Ethanol solution

P12 control and Gdf5 KO tibias were dissected, fixated overnight in 4% PFA in PBS, and dehydrated to 100% ethanol (dehydration sequence: 25%, 50%, 70%, and twice in 100% for 1 h each). According to ref. ^{97 (link)}, samples were soaked in 2% iodine in 100% ethanol solution (Sigma) for 48 h at 4 °C. Tissue was washed in 100% ethanol twice for 30 minutes each prior to scanning. Samples were then mounted in 100% ethanol and scanned by MicroXCT-400 (Xradia) at 30 kV and 4.5 W with the Macro-70 lens.

2,6-DCBQ (CH₃(CH₂)₃COCH(CH₃)₂, CAS: 13019-20-0, purity ≥ 98%) was purchased from ThermoFisher Scientific Co., Ltd. (Waltham, MA, USA). Ethanol solution (Sigma-Aldrich, Saint Louis, MO, USA) was used to make 10 g L⁻¹ stock solution and frozen at −80 °C before use. Different concentrations of test solutions were prepared by dissolving 2,6-DCBQ stock solution into 0.9% saline solution. The reagent kits used in biochemical, and assays were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The primers, reagents and kits for gene expression measurement were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and TaKaRa Co., Ltd. (Dalian, China).

Synthesis of DF-CMK-3: diclofenac sodium (Sigma-Aldrich, St. Louis, MO, USA) and distilled water (Merck, Darmstadt, Germany) were used during synthesis.
Electrochemical measurements: Appropriate volumes of 1 M CH₃COONH₄ and 1 M HCl (Sigma-Aldrich, St. Louis, MO, USA) were used to obtain 1 M buffer solution (CH₃COONH₄, CH₃COOH, and NH₄Cl) with a pH of 5.6. The standard solutions of Fe³⁺, Cd²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Sb³⁺, Pb²⁺, NO₂⁻, NO₃⁻, Cl⁻, and Triton X-100 (Merck, Darmstadt, Germany) were used to study the selectivity of the proposed procedure, and 96% ethanol solution (Merck, Darmstadt, Germany) was used to prepare 10 and 1 mM roxarsone (ROX, AK Scientific, Union City, CA, USA). The ROX solution with a concentration of 0.01 mM was prepared daily in 0.1 M PBS buffer with a pH of 7.2. The wastewater samples were obtained from the Municipal Water Supply & Waste Water Treatment Company Ltd. (Lublin, Poland), while river water samples were collected from the Vistula River (Sandomierz, Poland). The ROX spiked samples were filtered (0.22 µm Millipore filter).

Ultrapurified water (>18 MW cm, Milli-Q system, Millipore, UK) was used to prepare the solutions. To prepare 10 and 1 mM solutions of roxarsone (AK Scientific, Union City, CA, USA), 96% ethanol solution (Merck, Darmstadt, Germany) was used. Sodium dodecyl sulphate (SDS), Triton X-100 and cetyltrimethylamonnium bromide (CTAB) were obtained from Merck. An acetate buffer solution (NaAc–HAc) made with CH₃COONa and CH₃COOH (Merck, Darmstadt, Germany) of pH 5.6 was used for analysis of ROX. For interference studies, standard solutions of Fe³⁺, Ni²⁺, Pb²⁺, Cd²⁺, Zn²⁺, Sb³⁺, Cu²⁺, NO₂⁻, NO₃⁻, Cl⁻ and PO₄³⁻ (Merck, Darmstadt, Germany) were used. River water samples from the Bystrzyca River (Lublin, Poland) were spiked with appropriate concentrations of ROX and filtered using a 0.22 µm Millipore filter.
An µAutolab analyzer with GPES 4.9 and FRA 4.9 software (Eco Chemie, Utrecht, Netherlands) was applied to perform the electrochemical experiments. The CTAB-modified GCE (diameter of 1 mm), Ag/AgCl (3M KCl) and Pt wire were used as the working, reference and auxiliary electrodes, respectively. The electrode surface cleaning was performed using silicon carbide paper (SiC-paper, #2000, Buehler, Skovlunde, Denmark), alumina particle suspension (0.3 µm), a Buehler polishing pad and an ultrasonic bath (InterSonic, model IS-2, Olsztyn, Poland).

Cells were seeded in six-well plates (1.0 × 10² cells/well) and cultured in M10 medium for 15 days. For migration and invasion assays 5 × 10⁴ cells/chamber suspended in MEM were added to the upper compartments of the Transwells with 8 μm pores (Corning Incorporated, Corning, NY, USA). M10 was added to the wells for chemotactic attraction, followed by incubation of the plates for 12 h for migration and 36 h for invasion assay. Cell invasion was assessed by their ability to transpose both a Matrigel^® coat (BD Biosciences, San Jose, CA, USA) and barrier (Corning Incorporated, USA). Colonies, migrating and invading cells were fixed with 70% ethanol solution (Merck Millipore, Darmstadt, Germany), stained with 0.5% crystal violet (ThermoFisher, Waltham, MA, USA) in 10% ethanol solution and counted. For anchorage independent growth assay, 2.5 × 10² cells were suspended in 500 μL of M10 medium with 0.6% agarose and seeded in 24-well plates coated with the 1% agarose, and covered with M10 medium. After 30 days 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5mg/mL) was added for staining and counting of colonies.