Fv500

Confocal laser scanning microscopy was performed using LSM510 (Carl Zeiss) and Olympus FV500 (Olympus America Inc.) confocal laser-scanning microscopes. Images were acquired by sequential scanning and optimized by a two-frame Kalman filter and analyzed using the Fluoview SV500 imaging software 4.3 (Olympus America Inc., Melville, USA).

To determine the intracellular localization and changes of collagen-I, PMCs were incubated with TPE for 24 h. Then, the cells were stained using rabbit polyclonal antibody against collagen-I and mouse monoclonal antibody against calretinin at 4°C overnight and then incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse secondary antibody at room temperature for 60 min. The nuclei were stained for DAPI for 10 min in dark. The fluorescence-labeled cells were examined using a fluorescent microscope (Olympus FV500, Olympus, Tokyo, Japan).

MDA-MB-231 and BT549 cells (5 × 10⁴) were seeded after 6 h of starvation in serum-free DMEM onto Transwell™ filters coated with a collagen gel (1 mg/mL) and prepared as described in the collagen matrix preparation section. Medium containing 20% fetal bovine serum with EGF and HGF was present in the lower compartment as a chemoattractant. After 24 h, non-invading cells and collagen on the upper side of the filters were removed. Cells that had migrated through the membrane were fixed with 4% formaldehyde, stained with Hoechst 33342, and counted under a fluorescent microscope Olympus FV500 (Tokio, Japan). The results are presented as a relative invasion factor (%) versus the control. The number of control cells that invaded through the Transwell™ filters is set as 100%.

The cell apoptosis in brain slices and microglial climbing slices was assessed by TUNEL in situ cell death detection kit (Roche, Germany) according to the manufacturer’s instructions. Briefly, after permeabilized with 0.3% Triton-X100 and blocked with 10% bovine serum albumin, the slices were incubated with TUNEL reaction mixture for 1 h. Then, the cell climbing slices were mounted with an antifade mounting medium with DAPI, while the brain slices were further incubated with Iba1 primary antibody overnight at 4 °C and corresponding secondary antibody for 1 h before mounting. The images of stained cells were acquired with a laser scanning confocal microscope (Olympus FV500, Japan).

Human pleura slides were obtained from pleura biopsies of patients with TPE and MPE. To measure the level of calpain activation in pleura, spectrin breakdown product (SBDP) was measured using an antibody that specifically recognizes SBDP as described previously (10 (link), 15 (link)). Spectrin contains a specific calpain cleavage site, releasing SBDP. This method has been widely used to detect calpain activation in vivo (3 (link), 10 (link), 15 (link)). The slides were double-labeled for SBDP and calretinin (specific marker of PMCs) and counterstained using DAPI. The slides were mounted with anti-fade reagent and examined using a fluorescent microscope (Olympus FV500; Olympus, Tokyo, Japan). The yellow color indicates colocalization of SBDP and calretinin.

Cell migration tests were performed using Transwell™ filters (BD Biosciences, Franklin Lakes, NJ, USA) in a 24-well plate. Prior to the assay, cells were starved for 6 h in serum-free DMEM medium. The lower compartment of the Transwell™ contained 500 μL of medium consisting of 80% DMEM, 20% FBS, 5 nM EGF, and 30 ng/mL of HGF. Cells (5 × 10⁴) were seeded onto a rehydrated Transwell™ insert in medium without FBS in the absence (control) or presence of foretinib, lapatinib, or foretinib/lapatinib. After 24 h, non-migrating cells on the upper side of the filters were removed. Cells that migrated through the membrane were fixed with 4% formaldehyde, stained with Hoechst 33342 (Invitrogen), and counted under a fluorescent microscope Olympus FV500 (Tokio, Japan). The results are showed as a relative migration factor (%) where the number of control cells that migrated through the Transwell™ filters are presented as 100%. The experiments were performed three times. Each independent experiment consisted of three measurements.

In total, 1000 cells/well were added to 2 mL of DMEM containing 10% FBS and seeded in six-well plates. The cells were cultured for 24 h in media treated with various concentrations of HMH, followed by removal of the media. They were then replaced with fresh media without HMH every 3–4 days at 37 °C and grown in a 5% CO₂ incubator for 14 days to observe colony formation. Cell colonies were fixed in methanol for 15 min and stained with 0.1% crystal violet for 10 min. The colonies (>50 cells) were counted under a microscope (Olympus FV500; Olympus Corporation, Tokyo, Japan).