KC-4380

A549-Luc2-CPNE1-KO-1A2-Cell-Line

46502

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Background of A549-Luc2-CPNE1-KO-1A2-Cell-Line

Copine 1 (CPNE1), as a member of calcium-dependent phospholipid binding protein, is located on chromosome 20q11.21 region and expressed in various tissues and organs. CPNE1 is consisted of two tandem N-terminal C2 domains (C2A and C2B) associated with calcium-dependent phospholipid binding activities and a C-terminal A domain (von Willebrand factor) functioning as protein-binding domains. CPNE1 participates in various biological process. CPNE1 promotes neurite outgrowth via activating AKT phosphorylation and exerts an important role in the regulation of neural stem cell proliferation. In addition, proteomic profiling study demonstrated that CPNE1 may be a promising novel marker in ethanol-induced cardiotoxicity since expression of CPNE1 diminished after ethanol treatment in cardiomyocytes. CPNE1 regulates the half-life of NF-κB transcriptional responses through stimulating endoproteolysis of p65 protein. Moreover, downregulation of CPNE1 could induce senescence‐associated phenotypes. Recently, accumulating evidence indicates the key role of CPNE1 in tumorigenesis and malignant progression. It has been shown that high expression of CPNE1 predicts a poor prognosis in prostate cancer and triple-negative breast cancer. Furthermore, enhanced expression of CPNE1 promotes lung cancer growth and metastasis. CPNE1 also enhanced colorectal cancer cell growth, glycolysis and drug resistance via activating AKT-GLUT1/HK2 signaling pathway. Moreover, CPNE1 silencing can inhibit osteosarcoma cell migration and proliferation, suggesting a promising target for osteosarcoma therapy.

Specifications

Catalog Number	KC-4380
Cell Line Name	A549-Luc2-CPNE1-KO-1A2-Cell-Line
Host Cell Line	A549-Luc2
Description	Stable A549-Luc2 cell line with human CPNE1 gene knockout, No.1A2
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% F12K + 20% FBS + 10% DMSO
Propagation Medium	F12K+10% FBS+2μg/mL Puromycin
Selection Marker	N/A
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:3~1:6 every 2~3 days; seed out at about 1-3 x 10⁵ cells/ml
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

A549-Luc2-CPNE1-KO 1A2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A549-Luc2-CPNE1-KO 1A2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-Luc2-CPNE1-KO 1A2 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of A549-Luc2-CPNE1-KO 1A2 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (F12K + 10% FBS + 2μg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split the saturated culture at a ratio of 1:3 ~ 1:6 every 2~3 days; seed out at about 1-3 x 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% F12K + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Li Y, Li L, Liu H, Zhou T. CPNE1 silencing inhibits cell proliferation and accelerates apoptosis in human gastric cancer. Eur J Pharm Sci. 2022 Oct 1;177:106278. doi: 10.1016/j.ejps.2022.106278. Epub 2022 Aug 17. PMID: 35985444.