Mda mb 231

The human diffuse large B cell lymphoma cell lines (DLBCL) SuDHL8 and U2932 were obtained from Dr. Li Jia (Barts Cancer Institute, London, UK), and purchased from Deutsche Sammlung von Mikroorganismen and Zellkulturen GmBH (Braunschweig, Germany), respectively. HepG2 cells were purchased from ATCC (Manassas, USA). CXCR4 expression was modulated in a human triple-negative metastatic breast cancer cell line, MDA-MB-231 (PerkinElmer, Waltham, MA, USA), by using a doxycycline (DOX)-inducible lentiviral pTRIPZ vector encoding for shRNA targeting CXCR4 (pTRIPZ CXCR4 shRNA clone V3THS_346208; Dharmacon, Lafayette, USA) to obtain MDA-MB-231 shCXCR4 or non-targeting shRNA control (Dharmacon) to obtain MDA-MB-231 shSC. Stable clones were FACS sorted based on the expression of a DOX-inducible TurboRFP reporter gene within the same construct. SuDHL8, U2932, MDA-MB-231 shRNA and shSC were cultured in Roswell Park Memorial Institute Medium 1640 (RPMI) and HepG2 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). All media was supplemented with 10% Foetal Bovine Serum (FBS), 2mM L-glutamine (Invitrogen™, Thermo Fisher Scientific, Carlsbad, CA USA) and 100 U/mL penicillin-streptomycin (Invitrogen™) in a humidified atmosphere of 5% CO₂ at 37°C.

Human TNBC cell lines, MDA-MB-231, and BT549 were obtained from ATCC (Manassas, VA, USA), and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Gaitherburg, MD, USA) with 10% fetal bovine serum (FBS) (Gibco). All cell lines were used in the present experiments within 20 passages after the reception. All cell lines were routinely tested to rule out mycoplasma infection using PlasmoTest kit (InvivoGen, San Diego, CA, USA). All cell lines were cultivated in a humidified incubator at 37 °C in 5% CO₂.
Human PIEZO2 specific siRNA and non-targeting siRNA (Dharmacon, Lafayette, CO, USA) were transfected into the MDA-MB-231 using lipofectamine RNAiMAX, in accordance with the instructions of the manufacturer. The siRNA-treated cells were collected 48 h after transfection. PIEZO2 expression was determined by qPCR, and cells were utilized for further experiments. Either empty or PIEZO2-inserted pcDNA3.1 was transfected to MDA-MB-231 and BT549 cells utilizing jetPRIME (Polyplus transfection, Illkirch, France). The cells were selected by G418 treatment to generate stably PIEZO2 overexpressed cells. PIEZO2 level was confirmed by qPCR.

All cell lines (MDA-MB-231- RRID: CVCL_0062, HCC38- RRID: CVCL_1267, and MDA-MB-468- RRID: CVCL_0419) were purchased from American Type Culture Collection between 2015 and 2018 and maintained as directed. They were expanded, aliquoted, cryogenically stored, and used within 12 passages of thawing. Cell lines were further validated by STR mapping in 2022. MDA-MB-231, HCC38, and MDA-MB-468 cell lines were cultured in RPMI containing 10% fetal bovine serum and 1% Pen-Strep. All cells were incubated at 37 °C in 5% CO₂. MDA-MB-231 cells that stably express Cas9 were generated using Lentiviral Cas9 Nuclease Reagents from Dharmacon (VCAS10124) according to manufacturer’s instructions. Following transduction, cells were selected using Blasticidin (Gibco #A1113903-03). MDA-MB-231(Cas9) cells were passaged in Blasticidin continuously at 20 μg/ml. Cell lines were tested for Mycoplasma pulmonis and Mycoplasma spp using MycoAlert Plus Mycoplasma Detection Kit (Lonza, LT07-703).