iGEM 2023 — Team bibs-beijing-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# Log in July

Table1: reagent of WB (July13th-July16th)

| Product Name | Brand | Catalog Number |
| --- | --- | --- |
| DMEM Medium | Gibco | C11875500BT |
| Fetal Bovine Serum | Gibco | 42F7180K |
| Trypsin | Genview | 89040101100 |
| Antibiotics | Genview | 87020101100 |
| 24-Well Cell Culture Plate | Corning | 3524 |
| Transwell Chamber | Corning | 3422 |
| Crystal Violet | Bi Yun Tian | C0121 |
| 4% Paraformaldehyde | Bi Yun Tian | P0099-100mL |
| Matrigel | Corning | 356234 |

### 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)

| Reagent Name | Brand | Catalog Number |
| --- | --- | --- |
| DMEM Medium | Hyclone | SH30022.01 |
| Fetal Bovine Serum | Gibco | 42F7180K |
| Trypsin | Genview | 89040101100 |
| Antibiotics | Genview | 87020101100 |
| MTT | Shanghai Bio工 | A600799-0005 |
| DMSO | Sigma | RNBG8041 |

### 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.

# August Log

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

| Product Name | Brand | Catalog Number |
| --- | --- | --- |
| DMEM Culture Medium | Gibco | C11875500BT |
| Fetal Bovine Serum | Gibco | 42F7180K |
| Trypsin | Genview | 89040101100 |
| Antibiotics | Genview | 87020101100 |
| 4% Formaldehyde Solution | Bi Yuntian | P0099-100mL |
| Crystal Violet | Bi Yuntian | C0121 |

### 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)

| Product Name | Brand | Catalog Number |
| --- | --- | --- |
| DMEM Medium | Gibco | C11875500BT |
| Fetal Bovine Serum | Gibco | 42F7180K |
| Trypsin | Genview | 89040101100 |
| Antibiotics | Genview | 87020101100 |
| Crystal Violet | Biyuntian | C0121 |

### 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)

| Product Name | Brand | Catalog Number |
| --- | --- | --- |
| DMEM Medium | Gibco | C11875500BT |
| Fetal Bovine Serum | Gibco | 42F7180K |
| Trypsin | Genview | 89040101100 |
| Antibiotics | Genview | 87020101100 |
| 24-Well Cell Culture Plate | Coring | 3524 |
| Transwell Inserts | Coring | 3422 |
| Crystal Violet | Biyuntian | C0121 |
| 4% Paraformaldehyde | Biyuntian | P0099-100mL |
| Extracellular Matrix Gel | Corning | 356234 |

### 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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# Engineering of our new part

## Research and Brainstorm

In the initial phases of our project, we embarked on a journey to design an effective EIF6 siRNA. Our objective was to develop a sequence that could efficiently target and silence the EIF6 gene, a critical step in our efforts to enhance the drug sensitivity of apatinib against glioblastoma. To achieve this, we began with a comprehensive exploration of available tools and methodologies.

## Primitive Design

Our first step was to employ the DSIR website, a reliable resource for siRNA design.
This online platform utilizes an algorithm based on the work of Vert et al. (2006) to generate siRNA sequences.
We totally synthesized three siRNA (si-EFI6#1, si-EFI6#2, si-EFI6#3) and finally chose one sequence with the highest transfection efficiency. The sequences were as follows: si-EFI6#1: sense GCUACUGUGUCUUCAGCAAUC; anti-sense UUGCUGAAGACACAGUAGCUU; si-EFI6#2: sense GAGCUUCGUUCGAGAACAACU; anti-sense UUGUUCUCGAACGAAGCUCGG; si-EFI6#3: sense GGACAGGGAGACAGAAGAAAU; anti-sense UUCUUCUGUCUCCCUGUCCAA.

![](https://static.igem.wiki/teams/4717/wiki/engineering/engineering1.jpg)

Figure 1.Technical Route of Designing SiRNA

## Debugging through Discussion and Consultation

Recognizing the complexity and significance of siRNA design, we acknowledged the value of collaboration and consultation. We reached out to experts in the field of siRNA delivery, including Song Furan and medical editor Xu Zhongliang. They provided us with invaluable insights and guidance on siRNA-related knowledge. Based on their recommendations, we opted to select siRNA sequences that have demonstrated higher transfection efficiency in previous studies. These collaborative discussions served as a crucial debugging phase, allowing us to critically evaluate and refine our choice of siRNA sequences.

## Transfection efficiency comparison

The LN-229 glioblastoma cell line, obtained from ATCC (Manassas, VA, USA), was maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% Gibco® fetal bovine serum and 100 μg/mL penicillin-streptomycin (Thermo Fisher Scientific) at 37°C in a 5% CO2 environment. Cell transfection procedures were conducted utilizing Lipofectamine™ 3000 Transfection Reagent (Invitrogen™, CA, USA), strictly following the manufacturer's guidelines.
Upon comparing the outcomes of transfection with three distinct siRNAs, it was evident that siRNA #1 exhibited the highest transfection efficiency, as depicted in Figure 1.a.

## Learn and Rethink

To assess the impact of siRNA on the response to Apatinib, we need to examine the protein expression of the EIF6 gene.
Additionally, to evaluate cellular functionality, we must investigate the expression of three genes: GLUT1, HK1, and LDHA.
We designed three primers for each gene and selected one with the best specificity and efficiency. The designed primary was then synthesized by solid-phase oligonucleotide synthesis method using an automated DNA synthesizer.
From these, we will select the primer that exhibits the highest PCR efficiency for further analysis.

## Final Design

![](https://static.igem.wiki/teams/4717/wiki/engineering/engineering2.jpg)

Figure 2. Primer part of EIF6

![](https://static.igem.wiki/teams/4717/wiki/engineering/engineering3.jpg)

Figure 3. Primer part of GLUT1

![](https://static.igem.wiki/teams/4717/wiki/engineering/engineering4.jpg)

Figure 4. Primer part of LDHA

![](https://static.igem.wiki/teams/4717/wiki/engineering/engineering5.jpg)

Figure 5. Primer part of HK1

# Reference

1. Matveeva, O., Nechipurenko, Y., Rossi, L., Moore, B., Sætrom, P., Ogurtsov, A. Y., ... & Shabalina, S. A. (2007). Comparison of approaches for rational siRNA design leading to a new efficient and transparent method. Nucleic acids research, 35(8), e63.
2. Vert JP, Foveau N, Lajaunie C, Vandenbrouck Y. An accurate and interpretable model for siRNA efficacy prediction.BMC Bioinformatics. 2006 Nov 30;7(1):520.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Cell Culture and Transfection

The LN-229 glioblastoma cell line was purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 (Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% Gibco® fetal bovine serum (Thermo Fisher Scientific) and 100 μg/mL penicillin-streptomycin (Thermo Fisher Scientific) at 37℃ and 5% CO2. Cell transfection was performed using Lipofectamine™ 3000 Transfection Reagent (Invitrogen™, CA, USA) strictly according to the manufacturer’s instruction. After 48 h of transfection, the efficiency was determined by RT-qPCR and western blotting which are described below.

# Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

RT-qPCR was used for detection of EIF6 and glycolysis related genes GLUT1, HK1 and LDHA. Briefly, total RNA was extracted from LN-229 cells using TRIzol reagent (Tiangen Biotech, Beijing, China). The concentration of extracted RNA was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE, USA). Then reverse transcription was conducted and cDNA was obtained by a Prime-Script™ One Step RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China). A SYBR GREEN mastermix kit (Applied Biosystems, CA, USA) was used to perform the PCR reactions in Applied Biosystems 7500 Real Time PCR system (Applied Biosystems) with the following condition: 95 ℃ 15 min, 35 cycles, 4 ℃ denaturation for 15 sec, 55 ℃ annealing for 25 sec, 70 ℃ extension for 30 sec. The primer sequences were designed by Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/) and were listed in table 1. The reverse transcription system and PCR reaction system were listed in table 2-3.

Table 1. Primers in this study.

| Genes | Sequence (5' -> 3') Forward | Sequence (5' -> 3') Reverse |
| --- | --- | --- |
| EIF6 | TCTCATCTCTCCCTGCCACT | GCAACAAGAGCGAAAACTCC |
| GLUT1 | TCCCACTTGGCAGACTCTCT | ACTGGACCCAGTCCTGACAC |
| HK1 | CCTGAGCCCAGAACACTCTC | GTGGAAGCAACTCCTTGAGC |
| LDHA | AGGCCAACTCTGCAAAGAAA | GCACCTGGCCAAAATAAAAA |

Table 2. The reverse transcription system

| Reagent | Volume (μL) |
| --- | --- |
| Template RNA primer mixture | 10 |
| PrimeScript Reverse Transcriptase (200 U/μL) | 0.5 |
| RNase Inhibitor | 0.5 |
| 5×PrimeScript Buffer | 4 |
| RNase free dH2O | 5 |
| Total | 20 |

Table 3. PCR reaction system.

| Reagent | Volume (μL) |
| --- | --- |
| cDNA | 1 |
| 1×TaqMan Mix | 5 |
| TaqMan probe | 0.2 |
| Upstream primer | 0.5 |
| Downstream primer | 0.5 |
| RNase free dH2O | 3 |
| Total | 10 |

# Western Blotting Procedure

1. **Extraction of Total Protein:** Total proteins were extracted from LN-229 cells using radio-immunoprecipitation assay (RIPA) buffer (Vazyme Biotec Co., LTD, Nanjing, China).
2. **Protein Concentration Measurement:** The protein amount was quantitated using the BCA protein assay reagent from Bio-Rad (Hercules, CA, USA). A BCA working reagent was prepared according to the manufacturer's instructions.
3. **Electrophoresis Separation:** Samples containing an equal amount of protein (20-40 μg) were loaded into each well of a 10% SDS-PAGE gel. Electrophoresis was performed at a constant voltage (100-200 V) until the dye front reached the bottom of the gel.
4. **Transfer to PVDF Membrane:** The membrane was equilibrated in transfer buffer (Tris-glycine buffer with 20% methanol) for 15-20 minutes. A transfer sandwich (gel-PVDF-membrane) was assembled, and transfer was carried out using a semi-dry or wet transfer system following the manufacturer's recommendations.
5. **Blocking:** The PVDF membrane was incubated in 5% non-fat milk at room temperature for 1 hour.
6. **Primary Antibody Incubation:** The primary antibody against EIF6 (anti-EIF6 antibody from Abcam, Cambridge, MA, USA) was diluted in 1% non-fat milk or 1% BSA in PBS following the manufacturer's recommended dilution. The membrane was incubated with the primary antibody solution overnight at 4℃ with gentle shaking.
7. **Secondary Antibody Incubation:** The secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody from Abcam, Cambridge, MA, USA) was diluted in 1% non-fat milk or 1% BSA in PBS. The membrane was incubated with the secondary antibody according to the manufacturer's instructions at room temperature for 45 minutes.
8. **Chemiluminescent Detection:** The chemiluminescent reagent (Super Signal West Pico Chemiluminescent Substrate kit from Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was prepared according to the manufacturer's instructions. Chemiluminescent signals were captured using an imaging system. Relative protein expression was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

# MTT and Colony Formation Assay

The MTT assay was performed to assess cell viability following transfection at various time intervals. Initially, 5 × 10^3 cells were seeded into individual wells of 96-well plates and incubated for 48 hours at 37℃ in a 5% CO2 environment. Subsequently, 10 μl of MTT solution (5 mg/ml) was introduced to each well, and the cells were incubated for an additional 4 hours. Afterward, the MTT solution was carefully aspirated, and 100 μl of dimethyl sulfoxide (DMSO) was added to each well. Optical density (OD) values were measured at 490 nm to evaluate cell viability.

The clonogenic formation assay was carried out to assess cell proliferation following transfection, following established protocols. In summary, cells were initially seeded at a density of 5 × 10^3 cells per well in 6-well dishes. After a 14-day incubation period, cells were fixed using glutaraldehyde and subsequently stained with crystal violet. Positive colony formations were manually counted for each well, and all experiments were conducted in triplicate.

# Transwell Invasion Assay

The Transwell invasion assay was conducted to assess the invasive capacity of cells through a Matrigel-coated membrane (24-well insert; pore size, 8 μm; BD Biosciences). A total of 1.25 × 10^5 cells were seeded in the upper chamber with serum-free media, while the lower chamber contained media with serum as a chemoattractant. Following a 24-hour incubation period at 37℃, non-invading cells on the upper surface of the membrane were gently removed, and the invaded cells on the lower surface were fixed with glutaraldehyde and stained using 0.1% crystal violet. Subsequently, five randomly selected fields of view within each sample were imaged and counted under a microscope. The mean number of invaded cells from triplicate experiments was determined, providing a quantitative measure of the cells' invasive potential.

# Wound Healing Assay

Cell migration was assessed via the scratch wound assay, where cells were initially seeded into 6-well plates and cultured for 24 hours to achieve confluence. Subsequently, uniform wound gaps were generated within the cell monolayer using a 200 μL pipette tip. The cells were then cultured in DMEM supplemented with 10% FBS, and images were captured at both 0 and 48-hour time points to precisely document and quantify changes in wound width, providing insights into cell migration dynamics over the specified time frame.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Transfection efficiency of si-EIF6

Figure 1 shows the transfection efficiency of three designed si-RNAs by RT-qPCR (A-C). We chose the siRNA with the highest efficiency for further experiments.We chose the siRNA with the highest efficiency for further experiments. The protein level of EIF6 was also determined for the chosen siRNA (D-E). The result of PCR electrophoresis is also shown (F).

![](https://static.igem.wiki/teams/4717/wiki/results/results/results6.jpg)

# si-EIF6 enhances the inhibitory effects of apatinib on cell proliferation of LN-229 cells

Figure 2 shows the cell proliferation by clonogenic formation assay (A-B) and viability by MTT (C) of different LN-229 cells. It was found either si-EIF6 and apatinib significantly decreased cell proliferation and viability of LN-229 cells, while the effects were stronger when cells were treated with both si-EIF6 and apatinib.

![](https://static.igem.wiki/teams/4717/wiki/results/results2.jpg)

# si-EIF6 facilitates the inhibitory effects of apatinib on cell invasion of LN-229 cells

Figure 3 presents the assessment of cell invasion ability through a Transwell assay. The results indicate a significant reduction in cell invasion of LN-229 cells upon treatment with either si-EIF6 or apatinib. Notably, the combined treatment with both si-EIF6 and apatinib exhibited more pronounced inhibitory effects on cell invasion, suggesting a synergistic impact.

![](https://static.igem.wiki/teams/4717/wiki/results/results3.jpg)

# si-EIF6 enhances the inhibitory effects of apatinib on cell migration of LN-229 cells

Figure 4 illustrates the assessment of cell migration using a wound healing assay. The results demonstrate a significant inhibition of cell invasion in LN-229 cells when treated with either si-EIF6 or apatinib. Notably, the combined treatment with si-EIF6 and apatinib exhibited enhanced inhibitory effects on cell invasion, indicating a synergistic impact.

![](https://static.igem.wiki/teams/4717/wiki/results/results4.jpg)

# si-EIF6 inhibits glycolysis of LN-229 cells

Figure 5 shows the cell glycolysis by testing the marker genes of GLUT1, HK1 and LDHA. It was found either si-EIF6 and apatinib remarkably down-regulated the gene expression of GLUT1, HK1 and LDHA, while the inhibitory effects were stronger when cells were treated with both si-EIF6 and apatinib.

![](https://static.igem.wiki/teams/4717/wiki/results/results5.jpg)

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