Rpmi 1640 medium

TRExBCBL-1-RTA (a gift from Dr. Jae U. Jung, herein termed iBCBL-1) and its derivative lines were cultured in RPMI 1640 medium (Quality Biological) supplemented with 15% heat-inactivated fetal bovine serum (FBS), stable L-alanyl-glutamine (Glutamine XL), and streptomycin and penicillin, at 37 °C and 5% CO₂. 293T, HeLa.Kyoto (a gift from Dr. Ron R. Kopito), and their derivative cell lines were cultured in DMEM supplemented with 10% FBS and antibiotics. The cell lines were tested for mycoplasma contamination (R&D systems) and if necessary cultured in plasmocin™ treatment or prophylactic (InvivoGen). Transient transfection with plasmids was performed using GenJet version II (SignaGen Laboratories). For stable and doxycycline (Dox)-inducible expression of short hairpin RNAs (shRNAs), culture cells were lentivirally transduced with shRNA-specifying sequences in the presence of 10 µg/ml polybrene overnight, and stably transduced cells were selected by growing in the presence of 1 µg/ml puromycin or 400 µg/ml geneticin for more than 1 month, and pooled clones were collected. For detection of mitochondrial superoxide, cells were incubated with 5 µM MitoSOX red indicator (Invitrogen) in Hank’s buffered salt solution containing calcium and magnesium for 10 min at 37°C just before cell fixation.

The I. scapularis IDE12 and ISE6 cell lines were obtained from Dr. Ulrike Munderloh at University of Minnesota. Cells were cultured in L15C300 medium supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma), 10% tryptose phosphate broth (TPB, Difco), 0.1% bovine cholesterol lipoprotein concentrate (LPPC, MP Biomedicals) at 34 °C in a non-CO₂ incubator. ISE6 cells were grown to confluency in capped T25 flasks (Greiner bio-one) and either seeded at 3 × 10⁶ cells/well in 6-well plates (Millipore Sigma) or 3 × 10⁵ cells/well in 24-well plates (Corning). IDE12 cells were also grown to confluency in T25 flasks and either seeded at 1 × 10⁶ cells/well in 6-well plates (Millipore Sigma) or 3 × 10⁵ cells/well in 24-well plates (Corning). The human leukemia cell line, HL-60, was obtained from ATCC and maintained in RPMI-1640 medium with L-Glutamine (Quality Biological) supplemented with 10% FBS (Gemini Bio-Products) and 1% GlutaMax (Gibco), in vented T25 flasks (CytoOne) at 37 °C and 5% CO₂. All cell cultures were confirmed to be Mycoplasma-free (Southern Biotech).