6, aASC, aHis, and aActin. (C) Summary of the interactions of GRA7 WT and its mutants with ASC, PLD1, and TRAF6. The binding activities of GRA7 WT and mutants are summarized based on the results of Fig 1D and 1E, S2E Fig, and [17]. (D and E) Binding mapping. Schematic diagram of the structures of ASC and PLD1 (upper). (D) At 48 hr post-transfection with mammalian GST or GST-GRA7 and truncated mutant constructs together with V5-ASC (left), V5, or V5-ASC constructs together with GST-GRA7 (right), 293T cells were used for GST pulldown, followed by IB with aV5. WCLs were used for IB with aGST, aV5 or aActin. (E) 293T cells were co-transfected with GST or GST-GRA7 and truncated mutant constructs together with AU1-PLD1 (left), or AU1 or AU1-PLD1 constructs together with GST-GRA7 (right), and subjected to GST pulldown, followed by IB with aAU1. WCLs were used for IB with aGST, aAU1 or aActin. The data are representative of four independent experiments with similar results (A, B, D, E).

Fig 4: PLD inhibitors impair cell growth and block PLD activity in PKD cells.Effect of PLD1-/2-inhibitors on (A) the cell viability, (B) DNA synthesis ability and (C) cell proliferation were determined by MTS, BrdU and cell counting, respectively. All types of cells were exposed to various concentrations of either PLD1 or PLD2 inhibitor for 48 h. An experiment which is representative of three independent experiments is shown.

Fig 5: Dendritic branching of neurons in neuron-glia mixed culture under treatment with vehicle, PLD1 inhibitor and Na-PA.(A) MAP2 staining of DIV 15 neurons in neuron-glia mixed culture treated with 2-butanol, 1-butanol or 1-butanol plus 1 µM Na-PA. Mixed culture was treated with 0.5% 2-butanol, 0.5% 1-butanol or 0.5% 1-butanol plus 1 µM Na-PA at DIV 9. After 6 days of treatment the cells were fixed and subjected to immunostaining with anti-MAP2 antibodies. Bar, 100 µm. (B–E) Quantification of primary dendrites (B), secondary dendrites (C), dendritic tips (D) and Sholl analysis (E) for three groups of neurons in panel A, n = 54 for neurons treated with 2-butanol , n = 63 for neurons treated with 1-butanol, n = 44 for neurons treated with 1-butanol plus Na-PA. (B–D) *p < 0.05, **p < 0.01, ***p < 0.001, E, F (2, 1064) = 142.4, p < 0.001.