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3 protocols using tacedinaline

1

Immunofluorescence and Western Blotting Protocols

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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)).
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2

Cell Culture and Reagent Acquisition

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Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Invitrogen (Carlsbad, CA.). Ultrapure Lipopolysaccharide (LPS) was purified from Salmonella minnesota R595 (List Biological Laboratories). TBWCL was obtained from BEI resources. RGFP966, tacedinaline, tasquinimod, HPOB and PCI34051 were purchased from Selleckchem.
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3

Characterization of Neuroendocrine Cell Lines

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The human pancreatic NET BON1 cell line (14) was kindly provided by Prof. Rüdiger Göke, University of Marburg. The pancreatic islet tumor QGP1 cell line (14) was originally obtained from the Japanese Collection of Research Bioresources Cell Bank. The human, bronchopulmonary neuroendocrine NCI-H727 cell line (14) was obtained from the American Type Culture Collection. The human midgut carcinoid cell line GOT1 (14, 15) was kindly provided by Prof. Ola Nilsson, Sahlgrenska University Hospital. All cell lines were certified for identity by short-tandem repeat (STR) typing, performed by the German Biologic Resource Centre DSMZ. All cell lines used in this study were cultured and incubated as previously described (16) .
5-FU, decitabine, and tacedinaline were purchased from Selleckchem, dissolved in dimethyl sulfoxide, and stored at 220°C. 68 Ga-DOTATOC was prepared using SomaKit TOC purchased from Advanced Accelerator Applications SA. 68 Ga was obtained by elution of a GalliaPharm 68 Ge/ 68 Ga generator by Eckert and Ziegler.
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