Ouabain octahydrate

Ouabain octahydrate (Sigma–Aldrich, Cat. No. O3125-250MG) was dissolved in water. Cells targeted at the ATP1A1 locus and being tested for small molecule or culture conditions were treated 48 h after EP with 1 mM Ouabain in StemFit AK02N medium supplemented with ROCK inhibitor until day 6, in order to selectively obtain HDR-edited colonies. Cells were then maintained normally with StemFit AK02N medium until day 13, and forming colonies were stained with Crystal Violet. Tissue culture plates were placed on ice, washed twice with ice-cold 1X DPBS, fixed with ice-cold methanol for 10 min, then cells were removed from the ice and incubated with 0.05-0.1% Crystal Violet (Nacalai, Cat. No. 09804-52) in 25–100% methanol for 10 min at RT. Excess Crystal Viotel was removed by washing with water, and the plates photographed for manual colony counting.

Ouabain octahydrate (catalog number O3125, Sigma-Aldrich, Oakville, ON), digoxin (catalog number D6003, Sigma-Aldrich), and oleandrin (catalog number 06069, Sigma-Aldrich) were initially dissolved in water or dimethylsulfoxide (DMSO), and further diluted in cell culture media to 1000 x working concentrations prior to treatments. For treatments of differentiated ReN VM cells spanning several days, CG levels were replenished daily along with a change of half of the cell culture medium.

MMCT was performed as described previously^{27 (link)}. The 10MAC2-HR3 cells were transferred to CHO (Hprt^−/−) cells. The CHO cells fused with DT40(10MAC2-HR3)-derived microcells were selected with 8 µg/mL of BS and ouabain octahydrate (Sigma-Aldrich), then we obtained BS-resistant and EGFP-positive clones. The clones were identified by PCR and FISH analysis.

Sprague-Dawley rats (230∼270 g) and C57BL/6 mice (25∼30 g) were purchased from Sino-British SIPPR/BK Laboratory Animals (Shanghai, China). α7^-/- mice were generated and genotyped by PCR analysis as described previously (Liu et al., 2009 (link)). Animals were housed at 22°C under a 12/12 light schedule (on: 08:00), with free access to tap water and standard rat chow. All experimental procedures were in accordance with institutional animal care guidelines and approved by ethics committee of Second Military Medical University. Ani (Ani hydrochloride: C₁₇H₂₄NO₄) was purchased from Fu-Ma Chemical and Engineering Company (Hangzhou, China). Mecamylamine hydrochloride, methyllycaconitine (MLA) citrate, hexamethonium chloride, ACh chloride, nicotine, PNU282987 (PNU), static, ouabain octahydrate, potassium chloride, and antibody against α7nAChR were purchased from Sigma–Aldrich (St. Louis, MO, USA). HNMPA-(AM)3 and antibody against p-Na/K-ATPase were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). antibody against insulin receptor, Alexa-488-labeled and Cy3-labeled second antibodies, and DAPI were purchased from Abcam (Cambridge, MA, USA). LY 294002 and rapamycin were purchased from Merck Millipore (Darmstadt, Germany).

3, 3′-dithiobis sulfosuccinimidylpropionate (DTSSP) and beta-D-Lactose were purchased from Thermo Scientific (Pittsburgh, PA). Sucrose was purchased from MP Biomedicals (Solon, OH). Ouabain octahydrate, cis-diammineplatinum dichloride (CDDP), doxorubicin hydrochloride (DXR) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Pyrazolo pyrimidine (PP2), a Src kinase inhibitor, was purchased from Tocris Bioscience (Bristol, UK).
Customized polyclonal rabbit anti-Gal-3 antibody was created by Pierce Biotechnology; mouse anti-V5 and purified mouse IgG were purchased from Invitrogen; polyclonal goat anti-Na⁺/K⁺-ATPase alpha1 and monoclonal mouse anti-Mdr-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal rabbit anti-Mdr was purchased from Oncogene Research Products (Cambridge, UK); mouse anti-beta-actin was purchased from Sigma-Aldrich; rabbit anti-Src and anti-p-Src were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA); monoclonal mouse anti-phosphoserine was purchased from abcam (Cambridge, MA).

E11.5 mouse embryos were dissected from timed pregnancies (Control line females). Fore- and hind limb buds were dissected on ice in Puck’s Saline A (PSA), and mesenchymal cells were dissociated in dispase (Dispase II, Sigma Aldrich) in chick serum at 37°C in an orbital shaker for 30–45 min. Cells were then resuspended in medium containing 36% Dulbecco’s Modified Eagle’s Medium (DMEM), 53% Ham’s nutrient mixture F12, 10% fetal bovine serum (FBS), 0.5% L-glutamine and 0.5% streptomycin-penicillin, and plated at high density (2 × 10⁷ cells/mL) in 10 µl droplets in 6-well culture plates (7 droplets/plate), with daily media changes as previously described (Stanton et al., 2004 (link); James et al., 2005a (link); Woods et al., 2005 (link)), On day 3, media was supplemented with β-glycerophosphate and ascorbic acid to achieve final concentrations of 1 and 0.25 mM, respectively. Serial dilutions of ouabain (ouabain octahydrate, Sigma-Aldrich) or control vehicle (ddH₂O) were introduced at 6, 8 or 10 days to the medium to achieve final concentrations of 1 µM, 10 µM, 100 µM and 1 mM ouabain. These time points were chosen to determine the impact of NKA pump inhibition at different stages of chondrocyte differentiation. Micromasses were harvested at selected time points for RNA extraction or histology (6, 8, 10 and 12 days post-plating, day 15 for histology).