Tubacin

A full list of the antibodies used in this study is provided in S1 Table. Thymidine, propidium iodide, psoralen, nocodazole, monastrol, ciliobrevinD, MG132, cycloheximide, tubacin and sodium butyrate were purchased from Sigma-Aldrich (St Louis, MO, USA). Cytochalasin B was purchased from AppliChem GmbH (Darmstadt, Germany). Lactacystin and purvalanol were purchased from Santa Cruz Biotechnology, Inc. UBEI-41 was acquired from Biogenova, USA.

50,000 J-Lat 10.6 and JNLGFP cells were aliquoted into 96-well plates in 200 μL medium. Each of the following drugs were added to the cells in triplicate at the indicated concentrations: BMH-21 (MedKoo, 406586), RA190 (Calbiochem, 5.30341.0001), ERW1041E (Sigma, 509522), ZDON (Calbiochem, 616467), ZDON (Calbiochem, 15227), tubacin (Sigma, SML0065), b-AP15 (Cayman chemical, 11324; AdooQ Bioscience, A15391; MedKoo, 406452), capzimin (Glix labs, GLXC-09966), thiolutin (MedKoo, 525293; Cayman chemical, 11350), WP1130 (Cayman chemical, 15227), P005091 (MedKoo, 406577; Cayman chemical, 15224), IU1 (AdooQ, A13209; Cayman chemical, 10617), TCID (Cayman chemical, 16353; Sigma, SML1402), acitretin (Sigma, 44707), prostratin (Sigma, P0077), ingenol (Sigma, SML1318), vorinostat (Sigma, SML0061), JQ1 (Sigma, SML1524), I-BET151 (Tocris, 4650), 5-azacytidine (Sigma, A3656), romidepsin (Sigma, SML1175), PR619 (AdooQ Bioscience, A13190). For control groups, 0.1% DMSO was used. After 48 or 72 h of treatment, GFP expression in the cells were measured by flow cytometry. Cell viabilities were determined by forward scatter vs. side scatter gating using untreated cells as the control. The data were analyzed with FlowJo software and plotted as bar graphs.

Tubacin (Sigma-Aldrich, #SML0065) at 2 μM in DMSO; dibucaine hydrochloride (Sigma-Aldrich #285552) at 190 μM in DMSO. The following were used at 1 μM in DMSO: Cytochalasin D (Sigma-Aldrich #PHZ1063), Thapsigargin, (Sigma-Aldrich #T9033). BAPTA-AM (Sigma-Aldrich #A1076). Ionomycin 10-mM stock was a gift from the Rich Lewis Laboratory, Stanford University.

Transfections of various plasmids were done by the use of Polyethylenimine (PEI MAX 40000) (Polysciences, Inc.). In MCF7, transfection was performed at 70–80% confluency and the DNA:PEI ratio used for transfection was 1:3. In MDA-MB-231, reverse transfection was performed in which DNA: PEI mix was added before cell seeding. Tubacin (Sigma), a selective HDAC6 inhibitor, was used at 5 μM concentration for 5 h for optimum HDAC6 inhibition and an equal amount of DMSO was used as vehicle control.

BZ was purchased from Selleck Chemicals (Houston, TX, USA). CAM was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), SAHA was from Cayman Chemical Co. (Ann Arbor, MI, USA), and tubacin was from Sigma-Aldrich (St. Louis, MO, USA). BZ, SAHA and tubacin were dissolved in dimethyl sulfoxide to make stock solutions at concentrations of 1, 10 and 1 mM, respectively. CAM was dissolved in ethanol to prepare stock solutions of 5 mg/ml.

All animal procedures were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Use and Care Committee of Zhongshan Ophthalmic Center at Sun Yat‐Sen University. Whole lenses of 21‐day‐old Wistar rats were cultured in 4 mL serum‐free M199 medium with 0.1% BSA, 0.1 µg/mL l‐glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. Different concentrations of Tubacin (SML0065; Sigma‐Aldrich) were added to the medium with or without 5 ng/mL TGF‐β2 for up to five days. On Day 5, all lenses were fixed with 10% formaldehyde and embedded in paraffin for further immunofluorescence analysis.