Ethylene glycol tetraacetic acid (egta)

Osteoclasts derived from Ano1^fl/fl, Ctsk-Cre;Ano1^fl/fl, WT, and Ano1 TG mice were seeded on confocal dish and induced by RANKL for 5 days to measure intracellular Ca^{2+51 (link)}. The cells were loaded with 5 μM fluo-4, AM (Molecular Probe) for 20 min at 37 °C in Tyrode solution, then rinsed twice with Tyrode solution and mounted on the inverted stage of a confocal scope. Fluorescence excitation was performed using 488 nm laser, and detection filters were set at 530 nm. Images were acquired every 3 s and analyzed using Interactive Data Language (IDL, Research Systems) software. Cells were scanned for 20–30 s to obtain F_resting (F), then replaced the solution with 0 Ca²⁺ Tyrode solution including 4 mM EGTA (Invitrogen), 5 μM thapsigargin (Molecular probes), and 10 μM A23187 (Sigma). Stored calcium was released to the cytoplasm immediately. We defined the peak value as F_{ER release}. Added 100 μM BAPTA, AM into solution to obtain F_min. Then replaced the solution with 10 mM Ca²⁺, 5 μM thapsigargin, 12 μM A23187 (Sigma), 3 μM FCCP (Sigma), and 20 mM 2-DG (Sigma) in Tyrode solution. The stable value was F_max. Finally, [Ca²⁺]_i was calibrated using the equation [Ca²⁺] = K_d×(F–F_min)/(F_max–F).

All cell lines used in this work were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured as we previously described [42 (link)].
TGF-β was purchased from R&D systems (Minneapolis, MN, USA). SKF96365 was bought from Abcam (Abcam, UK). The calcium dye Fura 2-AM and EGTA were obtained from Invitrogen (Carlsbad, CA, USA). The antibodies against ERK1/2, p-ERK1/2 were bought from Bioworld Technology (Louis Park, MN, USA) and anti-STIM1 antibody was from Abcam (Abcam, UK). The anti-GAPDH antibody was bought from KANGCHEN BIO-TECH (Shanghai, China). HRP-conjugated goat anti-rabbit antibody was purchased from Thermo Scientific (Waltham, MA, USA).

Brains were homogenized using a glass‐Teflon homogenizer (w/v = 100 mg tissue/1 ml buffer) in 250 mM sucrose, 20 mM Tris‐base pH 7.4, 1 mM EDTA, 1 mM EGTA plus protease, and phosphatase inhibitors (ThermoScientific), with all steps carried out on ice or at 4°C. Homogenates were centrifuged at 800 g for 10 min. Supernatant was collected and labeled S1 and used for Western analysis. Soluble fractions were generated by ultracentrifugation of S1 at 70 000 g for 1 hr to obtain S70 and P70. S70 was used for Western analysis of soluble APP content. Soluble fractions for ELISA were generated by solubilization of S1 with 0.1% SDS and 1% NP‐40 for 30 min rotating. Solubilized S1 was spun at 20 000 g for 10 m and analyzed by ELISA.

To induce spheroid formation, cells were cultured in a U-bottom low-adhesion 96-well plate (S-Bio, Hudson, NH, USA). All spheroid assays were performed in complete αMEM (10% FBS, 1% antibiotics). To observe spheroid fusion, spheroids were formed in separate wells for 48 h, then one spheroid was carefully transferred to the other’s well. To observe the effects of conditioned media, spheroids were formed in separate wells for 48 h, and the media was removed and replaced with conditioned media. To observe the spheroid reaction to chemical treatment, after 48 h of spheroid formation, media was removed and fresh media with the relevant chemicals were added. Conditioned media were treated with EGTA (Cat No. S311, Thermo Fisher Scientific, Waltham, MA, USA), type I collagen (Cat No. 344236, BD Biosciences, San Jose, CA, USA), or collagenase (Cat No. C9891, Sigma, St. Louis, MO, USA). Every 24 h, microscope images were taken of the spheroids to be analyzed with ImageJ. A threshold was applied and the spheroid area was identified with the “Analyze Particle” function. Area and circularity of the identified object were measured directly with ImageJ. Roughness was calculated by fitting an ellipse to the spheroid and adding the areas of the spheroid outside the ellipse and the areas of the ellipse not within the spheroid.

Rats were anesthetized with isoflurane and perfused via intracardiac catheterization with ice-cold PBS. Brains were extracted and homogenized using a glass–Teflon homogenizer (100 mg tissue/1 ml buffer (w/v)) in 250 mm sucrose, 20 mm Tris-base (pH 7.4), 1 mm EDTA, and 1 mm EGTA plus protease and phosphatase inhibitors (Thermo Scientific), with all steps carried out on ice or at 4 °C. Total lysate was solubilized with 0.1% SDS and 1% NP-40 for 30 min while rotating. Solubilized lysate was spun at 20,000 × g for 10 min, the supernatant was collected and analyzed by ELISA and Western blotting.