Tf1 cells

The human mast cell line, HMC1 cells and erythroleukemic cell line, TF1 cells (ATCC, Manassas, VA) were cultured in IMDM (HyClone, Logan, UT). Human embryonic kidney HEK293T (ATCC, Manassas, VA) and human melanoma cell, MML1 cells were cultured in RPMI1640 (Sigma Aldrich, St Louis, MO) media. Media was supplemented with 10% EV-depleted FBS (Sigma Aldrich), 2 mM L-glutamine (HyClone), and 1.2 U/ml 1-thioglycerol (Sigma Aldrich). The human MSCs from bone marrow were obtained as passage 1 from the MSC distribution of the Institute of Regenerative Medicine at Scott and White, USA, and cultured in alpha minimum essential medium (GIBCO® GlutaMAX™, Invitrogen, Carlsbad, CA) supplemented with 15% EV-depleted FBS (Sigma Aldrich). Three to four passages of MSCs were used for EV isolation. All media contained 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone). For the EV depletion, FBS was ultracentrifuged at 118,000 × g_avg (Type 45 Ti rotor, Beckman Coulter, Miami, FL) for 18 h and filtered through a 0.22 µm filter as previously described [16].

HEK-293T cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. TF-1 cells (ATCC) were maintained in RPMI containing 10% FBS, 1% penicillin/streptomycin, and 2 ng/mL recombinant human GM-CSF (R&D Systems). Lentiviral particles were generated by Lipofectamine 2000 (Invitrogen) co-transfection of HEK-293T cells with control or IDH1 expression vectors and the lentiviral packaging constructs psPAX2 (GAG-Pol) and pMD2.G (VSV-G) in a 2:2:1 ratio. Lentiviral infections were performed as previously described in ref. ^{21 (link)}, and the infected cells were selected with 1 μg/mL puromycin starting 24 h after infection. TF-1 cells were treated with ivosidenib (Selleck Chemicals, LLC) or LY3410738 (Eli Lilly) prior to immunoblot analysis, 2HG measurement, and growth factor deprivation. Cell lines were authenticated by Short Tandem Repeat (STR) profiling. Cell lines repeatedly tested negative for mycoplasma throughout the experimental period.

Monocyte suspension cells (THP-1, Public Health England Culture Collection) were cultured undifferentiated in RPMI-1640 (A10491, Gibco) supplemented with 10% BCS (30-2030, ATCC) (complete medium). For M0 differentiation, THP-1 cells were centrifuged and the conditioned media were replaced with fresh complete medium supplemented with 100 ng/ml phorbol 12-myristate-13-acetate (PMA, Sigma P8139). After 48 h, M0 macrophages were further differentiated into M1 or M2 macrophages as described [17 (link), 25 ]. Briefly, M0 differentiation medium was removed and adherent M0 cells were washed twice with serum-free medium. For further differentiation, complete medium supplemented with either 100 ng/ml LPS and 20 ng/ml IFN-γ (M1) or 20 ng/ml IL-4 and 20 ng/ml IL-13 (M2) was added to adherent M0 cells and then incubated for up to a further 48 h. A375 cells (ATCC) were cultured in DMEM (41966, Gibco) supplemented with 10% FBS (F7524, SIGMA). Cells were seeded at a density of 2 $e$ 4 cell/cm² and passaged every 3 days. TF-1 cells (ATCC) were cultured in RPMI-1640 supplemented with 2 ng/ml GM-CSF and 10% FBS. Cells were seeded at a density of 2 $e$ 5 cells/ml and passaged twice a week.