3 deazaneplanocin a hydrochloride dznep

T-ALL cell lines are held by the DSMZ (Braunschweig, Germany) and were cultivated as described previously [64 ]. PER-117 cells were kindly provided by Dr. Ursula Kees (Perth, Australia) [65 (link)]. Treatments of cell lines were performed for 20 h with 5 μM 3-Deazaneplanocin A hydrochloride (DZNep) (Sigma, Taufkirchen, Germany), 10 μg/ml Trichostatin A (TSA) (Sigma), indicated concentrations of ICBP112 (Sigma), or 20 ng/ml recombinant human Interleukin-7 (IL7) protein (R&D Systems, Minneapolis, MN, USA). Gene specific siRNA oligonucleotides and AllStars negative Control siRNA (siControl) were obtained from Qiagen. Expression constructs for AUTS2, PCGF5 and RUNX1 were obtained from Origene (Wiesbaden, Germany). SiRNAs (80 pmol) and expression constructs/vector controls (2 μg) were transfected into 1×10⁶ cells by electroporation using the EPI-2500 impulse generator (Fischer, Heidelberg, Germany) at 350 V for 10 ms. Electroporated cells were harvested after 20 h of cultivation. Lentiviral transduction of the STAT5 gene into JURKAT cells was performed as described previously [28 (link), 66 (link)].

Human lung fibroblasts (LL29, AnHa), which were isolated from a 26‐year‐old Caucasian female, were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in F‐12K medium (Kaighn's modification of Ham's F‐12 medium, ATCC) supplemented with 1% penicillin/streptomycin and 10% heat‐inactivated fetal bovine serum (FBS). The cells (1.6 × 10⁵ per well) were cultured in complete F12‐K culture media in 12‐well plates. After reaching 70% confluence, the cells were treated with 4 μM 3‐deazaneplanocin A hydrochloride (DZNep; Sigma‐Aldrich, St. Louis, MO) for 24 h, followed by stimulation with 5 ng/mL of recombinant human TGF‐β1 (R&D Systems, Minneapolis, MN) for various amounts of time. The cells were then washed with ice‐cold phosphate‐buffered saline (PBS) and collected with either Tri Reagent (Molecular Research Center, Cincinnati, OH) to isolate the RNA or the M‐PER Mammalian protein extraction reagent (Thermo Fisher Scientific, Grand Island, NY) plus Halt protease and/or phosphatase inhibitor cocktail to extract the proteins.