Ant mpp

We used MeT-5A, M14K, and MSTO cell lines (kindly provided by Professor Ioannis Kalomenidis, National Kapodistrian University of Athens). Culture of cells was done in 10% Fetal Bovine Serum (FBS)-RPMI (F0804, Sigma, St Louis, MO, USA) that was supplemented with 2 Mm L-Glutamine (G7513, Sigma), 1% Penicillin/Streptomycin (P4333, Sigma), and 0.5% w/v Plasmocin (ANT-MPP, InvivoGen, Toulouse, France) in a 5% CO₂ incubator. Cells were synchronized for 24 h in 0.5% FBS-RPMI before experiments. For drug treatments, cells were incubated for 24 h with 30 Mm Ammonium Sulphate (AS; A4915-500G, Sigma), or 50 Mm Lithium Chloride (LC; L4408-100G, Sigma).
To generate 3D spheroids, we employed the hanging drop method [16 (link), 17 (link)]. Synchronized cells at a density of 4 × 10⁶ /ml were mixed with 250 ng/mL of sterile bovine plasma fibronectin (341,631-1MG, Sigma) in 10% FBS-RPMI. 25 μL microliter of this cell solution containing 10⁵ cells was used to generate individual spheroids on the underside of sterile petri dishes with 1 ml of sterile PBS in the bottom dish. After 24 h the cell medium was removed with a sterile filter paper and replaced with the appropriate drug treatments for another 24 h. Finally, to collect RNA for gene expression assessments 10 hanging drops were combined and centrifuged at 5000 rpm for 5 min followed by lysis for RNA collection.

Hep3B, HepG2, and BxPC-3 cells were purchased from Tongpai Biotechnology Co., Ltd. (Shanghai, China). Huh7, MCF-7, MDA-MB-231, and LO2 cells were purchased from Hunan Fenghui Biotechnology Co., Ltd (Changsha, Hunan, China). Hep3B, Huh7, HepG2, LO2, and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, C12430500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, 10,099-141C), penicillin (Shanghai Yuanye Bio-Technology Co., Ltd, B25911), and streptomycin (Sangon Biotech, A610494-0050). MCF-7 and BxPC-3 cells were cultured in RPMI Medium 1640 (Gibco, C11875500BT) with 10% FBS and penicillin/streptomycin. To avoid mycoplasma contamination, cells were treated with prophylactic plasmocin (InvivoGen, ant-mpp), according to the manufacturer’s instructions. For inhibitor treatments, cells were pretreated for 20 min with A66 (6 μM) and 30 min with TGX221 (4 μM) or cytochalasin D (10 μM). Lipofectamine 2000 (11,668,019; Thermo Fisher) was used for transfection according to the manufacturer’s protocol. Plasmid PH-Btk-GFP was purified using the TIANpure Midi Plasmid Kit (TIANGEN #DP107-02).

B16F10 cells negative for mycoplasma contamination were cultured and passaged in RPMI-1640 medium containing 10% fetal calf serum (FCS) (Gemini, USA) at 37 °C/5% CO₂. For animal inoculations, cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Hyclone, USA) supplemented with 10% FCS and 5 µg/ml plasmocin prophylactic reagent (InvivoGen, Cat.: ant-mpp, USA) at 37 °C/5% CO₂ to induce better melanin production. B16F10 cell line was generously provided by Dr Hui Zhang from Institute of Human Virology, Sun Yat-sen University and originally purchased from the ATCC.

Ovarian cancer cell lines (A2780, IGROV1, OVCAR8, and SKOV3), endometrioid cancer cell lines (EFE-184 and KLE) were purchased from the American Type Culture Collection (ATCC, VA, USA) and ACI-98 was kindly provided by Carrie D. House (San Diego State University). Breast cancer cell lines (T47D and MCF-7) were kindly provided by Stanley Lipkowitz. Ovarian cancer cell lines (1A9CP80 and CP80) were kindly provided by Antonio Tito Fojo (Columbia University). Cells were cultured at 37 °C in a 5% CO₂ environment. For 2D-cultured cells, RPMI 1640 (#11875093, Thermo Fisher Scientific, USA)　medium supplemented with 10% fetal calf serum (FCS) (#100-106, GeminiBio, CA, USA), penicillin (100 units/mL) and streptomycin (100 units/mL) (#15140-122, Thermo Fisher Scientific) was used. For 3D-spheroids, ultra-low attachment plates (Corning, NY, USA) were used with Stem Cell culture Media, consisting of 1% KnockOut serum replacement (#10828-010, Thermo Fisher Scientific), 1% penicillin/streptomycin, 0.1% Insulin-Transferrin-Selenium (#41400-045, Thermo Fisher Scientific) and 0.4% Bovine Serum Albumin (#A9418, Millipore Sigma). Cultures were grown for 3 days prior to drug experiments. Mycoplasma infection was addressed using Plasmocin^TM prophylactic (#ant-mpp, InvivoGen, CA, USA) treatment, with confirmation of its absence.