Ars solution ph 4.2

For the validation of the VC cellular model, primary RVSMCs were seeded in a 12-well plate. When cells reached about 80% confluence, 2 mM Pi was treated for 6 h, 3 days, and 6 days, respectively. For the confirmation of the effects of circSamd4a knockdown or its overexpression, A10 cells were seeded in a 24-well plate. After a day, cells were treated with either siRNA or overexpression vector. Then, 4 mM Pi was treated for an additional 3 days. For the ARS staining, cells were washed with PBS and fixed with 10% formalin at room temperature for 1 h. After removing 10% formalin, cells were washed three times with distilled water. When the samples were exposed to air and dried, 40 mM ARS solution (pH 4.2) (Sigma) was treated, and the samples were incubated overnight with rotation at room temperature. After removing the ARS solution, samples were washed with distilled water and PBS.
For ARS staining quantification, 300 μL 10% cetylpyridinium chloride (Sigma) was added to samples and incubated for 30 min. Then 200 μL dissolved solution was used to measure their absorbance at 562 nm by utilizing a microplate spectrophotometer (BioTek Instruments).

We verified the effects of both circSmoc1-2 knockdown and overexpression using A10 cells cultured in 24-well plates. Cells with 40% confluence were transfected with either 30 nM siRNA or 0.25 μg overexpression vector. After 1 day, cells were treated with 4 mM Pi for 3 days. The cells were washed in PBS, fixed in 10% formalin for 1 h, washed three times with deionized water, and air-dried. These samples were then treated with 40 mM ARS solution (pH 4.2) (Sigma-Aldrich) and kept at 20°C–25°C. Then the ARS solution was removed, and samples were washed using PBS and deionized water. ARS staining was then quantified by dissolving the ARS precipitate in 10% cetylpyridinium chloride (Sigma-Aldrich) and evaluating the absorbance of this solution at 562 nm using a microplate spectrophotometer (BioTek).