Table1: reagent of WB (July13th-July16th)

July 13th

Cell Treatment:

1. Cell Seeding: Cells in the logarithmic growth phase and in good condition were seeded into 6-well plates at a density of 2×10^5 cells per well.
2. Culture: Cells were cultured at 37°C for 24 hours. Afterward, the medium was replaced, and different plasmids were transfected into the cells.
3. Transfection: After 24 hours of transfection, the culture supernatant was removed. Cells were briefly treated with 0.25% trypsin at 37°C for about 2 minutes, and the digestion was stopped by adding an appropriate amount of culture medium. Cells were collected by centrifugation at 900rpm for 4 minutes and the cell pellet was used for Western Blot (WB) analysis.

July 14th

Protein Extraction:

1. Washing: Cells collected by centrifugation were washed with 1mL of physiological saline and transferred to 1.5mL centrifuge tubes.
2. Lysis: To each tube, add 10μL PMSF (100mM) to 1mL of lysis buffer, and gently shake on ice (PMSF should be shaken until it's crystal-free before mixing with the lysis buffer).
3. Cell Lysis: Add 100-500μL of lysis buffer containing PMSF based on the amount of cells. Incubate on ice for 10 minutes.
4. Centrifugation: Centrifuge at 12,000rpm for 30 minutes at 4°C (pre-cool the centrifuge).
5. Transfer Supernatant: Transfer the supernatant to 1.5mL centrifuge tubes for protein quantification. If protein quantification cannot be performed promptly, store at -80°C.

July 15th

Protein Quantification and Processing (BCA Method):

1. BCA Working Solution: Shake Solution A in the BCA assay kit and, depending on the number of samples, mix 50 volumes of Solution A with 1 volume of Solution B (50:1) to prepare the BCA working solution. Mix thoroughly to form a light green working solution. The BCA working solution is stable at room temperature for up to 24 hours.
2. Standard Curve: Add 0, 1, 2, 4, 6, 8, and 10μL of 1mg/mL BSA standard into a 96-well plate and make up to 10μL with deionized water for each standard.
3. Sample Preparation: Add 1μL of each sample to the 96-well plate and make up to 10μL with deionized water.
4. BCA Reaction: Add 200μL of BCA working solution to each well, gently mix by pipetting (avoid introducing bubbles), and incubate at 37°C for 30 minutes.
5. Absorbance Measurement: After cooling to room temperature, measure the absorbance at 562nm using a microplate reader.
6. Calculate Protein Concentration: Calculate the protein concentration in the samples using the standard curve. If necessary, perform protein denaturation: mix the protein solution with 5× loading buffer at a volume ratio of 4:1. Boil for 10 minutes in boiling water, then cool for SDS-PAGE electrophoresis or store at -80°C to avoid repeated freeze-thaw cycles.

July 16th

SDS-PAGE Electrophoresis:

1. Gel Preparation: Prepare a 12% separating gel and a 5% stacking gel according to the target protein molecular weight.
2. Sample Loading: Calculate the volume of the solution containing 50μg of protein, which is the sample volume. Add 1× loading buffer to each sample well to make the total volume consistent.
3. Electrophoresis: Run electrophoresis at 80V for 30 minutes for the stacking gel and then at 120V for 30-50 minutes for the separating gel. Stop electrophoresis when the Bromophenol Blue reaches the bottom of the gel. Proceed to membrane transfer.
4. Membrane Transfer: Transfer at a constant voltage of 100V for 60 minutes to a 0.45μm PVDF membrane.
5. Blocking: Immerse the PVDF membrane completely in 5% BSA-PBST and shake gently at room temperature for 60 minutes.
6. Primary Antibody Incubation: Dilute the primary antibody in 5% BSA-PBST and incubate at 4°C overnight.
7. Membrane Washing: Remove the PVDF membrane and wash with PBST for 5 times, each for 6 minutes.
8. Secondary Antibody Incubation: Dilute the secondary antibody in PBST and incubate at room temperature for 60 minutes.
9. Membrane Washing: Wash the PVDF membrane with PBST for 5 times, each for 6 minutes.
10. Visualization: Mix ECL A and B solutions in a 1:1 volume ratio and evenly add to the membrane. Expose as needed, save the images, and export.

Table2: reagent of MTT (July28th-July31st)

July 28th

Cell Resuscitation: Pre-warmed ddH2O to 37℃.

Cell Resuscitation: Wore gloves, masks, and caps. Removed LN-229 cell vials from the liquid nitrogen tank (containing 1 mL cell mixture) and immediately placed them in 37℃ ddH2O, gently shaking the vials for rapid thawing.

Disinfecting Vials: After complete thawing, wiped the outer surface of the vials with 75% alcohol for disinfection.

Cell Centrifugation: In a sterile hood, added 10 mL of freshly prepared culture medium to a 15 mL sterile centrifuge tube containing thawed cells. Centrifuged at room temperature (1000 rpm) for 3 minutes to remove the supernatant.

Cell Resuspension: Added 1 mL of culture medium to resuspend the cell pellet, gently mixed, and transferred it to a T25 cell culture flask. Topped up with medium to reach 5 mL and placed in a 37℃, 5% CO2 incubator.

Cultivation (12 h): Checked cell culture at 12 hours to monitor progress.

July 29th

Cultivation (24 h): Checked cell culture at 24 hours to monitor progress.

July 30th

Cultivation (48 h): Checked cell culture at 48 hours to monitor progress.

July 31st

Cultivation (72 h): Checked cell culture at 72 hours to monitor progress.

Table3: reagent of cell clone (August 1st-August 19th)

August 1st

Cell Culture: TJ861 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2.

August 2nd

Cell Seeding and Transfection Preparation: TJ861 cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and seeded into 6-well plates at a density of 2×10^5 cells per well. Cells were allowed to attach for 18-24 hours, not exceeding 24 hours to avoid affecting transfection efficiency. Transfection was performed when cells reached 65-75% confluence. In a typical 6-well plate:

1. Added 2 μl lipofectamine 2000 to 100 μl serum-free, antibiotic-free basic culture medium, forming pre-mix 1, and left it at room temperature for 5 minutes.
2. Mixed 2 μg of plasmid with 100 μl of serum-free, antibiotic-free basic culture medium, forming pre-mix 2.
3. Combined the plasmid and lipofectamine 2000 mixture (pre-mix 1 and pre-mix 2), left it at room temperature for 20 minutes.
4. Replaced the complete culture medium in the 6-well plate with serum-free, antibiotic-free basic culture medium.
5. Slowly added the transfection mixture to the 6-well plate, gently rocked it back and forth for even distribution.
6. Cultured at 37°C with 5% CO2 for 4-6 hours and then replaced the medium with complete culture medium.

August 3rd

Plate Cloning: After transfection, TJ861 cells were digested with 0.25% trypsin, counted, and seeded into 6-well plates at a density of 200 cells per well. The culture medium used was DMEM with 10% FBS. The next day, the medium was changed, and cells were regularly observed.

August 4th

Observe: The culture medium was replaced with fresh DMEM medium containing 10% FBS, and the cells were systematically examined under a microscope.

August 18th

The culture was terminated, and the culture medium was removed. The cells were fixed with 4% paraformaldehyde for 15 minutes. After removing the fixation solution, an appropriate amount of crystal violet staining solution was added, and staining was carried out for 10-30 minutes. Then, the staining solution was slowly washed away with running water, air-dried, and photographed for counting.

August 19th

Cell Transfection:

1. Cell Seeding: Cells in logarithmic growth phase were digested with trypsin to obtain single-cell suspensions and were seeded into 6-well plates (1-5×10^5 cells per well) according to experimental requirements.
2. Culture: Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours, allowing them to reach 65%-75% confluence for transfection.
3. Plasmid and Lipofectamine Preparation: For 6-well plates, 125μL of serum-free medium was used to incubate 2 μg of plasmid; 125 μL of serum-free medium was used to incubate 2μL of Lipofectamine 2000, left at room temperature for 5 minutes (note: do not vortex or shake Lipofectamine). The plasmid and Lipofectamine mixture were combined and left at room temperature for 20 minutes.
4. Washing: The plates were washed twice with serum-free medium, and each well was supplemented with 800μL of serum-free medium.
5. Add Mixture: The mixture was added to the cell culture plates, and after 4-6 hours, the medium was replaced with serum-containing complete medium for continued culture for 24-48 hours.

Table4: reagent of Cell Scratch Assay (August 20th-August 21th)

August 20th

Cell Scratch Assay:

1. Cell Seeding: Cells in logarithmic growth phase were digested with trypsin to obtain single-cell suspensions and seeded into 6-well culture plates. Cells were seeded at a density of 6×10^5 cells per well to ensure confluence by the next day, with a final volume of 2mL per well.
2. Cell Culture: Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours.
3. Scratch: The next day, a scratch was made using a straightedge ruler. Care was taken to make the scratch as vertical as possible, and the ruler was kept perpendicular to the plate surface (the same ruler was preferred for different wells).
4. Washing: Cells were washed with PBS three times to remove detached cells, and serum-free culture medium was added.
5. Photography: Pictures were taken under 4x magnification, ensuring the scratch was centered and vertical. Samples were taken at 0, 6, 12, and 24-hour time points, and photos were taken (specific times depended on experimental requirements).
6. Data Analysis: Using AI software, the width of scratches at different time points and in different groups was measured. Migration rate was calculated as: Migration Rate = (Width at 0h - Width at 24h) / Width at 0h * 100%.

August 21th

Data Analysis:

1. Continued data analysis using AI software to determine migration rates and gather experimental results.

Table5: reagent of transwell (August 22th-September 3rd)

August 22th

Lab Preparation: Pre-warmed the lab to 37℃ to ensure the desired temperature.

Cell Resuscitation: Wearing lab gloves, masks, and caps, removed LN-229 cell vials from the liquid nitrogen tank and immediately placed them in pre-warmed 37℃ ddH2O, gently shaking the vials to facilitate rapid thawing.

Disinfecting Vials: After complete thawing, wiped the outer surface of the vials with 75% alcohol to ensure disinfection.

Cell Centrifugation: In a sterile hood, added fresh culture medium to the centrifuge tube containing thawed cells, centrifuged for 3 minutes at room temperature to remove the supernatant.

Cell Resuspension: Added 1 mL of culture medium to resuspend the cell pellet, gently mixed, and transferred it to a T25 cell culture flask, topped up with enough medium, and placed in a 37℃ CO2 incubator.

Cultivation: Waited for 48 hours before proceeding to the next step.

August 23th

Cell Culture: Continued the culture of LN-229 cells in a 37℃, 5% CO2 incubator.

August 24th

Cell Culture: Continued monitoring cell growth in the incubator to ensure normal growth.

August 25th

Cell Culture: Continued the culture of LN-229 cells, monitoring their growth status.

August 26th

Cell Culture: Continued observing cell growth, ensuring that the cell confluence reaches 50%~60%.

August 27th

Cell Transfection: Prepared for cell transfection by digesting cells into a single-cell suspension and seeding them into 6cm dishes, culturing overnight.

August 28th

Cell Transfection: After the cell confluence reached 50%~60%, prepared plasmids and liposomes.

August 29th

Cell Transfection: Performed cell transfection, ensuring cells were cultured for 48 hours in medium containing plasmids and liposomes.

August 30th

Cell Invasion Assay: Prepared Matrigel and allowed it to solidify at 37℃ in the incubator before conducting the invasion assay.

August 31th

Cell Invasion Assay: Conducted the Transwell invasion assay, incubating for 48 hours.

September 1st

Cell Invasion Assay: Continued with the Transwell invasion assay.

September 2nd

Cell Invasion Assay: Concluded the Transwell invasion assay, fixed and stained the cells.

September 3rd

Data Collection: Captured photos using a microscope and counted the number of cells that passed through the Matrigel.