Dm4000

For adherence analysis, 1 ml RPMI medium and 1 ml cell suspension at a density of 4×10⁵/ml were added to 6-well plates coated with collagen, followed by incubation at 37 °C for 1 h, then fixed with 4% PFA. Images were taken by Leica DM4000 (Germany) for statistics analysis. For abscission assess, cells at 80% confluence density were stimulated with 100 μg/ml puromycin aminonucleoside at 37 °C for 48 h, then counted the adherent cells under Leica DM4000.
For migration analysis, 8-μm Transwell kit (Falcon) coated with collagen were hanged on 24-well plates containing 500 μl RPMI medium with 10% FBS. 100 μl cell suspension at a density of 2×10⁵/ml were added to the upper layer of the chamber, followed by incubation at 37 °C for 24 h. Cells from the bottom side of the chamber (migrated cells) were fixed with 4% PFA and stained with 1% crystal violet solution. Migrated cells were observed and counted by Leica DM4000 (Germany).
For endocytosis analysis, MPC5 was cultured in PBS containing FITC-transferrin (50 µg/ml) at 37 °C for 5 h. After washed with PBS and citrate buffer, cells were fixed with 4% PFA and stained with DAPI. Images were taken by a Leica sp8 laser (TCS SP8, Germany) scanning confocal microscope. The endocytosis abilities were indicated by the mean fluorescence intensity of FITC-transferrin.

Immunohistochemistry: Mucosal sections were processed for histology. The Diva Decloaker reagent was used for antigen retrieval in formalin-fixed, paraffin-embedded tissues. α-SMA and vimentin were detected with monoclonal rabbit anti-α-SMA (ab32575, Abcam, Cambridge, MA) (1:300) and anti-vimentin (ab92547, Abcam) (1:300), followed by treatment with goat anti-rabbit IgG H&L (HRP, ab6721, Abcam) and diaminobenzidine.
Immunocytochemistry: Primary cells were seeded on glass coverslips in six-well plates. At least five randomly selected visual fields of the immunostained cells were examined under a light microscope (Leica, DM4000, Tokyo, Japan). The obtained images were compiled using Adobe Photoshop CS6 software.
Immunofluorescence staining was performed on paraffin-embedded sections of normal and diseased human oral mucosal specimens. Rabbit polyclonal antibodies to CD31 (ab28364, Abcam) (1:25) or mouse monoclonal antibodies to α-SMA (ab7817, Abcam) (1:200) were applied overnight, followed by incubation with goat anti-rabbit Cy2 (111–225-144, Jackson ImmunoResearch, West Grove, PA) or a mouse monoclonal antibody to α-SMA (ab205719, Abcam) (1:250), respectively. Cells were examined with a fluorescence (Leica, DM4000, Instruments, Melville, NY) or confocal (Leica, tcssp8, NY) microscope.

24 h post-transfection, cells were treated with colchicine (NSC 757, Selleck) at 0.2 μg/mL for 2 h to arrest in metaphase. The cells in metaphase were harvested and washed with cold PBS twice, and then incubated in 0.075 M KCl at 37 °C for 20 min. Hypotonic cells were then centrifuged for 10 min at 1000 rpm and fixed in Cornoy’s fixative (methanol:acetic acid = 3:1) for 20 min at room temperature twice. Cells were resuspended with 0.5 mL Cornoy’s fixative and the suspension was dropped on a lean slide to release chromosomes. These slides were stained with 1% Giemsa solution for 5 min and mounted with neutral balsam (Sangon Biotech). All metaphase spread plates were imaged at a magnification of 1000x using a fluorescence microscope (Leica DM4000). Different types of chromosomal aberrations such as chromatid breaks and radial chromosomes were scored and calculated as percentage of chromosomal aberrations.
For micronucleus preparation, cells were grown on glass coverslips in a 6-well plate and washed gently with cold PBS twice after 24 h post-transfection. Cells were fixed with 4% paraformaldehyde and stained with DAPI. All plates were imaged at a magnification of 1000x using a fluorescence microscope (Leica DM4000). Micronuclei were defined as discrete DNA aggregates juxtaposed to primary nuclei in cells and the frequency of micronucleated cells was calculated.