Journal: Scientific Reports

Article Title: Long-chain bases of sphingolipids are transported into cells via the acyl-CoA synthetases

doi: 10.1038/srep25469

Figure Lengend Snippet: ( a , b ) IEC-6 cells were incubated with 5 μM [ 3 H]DHS ( a ) or [ 3 H]palmitic acid (PAL) ( b ) for the indicated time period. Radioactivities associated with cells and medium were counted by a liquid scintillation counter, and those associated with cells are expressed as a percentage of the total radioactivity. ( c , d ) IEC-6 cells were labeled with 5 μM [ 3 H]DHS ( c ) or 5 μM [ 3 H] palmitic acid (PAL; d ) in the presence of cold palmitic acid or DHS, respectively, at the indicated concentration at 37 °C for 15 min. Transport activity was determined as in ( a ). Values represent the means ± SDs of three independent experiments, and statistically significant differences are indicated ( t -test; * p < 0.05; ** p < 0.01). ( e , f ) BY4741 (wild-type; WT) or AOY13 ( faa1 Δ faa4 Δ; ΔΔ) cells harboring the pAKNF316 (vector; vec), pAO15 ( 3xFLAG-ACSL1 ; L1), pAO16 ( 3xFLAG-ACSL3 ; L3), pAO17 ( 3xFLAG-ACSL4 ; L4), pAO19 ( 3xFLAG-ACSL5 ; L5), or pAO21 ( 3xFLAG-ACSL6 ; L6) plasmid were grown in SC-URA medium at 30 °C. ( e ) Cells were labeled with 20 μM [ 3 H]DHS at 30 °C for 5 min. Radioactivities associated with cells, medium, and glass test tubes were counted by a liquid scintillation counter, and those associated with cells are expressed as a percentage of the total radioactivity. Values represent the means ± SDs of three independent experiments. Statistically significant differences with respect to the value of AOY13 ( faa1 Δ faa4 Δ) cells harboring vector are indicated ( t -test; ** p < 0.01). ( f ) Total cell lysates were prepared, separated by SDS-PAGE, and subjected to immunoblotting with anti-FLAG or, to demonstrate equal protein loading, anti-Pgk1 antibody. ( g , h ) IEC-6 cells were treated with DMSO or 20 μM triacsin C (Tri C) at 37 °C for 1 hr. Cells were then labeled with 5 μM [ 3 H] palmitic acid ( g ) or 5 μM [ 3 H]DHS ( h ) at 37 °C for 15 min. Transport activity was determined as in ( a ). Values represent the means ± SDs of three independent experiments, and statistically significant differences are indicated ( t -test; * p < 0.05; ** p < 0.01).

Article Snippet: Triacsin C was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Radioactivity, Labeling, Concentration Assay, Activity Assay, Plasmid Preparation, SDS Page, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis

doi: 10.1073/pnas.1921619117

Figure Lengend Snippet: E. chaffeensis is dependent on host-derived lipids and incorporates exogenous phospholipids and cholesterol. ( A ) E. chaffeensis- infected THP-1 cells were seeded in a six-well plate. At 1 hpi, cells were incubated with 0, 0.5, or 1 μM triacsin C (TC) for 2 d at 37 °C. DNA was extracted from treated samples, and quantitative PCR was performed for the E. chaffeensis 16S rRNA gene and normalized against human ACTIN . Results are shown as the mean ± SD from three independent experiments. ** P < 0.001; * P < 0.01 (ANOVA). ( B – D ) RF/6A cells were seeded onto cover glasses in a six-well plate for 3 h and then infected with E. chaffeensis ( Ech ). Cells were incubated with 25 μM NBD-PC for 1 d at 1 dpi ( B ), or with 5 μM Bodipy-PE for 4 h at 2 dpi ( C ); Alternatively, infected cells at 1 dpi were washed and replaced with AMEM containing lipoprotein-depleted serum for 8 h, then incubated with 1 μM TF-Chol for 1 d ( D ). Cells were fixed and DNA was stained with Hoechst 33342 to label host and bacterial DNA (pseudocolored in red). Samples were observed under a DeltaVision microscope. The boxed area in the merged image is enlarged 3× on the Right . DC, dense-core forms with diameter <1 μm and tightly packed chromosomes; DIC, differential interference contrast; RC, reticulate cell forms of E. chaffeensis with larger diameter (≥1 μm) and more loosely-packed chromosome DNAs. Open arrow, inclusion membrane; solid arrow, E. chaffeensis membrane; N, nucleus. Images are representative of at least three independent experiments. (Scale bars, 10 μm.)

Article Snippet: For treatment with the ACSL inhibitor triacsin C (StressMarq Biosciences), E. chaffeensis- infected THP-1 cells at 1 hpi were incubated with 0.5 or 1 µM triacsin C and cultured for an additional 2 d at 37 °C.

Techniques: Derivative Assay, Infection, Incubation, Real-time Polymerase Chain Reaction, Staining, Microscopy

Journal: bioRxiv

Article Title: Altering lipid droplet homeostasis affects Coxiella burnetii intracellular growth

doi: 10.1101/112300

Figure Lengend Snippet: Macrophages were infected with C. burnetii and at different times post-infection, cells were stained for PLIN2 (LDs; green), C. burnetii (red) and nucleus (blue). LD number per cell were quantitated by fluorescence microscopy. A) LD numbers in wild-type C. burnetii and dotA mutant-infected MH-S macrophages. B) LD numbers in MH-S macrophages infected with wild-type C. burnetii and treated with chloramphenicol (3ug/ml). C) LD numbers in wild-type C. burnetii and dotA mutant-infected T HP-1 macrophage-like cells. D) Images of LDs and bacteria in MH-S cells at day 1 post-infection imaged at lO0X. Scale bar = 10 μm. Error bars show the mean of 3 independent experiments +/- SEM * =p<0.05, ** =p<0.01, *** =p <0.001 as determined by ordinary one-way ANOVA with Tukey post-hoc test. Wild-type C. burnetii growth in infected MH-S cells treated with different inhibitors was measured at 2 and 4 days post-infection by FFU assay. A-E) Representative images for wild-type MH-S macrophages treated with inhibitors, fixed, stained for PLIN2 (LDs; green) and C. burnetii (red) and imaged day 4 post-treatment at lO0X. Scale bar = 10 μm. F) Growth while inhibiting LD formation with triacsin C (10 μM) in wild-type MH-S macrophages. Error bars represent the mean of 4 independent experiments +/- SEM. **= p <0.01 compared to vehicle-treated cells as determined by two-way ANOVA with Bonferroni post-hoc test. G) ACATl protein expression in wild-type and acat-J 1 macrophages. Cell lysates were immunoblotted and ACATl protein levels were compared with GAPDH as loading control. H) C. burnetii growth in vehicle-treated wild-type and acat-1 1 MH-S macrophages and (I) T863-treated acat-J-1 MH-S macrophages. Error bars represent the mean of at least 3 independent experiments +/SEM., * =p<0.05, *** =p <0.001 as determined by two-way ANOVA with Bonferroni post-hoc test.

Article Snippet: The optimum concentrations determined for each inhibitor was: Triacsin C (Enzo Life Sciences, Farmingdale, NY, USA) – 10 μM, T863 (Sigma-Aldrich) – 10 μM, CAY10499 (Cayman Chemicals, Ann Arbor, MI, USA) - 10 μM, Atglistatin (Cayman Chemicals) – 20 μM.

Techniques: Infection, Staining, Fluorescence, Microscopy, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: Altering lipid droplet homeostasis affects Coxiella burnetii intracellular growth

doi: 10.1101/112300

Figure Lengend Snippet: Wild-type C. burnetii growth in infected MH-S cells treated with different inhibitors was measured at 2 and 4 days post-infection by FFU assay. A-E) Representative images for wild-type MH-S macrophages treated with inhibitors, fixed, stained for PLIN2 (LDs; green) and C. burnetii (red) and imaged day 4 post-treatment at lO0X. Scale bar = 10 μm. F) Growth while inhibiting LD formation with triacsin C (10 μM) in wild-type MH-S macrophages. Error bars represent the mean of 4 independent experiments +/- SEM. **= p <0.01 compared to vehicle-treated cells as determined by two-way ANOVA with Bonferroni post-hoc test. G) ACATl protein expression in wild-type and acat-J 1 macrophages. Cell lysates were immunoblotted and ACATl protein levels were compared with GAPDH as loading control. H) C. burnetii growth in vehicle-treated wild-type and acat-1 1 MH-S macrophages and (I) T863-treated acat-J-1 MH-S macrophages. Error bars represent the mean of at least 3 independent experiments +/SEM., * =p<0.05, *** =p <0.001 as determined by two-way ANOVA with Bonferroni post-hoc test.

Article Snippet: The optimum concentrations determined for each inhibitor was: Triacsin C (Enzo Life Sciences, Farmingdale, NY, USA) – 10 μM, T863 (Sigma-Aldrich) – 10 μM, CAY10499 (Cayman Chemicals, Ann Arbor, MI, USA) - 10 μM, Atglistatin (Cayman Chemicals) – 20 μM.

Techniques: Infection, Staining, Expressing