KC-3654

NCI-H358-SHOC2-KO-1B3 Cell Line

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Background of NCI-H358-SHOC2-KO-1B3 Cell Line

SHOC2 encodes a prototypical leucine-rich repeat protein that promotes downstream receptor tyrosine kinase (RTK)/RAS signaling and plays an important role in several cellular and developmental processes. Gain-of-function germ line mutations of SHOC2 drive the RASopathy Noonan-like syndrome, and SHOC2 mediates adaptive resistance to mitogen-activated protein kinase (MAPK) inhibitors. Similar to many scaffolding proteins, SHOC2 facilitates signal transduction by enabling proximal protein interactions and regulating the subcellular localization of its binding partners.

Specifications

Catalog Number	KC-3654
Cell Line Name	NCI-H358-SHOC2-KO-1B3 Cell Line
Host Cell Line	NCI-H358
Description	Stable NCI-H358 clone with human SHOC2 gene knockout, No.1B3
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:3 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 38 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

NCI-H358-SHOC2-KO-1B3 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of SHOC2 knockout in NCI-H358 using PCR sequencing.

Figure 2: Characterization of SHOC2 knockout in NCI-H358 using RT-PCR sequencing.

Figure 3: Characterization of SHOC2 knockout in Jurkat using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS)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 saturated culture 1:2-1:3 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Kwon JJ, Hahn WC. A Leucine-Rich Repeat Protein Provides a SHOC2 the RAS Circuit: a Structure-Function Perspective. Mol Cell Biol. 2021 Mar 24;41(4):e00627-20. doi: 10.1128/MCB.00627-20. PMID: 33526449; PMCID: PMC8088128.