Tubacin

A rabbit polyclonal antibody against DDDDK-tag was used as the anti-Flag antibody (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan). Mouse monoclonal antibodies against α-tubulin (DM1A (epitope; 426–450 residues), NeoMarkers, Fremont, CA, USA), Cy3-conjugated-β-tubulin (both antibodies used for general tubulin staining), and acetylated-α-tubulin (clone 6-11B-1) (Sigma, St. Louis, MO, USA), were used. Human anti-centromere antibody (Antibodies Inc., Davis, CA, USA) was also used. DAPI (4,6-diamidino-2-phenylindole dihydrochloride hydrate) (Sigma), and tubacin (Enzo Life Sciences, Farmingdale, NY, USA) were used. Salicylate (sodium salicylate) was purchased from Sigma. NAP peptide (Biorbit, San Francisco, CA, USA) was aliquoted and stocked as a 1 mM solution in 75% dimethyl sulfoxide at −20 °C.

Nocodazole, STLC, taxol (Paclitaxel), Triton X-100, phenylmethanesulfonylfluoride (PMSF), N-ethymaleimide and anti-FLAG M2 antibody conjugated agarose beads were purchased from Sigma; protein A/G-conjugated agarose beads, dithiobis(succinimidyl proprionate) and MG132 from ThermoFisher (Rockford, IL), tubacin from Enzo Lifesciences (Farmingdale, NY), rapalog-1 or A/C heterodimerizer from Clonetech (catalogue: 635057; Mountain View, CA) and digitonin from EMD Millipore (San Diego, CA). Antibodies used in this study are listed in Supplementary Table 1.

All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at The Johns Hopkins University School of Medicine. Eight to ten weeks old C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Unless otherwise stated, all reagents were obtained from Sigma (St. Louis, MO). Antibodies to hemagglutinin (HA; C29F4), HDAC6, NEDD8, p-eNOS (T495) and FLAG were purchased from Cell Signaling (Danvers, MA). NOS3 (A-9) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX). Acetylated α-tubulin antibody was purchased from Abcam (ab24610; Cambridge, MA), total α-tubulin antibody was purchased from Invitrogen (62204; Waltham, MA), and Lipofectamine 2000 was purchased from Life Technologies (Waltham, MA). Tubacin was purchased from Enzo Life Sciences (Farmingdale, NY). Fresh batches of oxidized low-density lipoprotein (OxLDL) was purchased from Alfa Aesar (a Johnson Matthew company).

Neuroblastoma SH-SY5Y cell line is a kind gift from Dr. Juma Mora at Sant Joan De Deu, Barcelona, Spain. SH-SY5Y cells were authenticated using AmpFlSTR® SGM Plus® PCR Amplification Kit (Applied BioSystems). SH-SY5Y and HEK293-T cells were cultured in RPMI and DMEM (Lonza) respectively supplemented with 10% FBS (Lonza). For induction of autophagy, cells were serum starved in Hank’s balanced salt solution (HBSS) (Lonza) for 2 and 6 hours. For HDAC6 inhibition, cells were treated with 20 μM tubacin or niltubacin (Enzo Life Sciences). For 5-aza-2′-deoxycytidine (5-AZA-dC) treatment (Sigma Aldrich), cells were treated with either DMSO or 10 μM 5-AZA-dC for four successive days.

MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [33 (link)]. Cells were periodically tested for mycoplasma contamination. PDX cells were cultured in MEMα medium (Thermo Fisher Scientific) supplemented with 10% human heat inactivated AB⁺ serum, 10% fetal calf serum (FCS), 1% Glutamax, 1% penicillin/streptomycin and with 20 ng/ml FLT3 ligand, 10 ng/ml IL7, 50 ng/ml SCF (Peprotech, Rocky Hill, NJ, USA), and 20 nM human insulin (Sigma Aldrich, Saint Luis, MO, USA).
The following drugs were used: 0.5 µM TSA (Sigma Aldrich), 500 µM cyclohexamide (Sigma Aldrich), 20 µM MG132 (Sigma Aldrich), 20 µM CHL (Sigma Aldrich), 2 µM tubacin (Enzo Life Science, Farmingdale, NY), 100 nM bafilomycin (Sigma Aldrich), 2 µM HDAC1i and HDAC8i (Italfarmaco, Milan, Italy), 20 µM Ciliobrevin D. At planned time points, cells were harvested and processed for assessment of cell viability, caspase assay, and RNA and protein extraction.