Icbp112

HL-cell lines HDLM-2, KM-H2, L-428, L-540, L-1236, SUP-HD1, U-HO1 and AML cell line THP-1 are held by the DSMZ (Braunschweig, Germany) and cultivated as described [70 ]. Cell stimulations were performed for 16 h by treatment with 20 ng/ml recombinant human proteins IGF1, IFNG, IL2, IL6, IL7, IL12, IL21, IL22 or IL23 (R&D Systems, Wiesbaden, Germany), with inhibitory antibodies directed against IGF1, IFNGR1, IFNGR2, IL7, IL22 (R&D Systems), with 10 or 100 nM 5-Azacytidine (AZA, Sigma, Taufkirchen, Germany), 5 μM DZNep (Sigma), 10 μg/ml Trichostatin A (TSA, Sigma), 50 μM Resveratrol (Sigma), 0.5 μM ICBP112 (Sigma), 1 μM Epinephrine (Sigma), 1 μM Propranolol (Sigma), Doxorubicine (Sigma), AG490 (Sigma), and Etoposide (Sigma). Gene specific siRNA oligonucleotides and AllStars negative Control siRNA (siControl) were obtained from Qiagen (Hilden, Germany). Expression constructs for HLX, MSX1, STAT3, and GFP-tagged STAT3 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. Transfected cells were harvested after 20 h cultivation.

BCLs were continuously treated for 72 h in adherent conditions with bortezomib (stock concentration SC = [10 mM], Selleckchem), carfilzomib (SC = [10 mM], Selleckchem), chloroquine (SC = [10 mM], Selleckchem), Cortistatin A (SC = [100 μM], a kind gift from Prof. P. Baran, The Scripps Research Institute, La Jolla, CA, USA), docetaxel (SC = [10 mM], GSK343 (SC = [1 mM], Active Biochemicals Co), I‐CBP112 (SC = [10 mM], Sigma), JQ1 (SC = [10 mM], Selleckchem), mifepristone (SC = [10 mM], Selleckchem), Ro5‐3335 (SC = [10 mM], Calbiochem), ruxolitinib (SC = [10 mM], Selleckchem), SGC‐CBP30 (SC = [10 mM], Selleckchem), salinomycin (SC = [200 μM], Selleckchem), spliceostatin A (SC = [10 mM], Adooq Biosciences), tazemetostat (SC = [5 mM], Selleckchem), and 8‐AZA (SC = [100 mM], Chembiotech). All these compounds were resuspended in dimethyl sulfoxide (DMSO; Sigma), except for chloroquine resuspended in H₂O. For the in vivo experiments, salinomycin (SC = [6 mg/ml], Medchemexpress) and JQ1 (SC = [100 mg/ml], Medchemexpress) were resuspended in a solution of DMSO/(2‐Hydroxypropyl)‐β‐cyclodextrin (HPCD) 10% (1:9, v/v).

FBP1 wildtype (wt) and FBP1 mutant (G260R) constructs were kindly provided by Dr. Haojie Huang from the Mayo Clinic [11 (link)]. The pCMV4a-Flag-c-Myc plasmid was purchased from Addgene. FBP1 nes mutants were generated by adding a nuclear export sequence (LALKLAGLDIGS) to the FBP1 c-terminal using the KOD-Plus- Mutagenesis Kit (Toyobo, Japan). Bacterial expression vectors for GST-TRIM28 recombinant proteins were generated using the pGEX-4 T-1 backbone vector. FBP1 antibody (ab109732)was purchased from Abcam (working dilution 1:1000); beta-Tubulin (2128S) was from Cell Signaling Technology - (working dilution 1:5000); TRIM28 (ab10483) was from Abcam (working dilution 1:3000); c-Myc (5605P) was from Santa Cruz Biotechnology (working dilution 1:1000); BRD2 (ab139690) was from Abcam (working dilution 1:1000); BRD3 (A302-368A) was from Bethyl Laboratories (working dilution 1:1000); BRD4 (ab128874) was from Abcam (working dilution 1:1000). JQ1, MG132, I CBP112, puromycin and cycloheximide (CHX), were purchased from Sigma-Aldrich (Shanghai, China); MK 2206, Trametinib, GSK126, Ku55933, SB203580 and Palbociclibwere from Selleckchem (Houston, USA). Gemcitabine was obtained from Eli Lilly and Company (Indianapolis, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, USA).

The synthesis and characterization of CPI203 have been published previously (Devaiah et al., 2012 (link)). SGC-CBP30 and I-CBP112 are commercially available (Sigma).

NK-cell lines KHYG-1, NK-92, NKL and YT have been reviewed by Drexler & Matsuo [16 ]. Together with T-cell lines JURKAT and LOUCY they are held by the DSMZ (Braunschweig, Germany). The NK-cell line IMC-1 was kindly obtained from Dr. I. Ming Chen (Albuquerque, NM, USA) and NK-cell line SNK-6 from Dr. N. Shimizu (Tokyo, Japan) [44 , 72 ]. These NK-cell lines tested all CD3-negative and CD56-positive. Cell lines were cultivated as described elsewhere [73 ]. Cell stimulations were performed by treatment with recombinant human BMP4 for 16 h at a concentration of 20 ng/ml (R&D Systems, Wiesbaden, Germany), 10 μg/ml Trichostatin A (TSA) (Sigma, Taufkirchen, Germany), and 0.5 μM ICBP112 (Sigma). Gene specific siRNA oligonucleotides, AllStars negative Control siRNA (siControl) and miRNA-Mimics were obtained from Qiagen (Hilden, Germany). Expression constructs for GATA3, HHEX, IRF4, MSX1, PCGF5 and PRDM1 were cloned in vector pCMV6 and obtained from Origene (Wiesbaden, Germany). SiRNAs (80 pmol), miRNA-Mimic for miR155 (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. Transfected cells were harvested after 20 h cultivation.