As the converging point of numerous oncogenic signaling pathways, signal transducer and activator of transcription 3 (STAT3) play a central role in regulating antitumor immune responses. STAT3 is extensively overactivated in both cancerous and non-cancerous cells within the tumor ecosystem and plays a vital role in suppressing the expression of key immune activation regulators and promoting the production of immunosuppressive factors. Mutations in human STAT3 are associated with diseases such as immunodeficiency, autoimmunity, and cancer. In recent years, many studies have shown that STAT3 is closely related to the occurrence and development of liver fibrosis caused by various factors. Activation of STAT3 may play an anti- or pro-inflammatory role in the pathogenesis of liver fibrosis. Furthermore, mitochondrial translocation of STAT3 inhibits oxidative stress-induced autophagy and may effectively protect mitochondria from degradation by mitophagy. Strikingly, however, either overactivation or inactivation of STAT3 leads to human disease, suggesting that tightly regulated STAT3 function is critical for health. Therefore, targeting the STAT3 signaling pathway has emerged as a promising therapeutic strategy for many cancers.
Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.
Cell Line Name:
Ba/F3 STAT3-Luc2 cell line
Host Cell Line:
Mouse Ba/F3 cell line
Stable Ba/F3 cell line expressing exogenous luciferase gene under the control of STAT3
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% RPMI1640+20% FBS+10% DMSO
RPMI1640+10%FBS+8ng/mL mouse IL-3 +1000μg/mL Hygromycin B
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3-STAT3-Luc2 cell line was generated using lentivirus methods.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (RPMI1640 + 10% FBS + 8ng/mL mouse IL-3 + 1000μg/mL Hygromycin B) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL.
1. Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage
1. Zou S, Tong Q, Liu B, Huang W, Tian Y, Fu X. Targeting STAT3 in Cancer Immunotherapy. Mol Cancer. 2020 Sep 24.
2. Hillmer EJ, Zhang H, Li HS, Watowich SS. STAT3 signaling in immunity. Cytokine Growth Factor Rev. 2016 Oct.
3. You L, Wang Z, Li H, Shou J, Jing Z, Xie J, Sui X, Pan H, Han W. The role of STAT3 in autophagy. Autophagy. 2015.
4. Zhao J, Qi YF, Yu YR. STAT3: A key regulator in liver fibrosis. Ann Hepatol. 2021 Mar-Apr.
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