1. Introduction to Cell Culture

Cell culture is a fundamental technique in biochemistry that allows the growth and maintenance of various cell types in vitro. Specifically, this article focuses on the protocols involving COS-7 and HEK293T cells.

1.1 Cultivation of COS-7 and HEK293T Cells

COS-7 cells (available from Sigma, catalog number 87021302) and HEK293T cells (Beyotime, C6008) are routinely cultured in a specialized medium known as Dulbecco’s Modified Eagle Medium (Thermo Fisher, 41965-039). This medium is enriched with:

• 10% fetal bovine serum (FBS; Sigma, F7524)
• 1% penicillin-streptomycin (Thermo Fisher, 15140-122)
• 1% l-glutamine (Thermo Fisher, 25030-081)
• 1% sodium pyruvate (Thermo Fisher, 11360-070)

Cell cultures are maintained at 37 °C in a humidified atmosphere containing 5% CO2, with passages every 2 to 3 days. All experimental procedures utilized cells within 20 passages.

1.2 Subculturing and Induction of U-2 OS-CRISPR-NUP96 Cells

The U-2 OS-CRISPR-NUP96-mEGFP cell line, acquired from Cytion (300174) and provided by Y. Li, is maintained in McCoy’s 5A medium (Procell, PM150710) supplemented similarly with FBS and penicillin-streptomycin. An important advancement is the generation of a doxycycline-inducible variant of this cell line, which expresses importin-β. This is achieved through the transduction of lentiviral particles produced in HEK293T cells.

Viral Particle Production and Transduction

Lentiviral particles containing the pTRIPZ-importin-β-3×FLAG-ALFA-P2A-tagBFP plasmid are co-transfected with psPAX2 and pMD2.G at a ratio of 4:3:1. Viral supernatants are harvested and concentrated using polyethylene glycol precipitation. U-2 OS-CRISPR-NUP96-mEGFP cells are subsequently transduced and selected with puromycin. Induction of importin-β expression is then achieved through the administration of doxycycline.

2. Transfection Techniques

Transfection is a critical process for introducing plasmids into cells. For effective results with COS-7 cells, the following protocol is employed:

2.1 Transfection of COS-7 Cells

COS-7 cells, seeded on glass-bottom imaging dishes, are transfected using jetPRIME reagent according to the manufacturer’s instructions. After 4 to 5 hours, the medium is refreshed and supplemented with HEPES and either BCN for experimental use or BOC for control purposes.

3. Live-Cell Labelling Methods

Live-cell labelling techniques allow researchers to visualize specific cellular components. Following transfection, cells are treated with specific fluorescent dyes under carefully controlled conditions:

3.1 Labelling Procedure

After a 20-24 hour incubation, the cells are bathed in medium to clear the excess dye before applying a serum-free medium containing HD653 for staining. Additional reagents can be included as necessary for experimental conditions.

Cell imaging occurs under meticulously maintained conditions to ensure the integrity of the data collected.

4. Analysis of Endogenous Proteins

The localization and expression levels of endogenous importin-β can be examined through various immunostaining techniques. Fixation is achieved with paraformaldehyde, followed by permeabilization and blocking to enhance antibody efficacy.

4.1 Confirmation of Protein Presence

Cells are treated with an anti-KPNB1 antibody, and subsequent imaging with an Alexa Fluor-conjugated secondary antibody facilitates the visualization of importin-β at the nuclear envelope, revealing its crucial role in cellular transport mechanisms.

5. Quantitative Approaches to Importin-β Studies

Estimating importin-β concentrations and their dynamics within the nuclear pore complex is fundamental for understanding their biochemical activities:

5.1 Measuring Concentration and Expression Levels

Utilizing immunoblotting techniques, the expression levels of exogenous importin-β can be compared over time under doxycycline treatment. This analysis highlights the inducibility of transgenes and their physiological relevance.

6. O-GlcNAcylation Assessment

The modulation of glycosylation patterns through the use of specific inhibitors helps elucidate the role of O-GlcNAcylation in protein function.

6.1 Immunoblotting for O-GlcNAc Levels

By subjecting lysates to SDS-PAGE techniques, the impact of glycosylation modulators can be visually confirmed using anti-O-GlcNAc antibodies, ensuring a comprehensive understanding of cellular signalling pathways.

7. Dynamic Imaging Techniques

Advanced imaging modalities such as MINFLUX and time-resolved fluorescence anisotropy (TRFA) provide enhanced capabilities for monitoring protein dynamics in live cells.

7.1 Application of TRFA Measurements

TRFA is employed to assess molecular dynamics within condensates, providing insights into the interactions and behavior of proteins under various conditions. Calibration and fitting procedures are systematically applied for accurate data analysis.

8. Conclusion

The combination of robust cell culture, transfection techniques, and advanced labelling methods is pivotal for the modern biochemist. These methods allow for the detailed exploration of protein dynamics, localization, and post-translational modifications, deepening our understanding of cellular processes.