Upright light microscope

Tumor tissues were dissected, paraffin-embedded and sectioned (4 µm thickness). Sections were stained with hematoxylin and eosin (H&E), and immunohistochemistry was performed on formalin-fixed tumor tissue sections. To expose target proteins, heat-induced epitope retrieval (HIER) was performed in sodium citrate buffer (pH 6.0) for 20 minutes at 100°C. Tissues were blocked in 10% BSA for 20 minutes at room temperature, and anti-Ly6G antibody (RB6-8C5, Cat#: ab25377, Abcam) was applied to sections and incubated overnight at 4°C. After washing, colorimetric reactions were performed using two-step histostaining reagent kits (Beijing Zhong Shan Qiao Biotechnology Co., Ltd) according to the manufacturer’s protocol. The immunoreaction was visualized when brown precipitates formed following incubation in diaminobenzidine. Sections were subsequently washed with water and counterstained with 0.5% hematoxylin for 3 minutes at room temperature. Finally, tissues were visualized by an upright light microscope (Olympus Corporation, Tokyo, Japan).

CD11b⁺GR1^hi cells were sorted from Renca tumors that had received vehicle or cabozantinib treatment using a BD FACSAria™ III Cell Sorter. Next, cell concentrations were adjusted to 5 × 10⁶ cell/ml in 200 μl PBS, and cell smears were prepared using a cell smear centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd), spinning for 5 minutes at 2000 rpm. Then, the cell smear was placed on a dyeing rack according to the manufacturer’s protocol (Cat#: KAG227, Wright-Giemsa Assay Reagent), 3-5 drops of reagent 1 were added, and cells were stained for approximately 1 minute to fix the cell smear. Reagent 1 was not discarded, 6-10 drops of reagent 2 were directly added, the slide was gently shaken to mix the dye solution thoroughly and washed after 5-8 minutes, and then, cell morphology was assessed using an upright light microscope (Olympus Corporation, Tokyo, Japan).

The effect of miR-429 and CRKL deregulations on the migration and invasion abilities of HepG2 cells were examined using 24-well-plate transwell chamber assay. Briefly, 1 × 10⁴ cells in 200 μl serum-free RPMI-1640 were seeded onto the upper compartment of transwell with 8 μm pore size polycarbonate filters (Corning, USA). The chambers were then placed into 24-well plates containing 600 μl RPMI-1640 with 20% FBS and incubated for 48 h at 37 °C with 5% CO₂. For invasion assay, the inserts were coated with 50 μl extracellular matrix gel (ECM, Sigma, USA) which 1:8 dilution with RPMI-1640, and incubated at 37 °C for 1 h. 1 × 10⁴ cells in 200 μl serum-free RPMI-1640 were seeded onto the upper compartment of transwell. The chambers were then placed into 24-well plates containing 600 μl RPMI-1640 with 20% FBS and incubated for 48 h at 37 °C with 5% CO₂. The non-migrated and non-invaded cells on the upper surface of the insert were removed by swabbing, the migrated and invaded cells onto the lower surface were fixed with methanol for 30 min, stained with 0.5% crystal violet for 1 h, washed with PBS, counted using an upright light microscope (Olympus, Japan) with 100x. Five field views were randomly counted and averaged.

The influences of miR-429 and CRKL level changes on HepG2 proliferation was determined by MTT assay. The cells from each group were seeded into a 96-well plate at 3 × 10³ cells/well in 100 μl RPMI-1640 supplemented with 15% FBS, incubated at 37 °C, 5% CO₂ for 24, 48, 72 and 96 h separately, incubated in MTT solution (5 mg/ml) by replacing culture medium at 37 °C, 5% CO₂ for 4 h in darkness. The supernatants were then removed and 150 μl DMSO was added to dissolve for formazan crystals. The absorbance at 492 nm were measured using a microplate reader (Thermo, USA) for cell density quantification. Results were obtained from triplicate measurements.
The deregulation effects of miR-429 and CRKL on HepG2 colony forming ability was performed by colony formation assay. The dissociated 1000 cells in 2 ml RPMI-1640 supplemented with 15% FBS were seeded in 6-well plate and then kept at 37 °C with 5% CO₂ for 10 d until visible cell colonies appeared. The colonies were washed with PBS, fixed with methanol for 30 min and stained with 0.05% crystal violet for 1 h at RT. The colonies were observed and larger than 50 cells were counted using an upright light microscope (Olympus, Japan) with magnification of 100x. Triplicate experiments were performed for each assay.

Both carotid artery and heart samples were fixed in 4% paraformaldehyde for 24 hrs at 4°C and washed in PBS before being incubated overnight in 30% sucrose at 4°C and then embedded in O.C.T. compound and frozen at -20°C. Serial cross sections with a 7 μm thickness were stained with hematoxylin-eosin (H&E) and ORO.
Corresponding sections were incubated with the following primary antibodies: anti-Galectin 3 (diluted 1:200), anti-SIRT1 (diluted 1:100), and anti-LC3 (diluted 1:100) from Abcam and Cell Signaling Technology Inc. Appropriate IgG conjugated with peroxidase was used as the secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). In negative controls, incubation with the above antibodies was omitted. These sections were examined with an upright light microscope (Olympus, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).