Tubastatin a

Cultures were treated post microinjection and analyzed after 3 days, unless otherwise stipulated. Drugs used were: Arimoclomol (Medkoo Biosciences, Morrisville, NC, USA) dissolved in water to prepare a stock solution of 2 mM; HDAC inhibitors: the pan HDAC inhibitor SAHA (Cedarlane), HDAC 1/3 inhibitors RGFP963 (BioMarin Pharmaceutical Inc., San Raphael, CA, USA) and RGFP109 (Selleck Chemicals LLC, Houston, TX, USA) dissolved in dimethyl sulfoxide for stock solutions of 2 mM, and the HDAC6 inhibitor Tubastatin A (Cayman Chemical, Ann Arbor, MI, USA) dissolved in dimethyl sulfoxide for a stock solution of 6 mM. Drugs were diluted to working concentrations in a modified N3 culture medium with gentamycin, with an equivalent concentration of dimethyl sulfoxide as control.

HCT116 and HT-29 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). DLD-1 cell was a kind gift from Dr. Er-Chieh Cho (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan). Cells were maintained in 10% fetal bovine serum (FBS)-supplemented RPMI 1640 medium and 1% penicillin–streptomycin (GIBCO, Grand Island, NY, USA) at 37 °C in a humidified incubator containing 5% CO₂. Compound MPT0G612 (3-[4-(3-dimethylaminomethyl-2-methyl-indole-1-sulfonyl)-phenyl]-N-hydroxy-acrylamide) was obtained from Professor Jing-Ping Liou (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan). Puromycin, ACY-1215, tubastatin A, rapamycin, and wortamannin were from Cayman Chemical (Ann Arbor, MI, USA). Anti-mouse and anti-rabbit IgGs were from Jackson Immuno Research Laboratories (West Grove, PA, USA).

Primary antibodies were: mouse anti-human HSP70, specific for stress-inducible HSPA1A (StressMarq, Canada, SMC-100B; 1:100 ICC; 1:1000 WB), mouse anti-Flag M2 (Sigma-Aldrich, #F1804; 1:400 ICC); rabbit anti-FUS (Proteintech, USA, 11570-1-AP; 1:400 ICC), mouse anti-human SOD1 (Sigma-Aldrich Canada, SD-G6; 1:100 ICC), rabbit anti-acetyl-histone H3K9/K14 (Cell Signaling, #9677; 1:400 ICC; 1:1000 WB), mouse anti-GAPDH (MediMabs, Canada, #MM-0163; 1:1000 WB), moue monoclonal anti-acetylated tubulin (Sigma-Aldrich #T6793; 1:1000 WB) and rabbit anti-α-tubulin (Abcam #ab15246; 1:1000 WB).
Secondary antibodies (Jackson Immunoresearch: Cedarlane, Canada): Alexa Fluor 488-conjugated Affinipure donkey anti-mouse IgG (1:300); Cy3-conjugated donkey anti-mouse IgG 1:300); Cy5-conjugated donkey anti-rabbit IgG (1:300), and HRP-conjugated goat anti-mouse (1/5000 for WB, 1/500 for IC) or donkey anti-rabbit IgG (Jackson Immunoresearch (1:2500).
HDAC inhibitors and HSP-inducing drugs: SAHA (suberoylanilide hydroxamic acid) and tubastatin A (Cayman Chemical: Cedarlane, Canada); tacedinaline, RGFP109 and RGFP966 (Selleckchem: Cedarlane, Canada); sodium phenylbutyrate (Selleckchem); arimoclomol (Toronto Research Chemicals, Canada); NXD30001 was previously supplied by NexGenix Pharmaceuticals (Cha et al. 2014 (link)).

ACY-738 was purchased from Selleckchem (Houston, TX, USA) and prepared as stock solution with the concentration of 50 mg/ml by DMSO. Tubastatin A was obtained from Cayman Chemical (Ann Arbor, MI, USA) and prepared as stock solution with the concentration of 10 mg/ml by DMSO. The stocks were further diluted by 0.9% saline solution before injection in the animals. DAPI, Dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against Acetyl-α-Tubulin at Lys40 (mAb 5335) and anti-α-Tubulin (mAb 3873) were purchased from Cell Signaling (Beverly, MA, USA). HDAC6 antibody (H-300) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), β-actin antibody (MA5–11869) was purchased from Thermo Fisher Scientific.

C1A was synthesized in house,^{13 (link)} SAHA and tubastatin A were purchased from Cayman Chemical (Ann Arbor, MI, USA). API-2 was from obtained from Tocris (Abingdon, UK). BEZ235 was from LC Laboratories (Woburn, MA, USA). Cycloheximide, actinomycin D and LY29004 were from Calbiochem (San Diego, CA, USA). Okadaic acid was purchased from Cell Signalling (Danvers, MA, USA). Fumitremorgin C, MK-571, vinblastine, rapamycin, wortmanin and LY29004 were from Sigma (St. Louis, MO, USA).

Patient-derived GNS166 and GNS179 stem cell lines, kindly provided by Dr. Steven Pollard^{22 (link)}, were cultured in GSC medium consisting of DMEM/F-12 (Sigma) supplemented by N2, B27 (Fisher), glucose (Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin and growth factors (20 ng/ml basic fibroblast growth factor (bFGF) and 20 ng/ml epidermal growth factor (EGF)) (Sigma). Glioma-cell lines U87-MG, U373-MG, U251-MG, A172, and T98-G were purchased from the ATCC and cultured in DMEM (Gibco), supplemented with 10% FBS (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin for monolayer cultures and in GSC medium for oncosphere studies as previously described^{23 (link)}. All cells were maintained at standard conditions of 37 °C and 5% CO₂ in humidified atmosphere. TMZ (Sigma), Vorinostat (Suberoylanilide hydroxamic acid, SAHA) (Cayman), Tubastatin A (Cayman) and our candidate compound were dissolved in DMSO. Information about structure, synthesis and characteristics of the novel HDAC inhibitor can be found in ref. ^{20 (link)}. The name was changed to JOC1 for strategy purposes.