Inverted light microscope

Cell invasion was assessed using a Transwell assay and cell migration was measured using a scratch/wound healing assay. For the Transwell assay, cells (1×10⁵ cells/well) suspended in serum-free DMEM supplemented with 1 mg/ml mitomycin C (inhibitor of cell proliferation) were seeded onto the upper chamber of the Transwell inserts (24-well insert, pore size 8 mm). The filter membranes were pre-coated with Matrigel at 37°C for 30 min. DMEM containing 20% FBS was added to lower chamber. After 36 h of incubation, the cells migrating into lower surface of the inserts were fixed for 30 min, stained with 1% crystal violet at 37°C for 30 min, and photographed under an inverted light microscope (Olympus Corporation; scale bar, 100 µm). Invasion was measured by counting the number of stained cells.
For the scratch/wound healing assay, cells (1×10⁵ cells/well) were seeded in 12-well plates and cultured until 75% confluence. Then, a wound was created by manually scratching the cell monolayer with a 200-µl pipette tip. The cultures were washed to remove floating cells, and then the adherent cells were incubated in serum-free DMEM. Cell migration into the wound was observed under an inverted light microscope (Olympus Corporation; scale bar, 200 µm) at 0 and 24 h for each group. The scratch area was calculated using ImageJ software (v1.8.0; National Institutes of Health).

Ninety-six-well plate was coated by 50% Matrigel (50 μl/well, BD Biosciences) and maintained at 37°C for 30 mins for solidification. SK-Hep1, 5×10⁴, cells were seeded into the matrix and incubated at 37°C for 6 hrs. The tube structures were visualized under an inverted light microscope (magnification 100×; Olympus Optical Co., Ltd., Tokyo, Japan), and the tube length was calculated using Image J (Media Cybernetics).

The differentiation potential of induced pluripotent cancer (iPC) were investigated by induction of embryonic body (EB) formation through a 3-dimensional culture system. In brief, iPC colonies (at passage 5) on feeder cells were treated with collagenase (1 mg/mL; Invitrogen), transferred to 60-mm culture dishes, and incubated for 30 minutes to eliminate contamination with feeder cells. Suspended cells were then cultured with human embryonic stem media for 5 days to form spheroids (EBs) on no coated petri dishes (Greiner Bio-One, Monroe, NC, USA). The media was changed per day. The shape of EBs was analyzed by using an inverted light microscope (Olympus Optical, Melville, NY, USA).

The aforementioned 3-µm-thick kidney tissue sections were stained immunohistochemically with antibodies against TGF-β1 (1:1,000; cat. no. ab92486, Abcam), Smad3 (1:500; cat. no. ab40845; Abcam) and α-SMA (1:500; cat. no. bs-10196R; BIOSS). After deparaffin (xylene and gradient ethanol) and rehydration, slices were boiled for 15 min using microwave irradiation for antigen retrieval in citrate buffer (0.01 mol/l; pH 6.0). Slices were then washed three times with PBS and blocked with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. The slides were incubated with the aforementioned primary antibodies for 2 h at room temperature. Goat anti-rabbit IgG peroxidase conjugate (1:500; cat. no. bs-0295G, BIOSS) was used as the secondary antibody. Cells were incubated with secondary antibodies for 1 h and room temperature. 3,39-diaminobenzidine, nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (all, Sigma-Aldrich; Merck KGaA) were used as enzyme substrates. Finally, after dehydration with gradient ethanol and permeabilization with xylene, the slides were sealed and photographed. Visual analysis was performed using an Olympus inverted light microscope (magnification, ×400; cat. no. CX71; Olympus Corporation). The mean optical density (MOD) was calculated using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).