To detect the loci of MMPs activity within invading H9c2 cells, a

To detect the loci of MMPs activity within invading H9c2 cells, an in situ zymography approach was employed ( Galis et al., 1995). Briefly, after incubation with T. theileri for one hour, H9c2 cells were washed with PBS five times, and then incubated with DQ-labeled gelatin (Invitrogen) substrate solution (20 μg/ml DQ-gelatin, 50 mM Tris, pH 6.8, 50 mM NaCl, 20 mM CaCl2) for 2 h at 37 °C in the dark. After the substrate solution was washed off, slides were incubated in 4% PFA for 10 min, washed with PBS, mounted with DAPI, and photographed with identical exposure settings using a laser confocal microscope (FV1000-D, Olympus). After fixation with 4% paraformaldehyde (PFA)

in PBS for 5 min, chamber slides with attached cells were washed three times in PBS. Nonspecific immunoglobulin EPZ-6438 nmr binding sites were blocked with www.selleckchem.com/EGFR(HER).html 5% BSA for 30 min at room temperature, and then cells were incubated with first antibody: rabbit anti-TWTth1 polyclonal antibody, MAP1LC3A antibody (ab64123, Abcam), or TGF-β pan specific polyclonal antibody (AB-100-NA, R&D Systems) with Tris-buffered saline-Tween 20 (TBS-T) containing 2% BSA at 4 °C

overnight. The sections were then incubated with FITC conjugated anti-rabbit IgG for 1 h at room temperature, followed by washing, mounting and examination by laser confocal microscopy. Analysis of transcripts of the TGF-β1 gene by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed as described in our previous study (Cheng et al., 2010). For tracing MTMR9 in live cell images, BHK, SVEC, and H9c2 cells and T. theileri were labeled with CellTracker™ Probes (Molecular Probes™) for long-term tracing: LysoTracker Red DND-99/Green DND-26 (L-7528, L-7526), and MitoTracker Red CMXRos/Green FM (M-7512, M-7514) within 30 min according to the manufacturer’s protocol (Invitrogen), followed by Hoechst 33342 (H-21492) staining for invasion assay. To identify the possible interaction between T. theileri and the host cell via the autophagic pathway,

two sets of experiments were performed. (A) After 3 h infection, cells were washed four times with PBS and fixed with 4% PFA for 10 min at room temperature. Autophagosomes were stained with anti-MAP1LC3 antibody as described in Section 2.6 above. (B) H9c2 cells were transfected in vitro with pSelect-LC3-GFP expression plasmid (psetz-gfplc3, InvivoGen) by jetPEI™ transfection reagent (PolyPlus Transfection), according to the instructions supplied by the manufacturer. For the in vitro infection assay, LC3-GFP-expressing H9c2 cells were plated onto an Ibidi 35 mm u-Dish (Ibidi, GmbH, Martinsried, Germany), at a density of 4 × 103 cells per well, and incubated overnight at 37 °C in a 5% CO2 humidified atmosphere. Before infection, cells were washed once with culture medium to remove nonadhered cells.

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