Copper sulfate

Poly(d,l-lactic-co-glycolic acid (PLGA: 50/50, Mw: 33,000 Da) was purchased from Birmingham Polymers, Inc. (Birmingham, AL, USA). Poly(vinyl alcohol) (PVA, 87–89% hydrolyzed, Mw: 85,000–124,000 Da), Dex, dimethyl sulfoxide (DMSO), maleic anhydride, fluorescein (FI), IR-783, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride (DMTMM), sodium azide, propargyl amine, copper sulfate, ascorbic acid, and pyridine were bought from Sigma-Aldrich (St. Louis, MO, USA). Ethyl acetate, acetonitrile (ACN), toluene, and ether were used as received from Samchun (Gyeonggi, Korea). Dimethylformamide (DMF) were acquired from Junsei Chemical Co. (Chuo-ku, Tokyo), whereas methyltetrazine-PEG4-amine (TET) and trans-cyclooctene-amine (TCO) were from Click Chemistry Tools (Scottsdale, AZ, USA). Pluronic F-127 was used as received from BASF SE (BASF, Ludwigshafen, Germany). All other chemicals were of analytical grade and used without further purification. HA (1.0 MDa) was used as received from Humedix (Gyeonggi, Korea).

Casamino acids and yeast extract were purchased from Merck A/S (Hellerup, Denmark). The following chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA): ammonium acetate, acetic acid, glucose, potassium phosphate, methylamine, ammonium sulfate, magnesium sulfate, ferric chloride, manganese(II) chloride, zink sulfate, copper sulfate, vanadyl sulfate, sodium molybdate, sodium tetraborate decahydrate, calcium chloride, and saccharose. Methanol and Chloroform (both HPLC-grade) were purchased from Rathburn (Walkerburn, Scotland). Main polar lipid (Hex-GDGT-PG) from Thermoplasma acidophilum was purchased from Matreya LLC (State College, PA, USA) and internal lipid standard PI 17:0/14:0 and IS PE O-20:0/O-20:0 were purchased from Avanti Polar lipids (Alabaster, AL, USA).

Copper sulfate, copper bisglycinate, 2-Thiobarbituric acid (TBA), 5, 5′-Dithiobis (2-nitrobenzoic acid) (DTNB), sulfosalicylic acid, and propionaldehyde diethyl acetal were purchased from Sigma Company (St. Louis, MO, USA). Copper proteinate was obtained from a commercial premix company (Bioplex^®, Alltech Inc., Nicholasville, KY, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12) was obtained from Hyclone (Logan, UT, USA). A BCA TM Protein Assay Kit was obtained from Pierce (Rockford, IL, USA). A Protein G agarose column was obtained from Upstate Biotechnology (Lake Placid, NY, USA). ELISA plates (96 wells) and other cell culture plastic wares were obtained from Costar (Cambridge, MA, USA). HPLC grade methanol and acetonitrile were obtained from Fisher Scientific International (Hampton, NH, USA). HPLC grade formic acid and ammonium acetate were purchased from Dima Technology (Richmond Hill, Canada). Water used was ultrapure water (Milli-Q, Millipore Corporation, Billerica, MA, USA).

All chemicals were of analytical reagent grade. Deionized water (18.2 MΩ cm, Labconco, USA), LC-MS-grade methanol, and acetonitrile (MeCN) from Sigma (Milwaukee, USA) were used throughout.
The following Sigma-Aldrich reagents were used: potassium dichromate (Cr(VI)), formic acid, nitric acid, hydrogen peroxide, and sodium hypochlorite. Stock standard solution of chromium (1000 mg/L) was from Sigma and inductively coupled plasma mass spectrometry (ICP-MS) internal standard mix was from Agilent Technologies.
Hoagland's nutrient solution containing calcium nitrate 0.35 mM, calcium chloride 2.1 mM, magnesium sulfate 0.91 mM, monobasic potassium phosphate 0.97 mM, potassium nitrate 1.22 mM, boric acid 23 μM, manganese chloride 3.9 μM, molybdenum trioxide 23 μM, ferric nitrate 10 μM, zinc nitrate 0.6 μM, and copper sulfate 0.44 μM, pH 5.8, was prepared from Sigma reagents [22 ].
Sunflower seeds (Helianthus annuus L.) were purchased at a local garden market as a product of Vita company, distributed in Mexico by Rancho de Molinos, S.A. de C.V.

For hair cell development analysis, the embryos at 5 dpf were used for hair cell staining and counting. For hair cell regeneration analysis, the embryos at 5 dpf were exposed to 10 μM of copper sulfate (Sigma, Cat# 451657) for 2 h, allowed to recover for 48 h except when otherwise indicated, and then used for hair cell staining and counting. Hair cell staining by YO-PRO-1 (Thermo Fisher Scientific, Cat# Y3603) was done as previously described [18 (link)]. The stained embryos were oriented for lateral views in a 96-well plate for counting and imaging with a fluorescent microscope. Hair cells per neuromast presented in graphs were obtained by averaging the hair cell counts from four neuromast per embryos at the P1, P2, P4, and P5 positions [45 (link)] from multiple embryos. P2 neuromast was not counted, since it had a greater variation in the number of hair cells. For studying genotype and phenotype correlation, 24–32 embryos obtained from a single pair of heterozygote incross were analyzed and then genotyped. For studying the effect of chemical treatment, the number of wild-type embryos used for each data point was 10, except when otherwise indicated.

Hair cell staining and counting were as described.^{63 (link)} For analysis of hair cell development, WT or mutant embryos at 5 dpf were placed in a cell strainer (BD Falcon, San Jose, CA, USA) and stained with 2 μmol/l YoPro-1 (Life Science) for 5–15 min and then lateral line neuromast hair cells were counted using fluorescent imaging (inverted Zeiss Axiophot, ×10 magnification, Bethesda, MD, USA). For hair cell sensitivity analysis, WT or mutant embryos at 5 dpf were treated with the ototoxic drugs copper sulfate (Sigma) at 10 μmol/l for 30 min or neomycin (Sigma) at 200 μmol/l for 30 min, and then were immediately stained with YoPro-1 and used for hair cell counting. For hair cell regeneration analysis, WT or mutant embryos at 5 dpf were treated with the ototoxic drugs copper sulfate at 10 μmol/l for 2 h or neomycin at 200 μmol/l for 1 h, allowed to recover for 2 days and then regenerated hair cells were counted at posterior lateral line positions of P1, P2, P4 and P5.^{64 (link)} For each of the above experiments, ~10 embryos were used for the analysis and repeated three times. Hair cells from four neuromasts in each embryo were counted. The average number of hair cells and the s.e.m. were presented in the graphs.