Bx50 bs fla

Cells were grown on four-well collagen type I-coated culture slides (BD BioCoat). The slides were incubated with fibroblast activation protein (FAP) antibody (ab53066, rabbit polyclonal IgG: diluted 1:100; Abcam), α-SMA antibody (1A4, mouse monoclonal IgG; diluted 1:100; DakoCytomation, Denmark), and E-cadherin antibody (H-108, rabbit polyclonal IgG; diluted 1:100; Santa Cruz Biotechnology, Inc., USA). The secondary antibodies were anti-rabbit IgG antibody conjugated with Alexa Fluor^® 488 (ab150073, donkey polyclonal IgG, diluted 1:1000; Abcam) and an anti-mouse IgG antibody conjugated with Alexa Fluor 594^® (ab150116, goat polyclonal IgG, diluted 1:1000; Abcam). The slides were observed using an immunofluorescence microscope (BX50/BS-FLA; Olympus, Japan).

For visualization of FAP, E-cadherin, and α-SMA in HPMCs, cells were grown on four-well collagen type I-coated culture slides (BD BioCoat) and fixed when preconfluent in a 1:1 mixture of methanol and acetone for 10 min. Briefly, slides were immersed in methanol containing 0.3% H₂O₂ for 30 min, blocked with 3.3% normal goat serum in PBS, and incubated with FAP antibody (ab53066, rabbit polyclonal IgG: diluted 1: 100; Abcam, Tokyo, Japan), E-cadherin antibody (H-108, rabbit polyclonal IgG; diluted 1:100; Santa Cruz Biotechnology, Inc., USA), and α-SMA (1A4, mouse monoclonal IgG; diluted 1:100; DakoCytomation, Denmark) at 4 °C overnight. Following three washes in PBS, immunoreactivity was visualized by incubating the slides with an anti-mouse IgG antibody conjugated with Alexa Fluor^® 488 and an anti-rabbit IgG antibody conjugated with Alexa Fluor^® 546 (1:400; Molecular Probes/Invitrogen, USA) for 1 h at room temperature. Cells were then incubated for 5 min with Hoechst 33258 for nuclear staining and mounted with propyl gallate containing phenylenediamine under glass coverslips. The slides were observed using an immunofluorescence microscope (BX50/BS-FLA; Olympus, Japan).

Transwell migration and invasion assays were performed using 8 μm Transwell inserts (Migration Chamber: 140629, Lab Unlimited, Dublin, Ireland; Invasion Chamber: 354480, Corning Inc., Corning, NY, USA). Cells were suspended in serum-free RPMI-1640 medium without glucose or glutamine, containing 2% BSA and cultured in the upper chamber. 1% FBS-conditioned serum-free, no glucose, no glutamine RPMI-1640 with 2% BSA containing either 0.1 mM PA, 0.1 mM PA with 50 or 100 μM etomoxir, or respective controls, was added to the lower chamber. The cells that migrated or invaded the lower chamber of the inserts were photographed under a microscope (BX50/BS-FLA; Olympus), and five visual fields were randomly chosen to calculate the number of cells.