Chaps

PIK3C3/VPS34 kinase activity assay was performed as described previously [11 ]. Briefly, PIK3C3 was immunoprecipitated using an anti-ATG14 antibody after PVT1 or miR-619-5p overexpression with or without gemcitabine treatment. Then the immunoprecipitates were incubated with kinase reaction buffer (100 mM HEPES [ThermoFisher, 15,630,080] [pH 7.5], 300 mM NaCl, 2 mM CHAPS [ThermoFisher, 28,300], 10 mM MnCl2, 2 mM DTT, 100 μM ATP [Sangon Biotech, A600020]) and phosphatidylinositol substrate (Echelon, K-3000) and incubated at room temperature for 2 h. After the addition of the PtdIns3P detection buffer (provided by the kit, K-3004) and PtdIns3P detector protein (provided by the kit), the reaction mixture was then added to the PtdIns3P-coated microplate. The amount of PtdIns3P detector protein bound to the plate was determined through colorimetric detection of the absorbance at 450 nm. The concentration of PtdIns3P in the reaction mixture was calculated as the inverse of to the amount of PtdIns3P detector protein bound to the plate.

B. pseudomallei wild-type (K96243) and SDO mutant cells were resuspended in lysis buffer containing of 8 M urea (OmniPur^®, Germany), 2 M thiourea (Merck, Germany), 4% CHAPS (Thermo Scientific, USA) and 50 mM dithiothreitol (DTT) (OmniPur^®). The lysates were ultrasonicated on ice and the supernatants were collected after centrifugation at 12, 000 × g for 5 min at 4 °C. A 2-D clean-up kit (GE Healthcare, Germany) and Quick Start Bradford protein assay (Bio-Rad, USA) were used for protein precipitation and quantification.

For the isolation of mitochondria from infected MRC-5 lung fibroblasts (MOI 10) and J774.2 Mφ (MOI 5) the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific) was used according to the manufacturer’s recommendations. Briefly, 5 and 24 h post infection 1 × 10⁷ cells were washed several times with PBS and detached from the culture dish using a cell scraper. Mitochondria were obtained using the reagent-based method. For separation of the mitochondrial and the cytosolic fraction, the sample was centrifuged at 12,000 × g for 15 min at 4 °C. The mitochondria were lysed with 2% CHAPS (Thermo Fisher Scientific) in Tris-buffered saline (TBS; 25 mM Tris, 150 mM NaCl, pH 7.2) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Both the mitochondrial and cytosolic fractions were stored at −80 °C and the purity of the mitochondrial fraction was analyzed by western blotting.

Mitochondria were isolated from 2 × 10⁷ primary human macrophages infected with Mtb Erdman (MOI 2) for 5 h using the Mitochondria Isolation Kit for Cultured Cells according to the manufacturer’s recommendations (Thermo Fisher Scientific). In detail, we used the reagent-based methods described in the manufacturer’s manual. The mitochondria pellet was lysed with 2% CHAPS (Thermo Fisher Scientific) in Tris-buffered saline (TBS; 25 mM Tris, 150 mM NaCl, pH 7.2), containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) for 1 min by vortexing. Upon centrifugation, supernatants were harvested and the protein concentration was measured with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and mitochondrial lysates were subjected to immunoblot analysis.

Ten S. mekongi adult male and female worms were separately snap frozen in liquid nitrogen and ground with a mortar and pestle. A 300 µl of lysis buffer containing 8M urea (OmniPur®, Germany), 2M thiourea (Merck, Germany), 4% CHAPS (Thermo Scientific, USA), and 50 mM Dithiothreitol (DTT) (OmniPur®, Germany) was added to each sample. The worm lysates were further ultrasonicated on ice. Cell debris was removed by centrifugation at 12,000 × g for 5 minutes at 4 °C. The supernatants were collected and performed protein precipitation using 2-D clean-up kit followed the manufactural protocol (GE Healthcare, Germany). Protein concentration was determined using Quick Start Bradford protein assay (Bio-Rad, USA).