the resuscitated cells were gently shaken and melted in a water bath at 37℃ and transferred into a centrifuge tube containing 10ml dulbecco's modified eagle medium (DMEM); Centrifuge at 1000rpm /min for 5min, pour out the supernatant, add medium to the centrifuge tube and suspend it, then transfer it to the culture flask; The cells were proliferated to 80% density for passage.
Cell slides were dipped and washed with PBS; which was fixed with 4% paraformaldehyde for 15min at room temperature. Fixed cells were permeabilize with 0.5%TritonX-100 for 20min at room temperature; The Cell slides were blocked at room temperature for 30min, and incubated with primary antibody at 4℃ overnight, followed by anti-rabbit secondary antibody conjugated with Alexa Flour 488 at room temperature for 1 h. DAPI was used to stain the core and seal the film, and the images were observed and collected.
Quantitative real-time PCR (RT-PCR)
Preparation of cDNA, amplification of the target gene and preparation of RT-PCR reaction solution: 2× Master mix 10.0 (ul), Forward Primer 1.0 (ul), Reverse Primer 1.0 (ul), nuclease-free H2O 1.0 (ul), Total per Reaction 18.0 (ul); The PCR reaction solution was divided into eight PCR tubes and 2ul cDNA prepared was added. The difference of gene expression was detected by fluorescence quantitative PCR.
Western Blot assay
Cells were cultured to the density reached 80%, cells were collected and resuspended with an appropriate volume of RIPA lysate for 30 min on ice. Pyrolysis liquid centrifuged with 13000rpm at 4°C for 20min, the supernatant was determined by BCA protein assay kit and diluted to the same concentration. The different treated samples with 30ug total protein content were separated on SDS-PAGE and transferred to PVDF membrane. Protein expressions were blocked with 5% skim milk for 1h at room temperature. Primary antibody, diluted in the blocking solution, was incubate overnight at 4°C; and the secondary antibody was for 1h at room temperature. Finally, the membranes were visualized using Pierce® ECL Western Blotting. Primary antibody: STAT3
(Abcam, ab32500), p-STAT3(Abcam, ab76315), FN(promab, 30506), E-cad(Ptgcn, 20874-1-AP), SENP3(Ptgcn,. 17659-1-AP), Sumo 2(Abcam, ab233222), PYCR1(Ptgcn,. 66510-1-Ig), β-actin(Ptgcn, 66009-1-Ig).
Cells were treated with drugs (or not) for a certain time. Add the serum culture medium containing 20% FBS into wells (600ul/well). Put trans well chambers in 24-well plates. Digest cells and resuspend cells with serum-free culture medium. Cell concentration should be determined according to specific conditions. Add cell suspension into trans well chambers (400ul/chamber). Put it in an incubator for a certain period of time. Determine the time by checking the literature. Take trans well chambers out and wipe cells that have not passed through the inside of the microporous filter with a cotton swab. Fix cells methanol with for 5 minutes. Dye cells with purple crystal and clean trans well chambers with pure water. Under the microscope, take five fields (top,bottom༌left༌right༌middle) and take pictures.
Tissue samples were deparaffinized at 60 ℃ for half an hour,then deparaffinized and rehydrated. Antigen retrieval were performed in antigen Repair Solution along the microwave program. Tissue were permeabilized with 0.5% Triton X-100 in PBS for 20min at room temperature, then shaking for 30 minutes with 1% H2O2. Blocking buffer (0.5%BSA + 5% goat serum) was used to block for 30min at room temperature. Primary Ab, diluted in blocking buffer (1:100 or 1: 200), was incubated for overnight at 4℃. Secondary Ab (Biotin-labeled secondary antibody, 1:200, diluted in blocking buffer) was incubated for 30min at room temperature. ABC dilution (1mlPBS + 10ulA + 10ulB) were configured half an hour. DAB Chromogenic solution were used for visualization.
Extraction of plasma/nucleoprotein
NucBuster TM Protein Exaction Kit (merckmillipore, 71183-3) was used to extract plasma/nucleoprotein as the flowing method. The collected cells were added with liquid nitrogen and quickly ground into a powder. Appropriate amount of powder were diluted with 150ul NucBuster Reagent 1, then vortexed with high speed for 15S. The samples were placed on ice for 5min and centrifuged with 16000g at 4°C for 20min.The supernatant was served as cytoplasmic protein, and floccule was added 1ul 100×Protease Inhibitor Coctail, 1ul 100mM DTT and 75ul NucBuster Exaction Reagent. The mixture was vortexed with high speed for 15S and placed on ice for 5min. Then the mixture centrifuged with 16000g at 4°C for 20min. Cytoplasmic protein and nucleoprotein can be used immediately or stored in separate packages at -80℃.
Seed 5×104 cells on 6-well plates to achieve 70–90% cell convergence within 24 hours. Prepare the overexpressed plasmid-Lipofectamine™ 3000 complex as follows: Dilute 7.5ul Lipofectamine™ 3000 with 125ul Opti-Mem ® I and mix gently; Dilute 5ul plasmid with 125ul Opti-Mem ® I, then add 5ul P3000TM reagent, and mix gently; Mix the diluted plasmid with the diluted Lipofectamine™ 3000, mix gently and incubate at room temperature for 5 min to allow the formation of the complex. Add plasmid-LipofectaminetM3000 complex to each well containing the cells and medium. Incubate for 3–5 hours for downstream experiments.
assay was applied to determine the effects of test reagents on cell growth. Briefly, cells were
seeded in a 96-well plate with a density of 1 × 105 cells per well. After 24 h, cells were treated with agents for indicated time. Then, 10 µL of MTT solution was added to each well and incubated for 1 h. 96-well were shake on the shaker for one minute. Finally, the absorbance was read at 550 nm using a microplate reader (Thermo Fisher Scientific, Franklin, MA, USA). Cell growth inhibitory ratio (%) was calculated as following formula: Cell proliferation rate % = (OD value of test well - OD value of background control well)/(OD value of control cell - OD value of background control) × 100%.
The cells were fixed in 1% formaldehyde and incubated at room temperature for 10 minutes. 1 mL of 10X Glycine was added to each dish to quench unreacted formaldehyde and incubate at room temperature for 5 minutes. These dishes were placed on ice. Medium was removed as much as possible. Each dish was added 2 mL cold PBS containing Protease Inhibitor Cocktail. The cells were collected with 800g at 4℃ for 5 minutes. During spin, each sample was combined with 0.5 mL of cell Lysis Buffer with 2.5 µL of Protease Inhibitor Cocktail II.
Tumor formation assay in nude mice
Male BALB/c nude mice (6-week‐old) were purchased from model animal research center of nanjing university and maintained in pathogen‐free conditions. The mice were injected subcutaneously into the right flanks with 1 × 106 cells/mL (0.1 mL) of T24 cells, which were stably transfected with pLentiCon/NC, plentiSENP3/OE, plentiSENP3 + si-STAT3/OE + si-STAT3 plasmid and siRNA. Tumor growth was examined every 7 days, and tumor volumes were calculated using the equation, V = 0.52 × length × width2. The tumor growth curve was plotted with time as the abscissa and tumor volume as the ordinate. Finally, each tumor was examined by immunohistochemical analysis. All animal care and experiments were conducted in accordance with national and institutional policies for animal health and well‐being. The protocol was approved by the Institutional Animal Care and Use Committee.
Sample size was determined upon availability, and chosen based on previous experience and previous studies. No exclusion criteria were pre-established. Samples or animals were randomly allocated among groups. Investigators were not blinded to group allocation during data collection and analyzes. No data were excluded. All statistical tests were performed with SigmaPlot 14.0. Data shown were presented by means ± Standard Deviation (S.D.) from triplicate experiments performed in a parallel manner unless other wise indicated. Statistical significance was performed using an unpaired two-tailed Student’s t test for two data sets when the data met the normal distribution tested by F-test. The variance between comparison groups was not similar. Detailed descriptions of all methods are provided in Supplementary Materials and Methods.