Iscove modified dulbecco media (imdm)

Approval was obtained from the CSIC Bioethics Review Board for these studies. All patients signed an informed consent before blood was drawn. B-lymphocytes were purified from the 48 CLL samples listed in Table 1, using Ficoll-Paque^TM PLUS (GE Healthcare, Uppsala, Sweden) centrifugation and, if necessary, negative selection with anti-CD3-conjugated Dynabeads (Invitrogen Dynal AS, Oslo, Norway). The resulting B cell population was mostly >90% CD19⁺, determined on a Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA). Primary stromal cells (BMSC) were obtained from a bone marrow sample of a CLL patient after 3 week culture in IMDM (Lonza, Amboise, France)/20% FBS, and maintained for up to 4 weeks in IMDM/15% FBS. The HS-5 stromal cell line, with fibroblastoid properties [17 (link), 18 (link)], was obtained from Dr. Atanasio Pandiella (Cancer Research Center, Salamanca, Spain). The HS-27A stromal cell line, with epithelioid properties and functionally different than HS-5 cells [17 (link), 18 (link)] was purchased from ATCC (Manassas, VA, USA). Both cell lines were cultured in RPMI/10% FBS.

NALM-6 isogenic knock-in cell lines including different hotspot mutations in the HEAT domain of SF3B1 (parental, K700E, K666N, and H662Q) were from a previous study (11 (link)) and mutations were confirmed by Sanger sequencing. UM cell lines 92.1 (SF3B1wt) and Mel202 (SF3B1mut) were acquired from Martine de Jager (department of ophthalmology LUMC, The Netherlands). Pancreas carcinoma (PDA) cell line panc1 (SF3B1wt) was obtained from the LEXOR group (Amsterdam UMC, The Netherlands) and panc05.04 (SF3B1mut) was directly bought from ATCC and CLL cell lines PGA (SF3B1wt) and CII (SF3B1mut) were a kind gift from Tanja Stankovic (Bournemouth, UK). Cell lines were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with HEPES and L-glutamine (92.1, Mel202, PGA, CII, panc05.04, and NALM-6 cell lines) or IMDM (Lonza, Basel, Switzerland) with HEPES, L-glutamine (panc1), and supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin (Invitrogen) and incubated in 5% CO₂ at 37 °C. Panc05.04 was cultured in the presence of 1% Insulin-Transferrin-Selenium (ITS -G) (Thermo Fisher Scientific, Waltham, MA, USA). Primary CLL cells were thawed and cultured in IMDM (Lonza, Basel, Switzerland) with HEPES, L-glutamine, 10% FCS, and penicillin-streptomycin (Invitrogen) for functional experiments and incubated in 5% CO₂ at 37 °C.

Macrophages were differentiated by culturing bone marrow progenitor cells in IMDM (Lonza) containing 10% (vol/vol) heat-inactivated FBS, 30% (vol/vol) L929 cell-conditioned medium, 1% (vol/vol) nonessential amino acids (Lonza), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂ for 6 d. BMDMs were then seeded into 96- or 24-well plates as needed, in IMDM containing 10% FBS, 1% nonessential amino acids, and antibiotics. On the next day, cells were changed to fresh media and either primed or not with 100 ng/ml LPS for 3 h before treatment with vehicle or sitagliptin (water), 1G244 (DMSO), UAMC39 (water), UAMC1110 (DMSO), VBP (0.1%TFA in DMSO), acetyl VBP (DMSO), cyclic VBP (0.1%TFA in DMSO), or KYP-2047/UAMC714 (DMSO) at a final concentration of 10 μM unless otherwise stated in the figure legends. Alternatively, cells were stimulated with 1 μg/ml PA combined with 0.5 μg/ml LF (LeTx).