Trichostatin a

PREX1 [D8O8D] rabbit monoclonal, PTEN [138G6] rabbit monoclonal, Phospho-PAK1 (Ser144)/PAK2 (Ser141) rabbit polyclonal, PAK2 [3B5] mouse monoclonal were purchased from Cell Signaling (Danvers, MA, USA). GAPDH [6C5] mouse monoclonal and CDC42 [M152] mouse monoclonal were purchased from Abcam (Cambridge, MA, USA). Phospho-PKCι (T555)/PKCλ (T563) rabbit polyclonal was purchased from Invitrogen (Carlsbad, CA, USA) and PKCι mouse monoclonal from (BD Transduction Laboratories (Mississauga, ON, Canada). Sox2 mouse monoclonal was from R&D Systems (Minneapolis MN, USA). Rac1[23A8] mouse monoclonal, anti-Flag M2 mouse monoclonal and Stem121 mouse monoclonal were purchased from Millipore (Temecula, CA, USA), Sigma-Aldrich (Oakville, ON, Canada) and StemCells Inc. (Newark, CA, USA), respectively. Human brain cerebral cortex protein medley was purchased from Clontech (Mountain View, CA, USA). The following inhibitors were used in the study: gallein (Santa Cruz Biotechnology, CA, USA), BKM120 (Sigma-Aldrich, Oakville, ON, Canada); trichostatin A (Cayman Chemical Company, Ann Arbor, MI, USA); haloperidol, L-741,626 and L-745,870 (Tocris Bioscience, Minneapolis, MN, USA).

Human esophageal squamous epithelial cells (keratinocytes) were a gift of Dr. Anil Rustgi (Columbia University), human cervical keratinocytes were a gift from Dr. Craig Meyers (Penn State College of Medicine), and human skin keratinocytes were purchased from ATCC. Primary keratinocytes were grown at 37 °C and 5% CO₂ in keratinocyte serum-free medium (K-SFM; ThermoFisher Scientific), supplemented with 40 μg/mL bovine pituitary extract (Invitrogen), 1.0 ng/mL epidermal growth factor (EGF; Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). The medium was changed every 24 h for 5 days. For UV experiments, cells were treated in a Spectroline UV crosslinker without filtering. Unless noted, cells were harvested 8 h after UV irradiation or hydrogen peroxide treatment. For treatment with trichostatin A (Cayman Chemical), cells were harvested after 16 h.

S. mansoni is maintained in the laboratory using the intermediate snail host Biomphalaria glabrata and as definitive host the golden hamster (Mesocricetus auratus). Cercariae were released from infected snails and mechanically transformed to obtain schistosomula in vitro [22 (link)]. Newly transformed schistosomula were maintained for 12 h in M169 (Vitrocell) medium supplemented with 2% fetal bovine serum (FBS) (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine, 1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO₂ [23 (link)], after which time the drug treatment was initiated, as described below. Adult worms were obtained from 7-week infected hamsters by left ventricular perfusion, and release of worms from the hepatic portal vein. Paired worms were maintained in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Vitrocell), penicillin/streptomycin, amphotericin (Vitrocell) at 37°C and 5% CO₂.
The parasites were treated with 1 μM Trichostatin A (Cayman Chemical), a concentration that has been shown by Dubois et al. [15 (link)] to be sub-lethal, and the negative controls with an equivalent amount of ethanol (vehicle of TSA), for 12, 24 and 48 h for microarray experiments, and for 12 h for ChIP-qPCR and western-blotting experiments.

Chemicals: Crystal Violet (Sigma), cycloheximde (Sigma), DMSO (Sigma), EdU (Invitrogen), entinostat (Selleckchem), Geneticin (Invitrogen), MG132 (Cayman), Noble Agar (Difco), puromycin (Sigma), trichostatin A (Cayman), tubastatin A (Cayman), tubacin (Selleckchem). Primary Antibodies: pH3Ser10 ab47297 (Abcam), p21/Kip1 610241 RB 554136 (BD), p53BP1ser25 A300-652A (Bethyl), cyclin D1 CC12 (Calbiochem), ac- α-Tubulin #5335, GAPDH #2118, HDAC6 #7612, PARP #9542, Ph-p53Ser15 #9284, pHistone H2A.X Ser139 #2577, pChk2Thr68 #2197, pERK1/2 Thr402/Tyr404 #4370, pRbSer780 #9307, pRbSer807/811#9308 (Cell Signaling Technology), FGFR3 sc-123, c-MYC sc-764, cyclin A sc-751, cyclin B1 sc-752, cyclin D2 sc-754, cyclin E sc-481, cyclin E2 sc-28351, ERK1 sc-94 (Santa Cruz Biotechnology), pATMSer1981 #05-740, ac-Histone H3 #06-599, ac-Histone H4 #06-866 (Upstate).

HMC-1.1 and HMC-1.2 cells were either left untreated or were treated with the indicated concentrations of NaBu, trichostatin A (TSA; Cayman Chemical, Ann Arbor, MI, United States), suberoylanilide hydroxamic acid (SAHA; Cayman Chemical), or DMSO (0.0004% final) for four days. The cells were collected in the culture media, pelleted at 200  ×  g for 5 min, washed with HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 5.6 mM Na₂HPO₄, 1.8 mM CaCl₂, and 1.3 mM MgSO₄ at a pH of 7.4), and pelleted again at 200  ×  g for 5 min. The cells were resuspended in HEPES buffer to a concentration of 1  ×  10⁶ cells/ml and 0.4–1  ×  10⁵ cells were lysed using 0.05% Triton X-100 for 20 min on ice. In a black microplate, 30 µl cell lysate and 30 µl HEPES buffer or 60 µl of histamine dihydrochloride standards in HEPES buffer (7.8–500 ng/ml; Sigma-Aldrich) were added to duplicate wells, combined with 12 µl 1M sodium hydroxide and 2 µl 10 mg/ml o-phthalaldehyde (Sigma-Aldrich) in methanol, and incubated for 4 min at room temperature. Subsequently, 6 µl 3M hydrochloric acid was used to stop the reaction and the fluorescence intensity was measured using 360 nm (excitation) and 450 nm (emission) settings with a BioTek Synergy H1M plate reader (Winooski, VT, United States). Lower limit of detection for this assay is approximately 5–7 ng/ml (34 (link), 35 (link)).