Tetrac

In all pharmacological assays one-cell stage embryos were placed in glass Petri dishes (50–80 embryos per dish) containing either a specific drug or 5×10⁻⁶ M NaOH diluted in zebrafish water for control groups. The exposure medium (25 ml per dish) was exchanged twice a day. Stock solutions of 100 µM T3, T4, TETRAC, TRIAC (Sigma-Aldrich, St. Louis, MO) and DITPA (Santa Cruz Biotechnology, TX), were prepared in 0.05 M–0.1 M NaOH and diluted in zebrafish water to the final administered concentrations. In order to choose the appropriate working dilution for each substance, a preliminary dose-dependent assay was performed using WT embryos. Four to five different concentrations in the range of 0.5 nM to 100 nM were tested for each substance. In addition, a control group of embryos was raised in 5×10⁻⁵ M NaOH, the highest NaOH concentration applied to the treated groups. During the experiments, embryos were screened for morphological developmental abnormalities, such as distorted body shape, pigmentation defects, and movement disabilities. The highest substance concentration that lacked morphological defects (0.5 nM for T3, T4, TETRAC, TRIAC and 5 nM for DITPA) was chosen as the working dilution for all pharmacological experiments.

Normal or fibrotic ECMs were prepared as described in Section 2.4. DF were cultured on the ECMs for 48–72 h with or without 0.5 μM tetrac (T3787, Sigma Aldrich, Rehovot, Israel) dissolved in dimethyl sulfoxide (DMSO)-KOH propylene glycol 1 mM (1% DMSO). As a control, the cells were cultured for the same period in medium with or without DMSO-KOH in the same concentration as the tetrac (1:200,000). Cells were later harvested and analyzed for the following: (1) cell number and death (using an automatic cell counter and trypan-blue); (2) levels of collagen-I, elastin, αSMA, cyclin-D1, αv, β3, phospo-SMAD3, and D3 (using protein extraction and Western blot); (3) the activity of secreted MMPs (using gelatin zymography); and (4) the level of hsa-21-5p (using RNA extraction and qPCR; hsa-16-5p served to normalize results).

Tetrac and triac were obtained from Sigma-Aldrich (Steinheim, Germany). T1AM was purchased from ABX, Radeberg, Germany. Z-VAD-FMK was purchased from Enzo life sciences (Laufen, Switzerland). For long incubation periods (days), the derivatives were added daily to existing media. Primary anti human antibodies against p21 (#2947), p27 (#3686), CycD1 (#2978), total/cleaved caspase 3 (#9665), PARP-1 (#9542) were from Cell Signaling technology (Leiden, The Netherlands). AIF (AB16501) and phycoerythrin (PE) conjugated αvβ3 antibody (clone LM609, MAB1976) were from Merck Millipore (Darmstadt, Germany). phospho ATM antibodies (phospho-serine1981, #2152-1) were from Epitomics (Burlingame, CA, USA). For protein loading normalization ponceau S solution (Sigma-Aldrich, Steinheim, Germany) or anti tubulin antibody (#2128, Cell Signaling technology) were used.