Ym 244769

Ouabain octahydrate (catalog number O3125) and Calpain Inhibitor I (catalog number A6185) were purchased from Sigma-Aldrich and dissolved in water and DMSO respectively to generate stock solutions with 1000 X working concentrations prior to the treatment. YM244769 was sourced from TOCRIS (catalog number 4544) and dissolved in DMSO. The treatments were renewed daily for the indicated duration.
When specified, differentiated ReN VM cells were treated with CGs for 3 or 7 consecutive days with daily renewal of half of differentiation media plus the inhibitors. When applicable, Calpain Inhibitor I or YM244769 were added 2h before the initial ouabain treatment at a final concentration of 20 μM and replenished daily along with half media changes on differentiated ReN VM cells.

The cell counting kit-8 (CCK8) and Annexin V-FITC/PI apoptosis assay kit were purchased from DOJINDO LABORATORIES (Tokyo, Japan). Bepridil (Cat# 4117), KB-R7943 (Cat# 1244), YM-244769 (Cat# 4544), SN-6 (Cat# 2184), and [1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM)] (Cat# 2787) were from Tocris Bioscience (Minneapolis, USA). The BRAF inhibitor vemurafenib (Cat# HY-12057) were from MedChemExpress (Monmouth Junction, NJ, USA). These compounds were dissolved in dimethyl sulfoxide (DMSO) to make stock solution. Propidium iodide (PI) and Ribonuclease A were from Sigma Aldrich (St. Louis, MO, USA). Cell-permeable Ca²⁺ indicator Fluo-4 AM was from Invitrogen Life Technologies (Waltham, MA, USA). The primary antibody for NCX1 isoform (Cat# ab177952) was from Abcam (Cambridge, MA, USA); anti-NCX2 (Cat# ANX-012) and NCX3 (Cat# ANX-013) antibodies were from Alomone Labs (Jerusalem, Israel). Mouse α-tubulin antibody (Cat# 2144) was from Cell Signaling Technology (Boston, MA, USA).