2.1 Materials Collagen I (C8062), D-Mannitol(M8140), Fibronectin (from human plasma, F8180), Rapamycin (R8140) and BSA(A8020) were obtained from Solarbio.RPMI-1640 (22400-089), FBS (10100147C), penicillin-streptomycin (15070063), 0.25%Trypsin-EDTA solution (R001100), glucose (15023021), 1%Triton X-100(85111) and Tween-20(28320) were purchased from Gibco. Rabbit polyclonal anti-integrin β1-MF (26918-1-AP), rabbit polyclonal anti-LC3(14600-1-AP), mouse monoclonal anti-p62/SQSTM1(66184-1-Ig), mouse monoclonal anti-phospho-mTOR(67778-1-Ig), β-actin monoclonal antibody(66009-1-Ig) were from Proteintech. Goat anti-mouse IgG (Dylight 488, E032210), goat anti-mouse IgG (Dylight 649, E032610), Goat anti-rabbit IgG (Dylight 488, E032220) were from EarthOX. Other laboratory consumables for cell culture and Genistein(G6649) were all from Sigma. Of note, all cell culture flasks and plates had been coated with type I collagen on the surface when the podocytes were inoculated.
2.2 Cell culture A conditionally immortalized human podocyte cell line was kindly provided by Prof. Moin Saleem (Bristol Royal Hospital for Children, Bristol, UK). Podocytes were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin and streptomycin, and 10 U/ml recombinant mouse interferon-γ at 33˚C with 5% CO2 and 95% relative humidity for proliferation. After 2 to 3 days, when the cells grew to approximately 70% -80% confluence, they were transferred to 37°C in RPMI 1640 medium to induce differentiation without interferon-γ for 10 to 14 days. Of note, the cell density should not exceed 80% - 90%, otherwise the cell activity will be affected. The differentiated podocytes were used between passages 7 and 10 for all subsequent experiments.
2.3 Cell treatment HG medium was made by supplementing RPMI 1640 medium (normal glucose concentration of 5.5 mmol/L; NG) with additional glucose for a final glucose concentration of 30.0 mmol/L. In addition, 19.5 mmol / L D-Mannitol was added to NG medium to maintain the hypertonic effect (HP) of HG concentration on cells. According to the solubility of Genistein, HG medium were treated with Genistein at various concentrations (2, 4, 6, and 8 µg/ml) to prepare the drug culture medium. Different mediums below were added to podocytes separately at 37˚C for cck-8 test to select optimum drug concentration: NG medium, HP medium, HG medium, HG and 2 µg/ml concentration Genistein medium, HG and 4 µg/ml concentration Genistein medium, HG and 6 µg/ml concentration Genistein medium, HG and 8 µg/ml concentration Genistein medium. According to the results of CCK-8, podocytes were divided into NG group (NG medium), HG group (HG medium) and TM group (HG and 4 µg/ml concentration Genistein medium) in subsequent experiments.
2.4 Podocyte viability assay Cell Counting Kit-8 (96992-100TESTS-F, sigma, USA) was used to detect podocyte viability following manufacturer’s instructions. Briefly, the differentiated podocytes were seeded into 96-well plates (1.0×10⁴ cells per well) and incubated at 37°C, 5% CO₂ overnight. We sequentially added 100µl of corresponding medium to each well, and then cultured for 6, 12, 24, 36, 48 and 72 h respectively. CCK -8 solution was added in the amount of 10µl per well, and then the cells were incubated for 2 h at 37°C and 5% CO₂. The absorbance values of each well were assessed in a microplate reader with 450 nm.
2.5 Immunofluorescence Podocytes were grown on glass coverslips in 24-well plates and subjected to various treatments, then were fixed with 4% paraformaldehyde at room temperature (RT) for 15 min. After washing with phosphate-buffered saline (PBS) for three times, the cells were permeabilized with socking buffer including 0.2% Triton X-100 for 20 min. Then cells were blocked with 5% BSA for 30 min at RT to block non‑specific binding. Podocytes were incubated with the primary antibodies overnight at 4°(anti-integrin β1-MF (1:300)、anti-LC3-II (1:200), anti-p62(1:400), anti-p-mTOR (1:300)). Washed with PBS three times, followed by above 1 h of incubation with secondary antibodies at RT. Finally, after washing three times with PBS, the cells were stained with DAPI for 5 min to visualize the nuclear and observed under Immunofluorescence microscope (Axioscope 5, Carl Zeiss, Germany).
2.6 Western blot analysis Differentiated podocytes, with various experimental conditions, were washed twice with cold PBS. The washed podocytes were drained and extracted membrane protein from them with Membrane Extraction Kit (ThermoFisher,89842). Subsequently, protein (30 µg/lane) was separated by 8% SDS‑PAGE and transferred onto a polyvinylidene fluoride membrane, which was blocked with 5% fat‑free milk in TBST (0.1% Tween-20) 1 h at room temperature and then incubated at 4˚C overnight with the following primary antibodies: anti-integrin β1 (1:1,000), anti-LC3-II (1:1,000), anti-p62(1:1,000), anti-p-mTOR (1:1,000), anti-mTOR (1:1,000) and anti-β-actin (1:1,000). Following primary incubation, the membranes were washed twice with PBS, then were incubated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG for 1 h at RT. Subsequently, the immunoblots were washed three times with TBST (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.05% Tween-20) and visualized in ECL Plus Western Blotting Detection Reagents (Beyotime Institute of Biotechnology). Results were quantified using Image J 1.52a software (Wayne Rasband national institutes of Health, USA) and with β-actin as the loading control.
2.7 Wound healing assay Cell migration was evaluated by wound healing test. Draw 5 lines on the bottom of the six well plate with a marker, and then inoculate an appropriate amount of differentiated and mature podocytes on the plate coated with type I collagen overnight. Scratched with 200 µl pipette gun head, and then cleaned with sterile PBS twice. Finally, added 2ml medium of different groups into each well and cultured in a 37°C incubator. Photographs were taken at the specified time 0, 6, 12, 24, 36, 48 and 72 h, and analyzed using ImageJ 1.52a software (Wayne Rasband national institutes of Health, USA) image processing program. The percentage of cell migration area is calculated as follows.
2.8 Cell adhesion assay The differentiated podocytes were inoculated in type I collagen-coated 96-well plates overnight. Podocyte of different groups were cultured at 37°C for 6, 12, 24, 36 and 48 h respectively. Subsequently, discarded the culture medium, cells sticking to the bottom were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The dye was then washed three times with PBS and the cellular stain was dissolved in 33% acetic acid. The absorbance was quantified with a spectrophotometer at an optical density of 620 nm. The experiment was repeated three times to compare the percentage of podocyte adhesion (normalized to 1).
2.9 Statistical analysis Data was repeated at least three times independently and appeared as mean ± SD. Statistical analysis were processed by utilizing SPSS 24.0 software. Histogram and line chart were analyzed by GraphPad Prism 8.0 (GraphPad Software, USA) using Student’s t test or One-way ANOVA analysis. p < 0.05 was regarded as statistically significant.