Fluoroshieldtm with dapi

Fluorescent staining on the lipid droplet in the ovarian cancer cells were performed using Nile Red (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s protocol^{59 (link)}. Cells were seeded in 6-well plate in which a sterile glass coverslip had been placed in advance. After 24 h starvation, cells were treated with OCM or DMEM with 1% FBS for 24 h in the same condition as cell culture. Four percent paraformaldehyde (PFA) was used to fix the cell, and followed by the incubation of Nile Red at a concentration of 1 μg/10 mL 150 mM NaCl for 30 min. Then the coverslip was placed on a microslide with a drop of Fluoroshield^TM with DAPI (Sigma-Aldrich, St. Louis, MO, USA) to stain cell nucleus. Inner lipid droplet of ovarian cancer cells was stained into red color while nucleus was stained into blue. Cells were captured by Nikon eclipse Ti-S (Nikon, Tokyo, Japan) fluorescent microscope or Zeiss LSM 780 confocal microscope (Carl Zeiss, Germany). To quantify the fluorescent signal of the lipid droplets, three cells per visual field were randomly selected. Measurements of fluorescence intensity were performed by Carl Zeiss ZEN 2.3 Version 13.0.0.0, with normalization of cytosolic intensity. Mean signal intensities were obtained from at least five visual fields.

For LSCM analysis, cells adhered on coverslips from T0 and T4 of both conditions were fixed with 4% formaldehyde in seawater for 1 hour and incubated with 10 μg/ml Nile Red (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 minutes, then washed twice in PBS (pH7.4). The coverslips were then mounted on slides with Fluoroshield^TM with DAPI (Sigma-Aldrich) and taken to a TCS SPE microscope (Leica Microsystems, Wetzlar, Germany). The excitation wavelengths were 405 nm and 488 nm and fluorescence emission peaks were: 440 nm (DAPI), 535 nm (Nile Red) and 650 nm (Chlorophyll a). Images acquisition resulting resolution was 2,048 x 2,048 and, to improve the image quality, they were processed by 3D deconvolution with LAS AF software (Leica Microsystems Company).

The doxorubicin hydrochloride was purchased from BLD Pharmatech (#BD32885, Shanghai, China). The l-α-phosphatidylcholine (#441601, Soy PC), Avanti^® Mini Extruder (#610000), and 1-oleoyl-2-[12[7-nitro-2-1,3-benzoxadiazol-4-yl]amino]-sn-glycero-3-phophocholine (#810133C, NBD-PC) were obtained from Avanti Polar Lipid Inc. (Alabaster, AL, USA). Dulbecco’s modified Eagle’s medium (#10-013-CV, DMEM), fetal bovine serum (#35-015-CV, FBS), and penicillin–streptomycin (#30-002-Cl) were purchased from Corning (Corning, VA, USA). Dulbecco’s Phosphate Buffered Saline (#14200075, DPBS 10X) was purchased from Gibco (Waltham, MA, USA), while the Cell Counting Kit-8 (CCK-8), a colorimetric assay for measuring water-soluble tetrazolium salt, was acquired from DoGenBio (#EZ-3000, EZ-Cytox, Seoul, Republic of Korea). The bicinchoninic acid assay kits (#23225, Pierce™ BCA Protein Assay Kits), LysoTracker^TM Deep Red (#L12492), and 4′,6-diamidino-2-phenylindole (#F6057, DAPI) were purchased from Thermo Scientific Inc. (Waltham, MA, USA), Invitrogen (Waltham, MA, USA), and Sigma-Aldrich (Fluoroshield^TM with DAPI St. Louis, MO, USA), respectively. Ultrapure water was obtained from Welgene Inc. (#ML109-02, Gyeongsan, Republic of Korea). All the other reagents and chemicals used were of analytical grade.

Immunofluorescence staining was performed to visualize possible translocal alteration of NF-κB proteins and β-catenin by dexamethasone in cells. The PFA-fixed cells were permeabilized with 0.1% Triton^TM X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for 45 min at ambient temperature. Afterwards, the cells were labeled with primary NF-κB p65 rabbit polyclonal antibody #PA1-186 (Invitrogen, Carlsbad, CA, USA) at 1 µg/mL or β-catenin mouse monoclonal antibody #MA1-300 (Invitrogen) at a dilution of 1:200 in 0.1% BSA and incubated overnight at 4 °C in a moist chamber. The next day, cells were washed three times with PBS before incubation with the secondary Alexa Fluor 488 (AF488)-conjugated anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) or anti-mouse antibody (Invitrogen) at a dilution of 1:1000 for 1 h at ambient temperature. Cells were washed again three times with PBS and mounted with Fluoroshield^TM with DAPI (4’,6-diamidino-2-phenylindole) (Sigma-Aldrich). The slides were subsequently investigated with a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss) [35 (link)].

Deparaffinized liver sections (5 µm) were subjected to heat-induced epitope retrieval (10 mM sodium citrate, 0.1% Tween 20, pH 6.0) in a microwave for 30 min. After treatment with 5% BSA and 0.1% Tween 20 on Tris-buffered saline (TBS) to reduce non-specific reactions, the liver sections were incubated with anti-proliferating cell nuclear antigen antibody (anti-PCNA, 1:50, sc-25280, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Following washing with TBS, the sections were incubated with anti-mouse antibody conjugated to FITC (1:100, F-0257, Sigma-Aldrich, St. Louis, MO, USA) for 2 h, and slides were mounted by addition of Fluoroshield^TM with DAPI (Sigma-Aldrich, St. Louis, MO, USA). In the negative control, the primary antibody was omitted. The hepatocytes with fluorescence-marked nuclei were considered positive. Results are expressed as the mean of the percentage of marked hepatocytes in relation to the total number of hepatocytes per field. The images were obtained using the AxioImager A2 microscope (Zeiss, Oberkochen, Germany) with 200× magnification. For this analysis, 10 non-overlapping microscopic fields containing portal tracts or centrilobular veins were counted per specimen from at least 5 independent animals per group.

To determine mAb-1E7 binding to PCV2-Cap in the PCV2-infected PK‐15 cells, PK‐15 cells were infected with 100 TCID₅₀ of PCV2b SH strain. Then, the infected PK‐15 cells were fixed with 4% Paraformaldehyde (Sigma-Aldrich) for 15 min at 37°C and permeated with 0.25% Triton X-100 (Sigma-Aldrich) for 5 min. After washing three times with PBS, the fixed cells were blocked by 1% BSA for 30 min at room temperature. After being washed again, the mAb-1E7 (1 µg/mL) was added and incubated for 1 h at room temperature. Then, Alexa flour 488 conjugated-goat anti-mouse IgG (H + L) as a secondary antibody was incubated for 1 h at room temperature. Finally, cells were stained with FluoroshieldTM with DAPI (Sigma-Aldrich) and observed with a Leica SP8 confocal system (Leica, Wetzlar, Germany). All the images were captured and processed using Leica Application Suite X (Version 1.0. Leica Microsystems).

BMMs were cultured on 12 mm circle cover slips (Thermo Fisher Scientific) in 24-well plates with the above described cell density and stimulation protocol. At day 4 the cells were fixed and stained with Acti-stain 670 phalloidin (Cytoskeleton, Inc.), according to the manufacturer's instructions, to visualize the F-actin ring formation. After washing with PBS, cover slips were mounted with Fluoroshield^TM with DAPI (Sigma), and transferred upside down on object slides.