Acc 003

Fresh patient samples (AM and OKC) or 21-day cultured-tumoroids were immersed in 4% paraformaldehyde (PFA) for fixation. Paraffin-embedded specimens were sectioned into 5-µm thick sections. Hematoxylin and eosin (HE) staining was performed after deparaffinization. For immunological staining of histological sections or cells, the following antibodies were used, as previously described [46 (link)]. Rabbit anti-CACNA1C (1:200, Alomone Labs, ACC-003), mouse anti-CK14 (1:500, Abcam, ab7800), mouse anti-CK10 (1:500, Invitrogen, MA5-13705), mouse anti-E-cadherin (1:500, BD Biosciences, AF748), rabbit anti-MMP-9 (1:200, Merck, AB19016), rabbit anti-LGR5 (1:200, Abcam, ab75732), mouse anti-Ki67 (1:200, Abcam, ab16667), mouse anti-PCNA (1:500, Abcam, ab29), mouse anti-NFATc1 (1:200, Santa Cruz, SC-7294), and mouse anti-β-catenin (1:500, Santa Cruz, SC-7963). Target retrieval solution (DAKO, S2369) was used for antigen retrieval prior to blocking. TO-PRO-3™ Iodide (1:1000; Invitrogen, T3605) was used for counterstaining. Images were acquired using a confocal microscope (Leica DMi8, Wetzlar, Germany). Quantification analyses of Ki67, CACNA1C and NFATc1 positive cells were performed with five images (294 × 294 μm) acquired at different culturing wells, respectively (biological replication).

Total protein was extracted from cells or left ventricular tissues. The whole operation was carried out on ice. The collected cells or the ground tissues were lysed with RIPA lysis buffer, which added protease inhibitors. Lysates were centrifuged for 15 min at 12,000 rpm under 4℃. The supernatants were used for Western blotting after determining its protein concentration by Bradford assay (Yeasen, China). The protein was separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking, PVDF membranes were incubated with different primary antibodies overnight at 4℃ or 2 h at 25℃.
Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca_V1.2 α_1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus). The secondary antibodies used were HRP-conjugated goat anti-mouse (0.02 µg/mL, SA00001-1, Proteintech) or anti-rabbit IgG (0.02 µg/mL, SA00001-2, Proteintech), or rabbit anti-goat IgG (A21030, Abbkine). We used an imaging system (Tanon 5200, China) to visualize the blot with the enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific).

Antibodies against Cav1.2 (ab84814 and ACC‐003) were from Abcam (Cambridge, UK) and Alomone, respectively, which produced identical Western blots. Those against β‐actin (ab6276), MAP‐2 (ab32454), ERα (ab3573), ERβ (ab104804), calcineurin (ab3673), Mdm2 (ab16895), and ubiquitin (ab19247) were purchased from Abcam. pSer1928‐Cav1.2 (A010‐70) was from Badrilla (United Kingdom). 17β‐estradiol (E2758), propylpyrazoletriol (PPT, H6036), diarylprepionitrile (DPN, H5915), E2‐BSA (E5630), ICI 182,780 (V900926), nifedipine (N7630), FK506 (F4679), MG132 (C2211), ammonium chloride (A0171), chloroquine (C6628), carfilzomib (791938), and other reagents were purchased from Sigma (St. Louis, MO, USA). Lipid solvents were made in stock solutions in DMSO (1:1,000 ~ 2,000).