2 apb

To inhibit Gli1 expression, GANT61 (HY-13901; MedChemExpress, South Brunswick, NJ, USA) was dissolved according to the manufacturer's instructions and was intraperitoneally injected at 40 mg/kg of body weight every second day until the mice were sacrificed. For in vitro experiments, 10 μM GANT61 was administered for approximately 6 days after incubation of primary cells. To induce Cre activity, tamoxifen (T5648; Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally injected at 100 mg/g of body weight for 4 consecutive days. Follow-up experiments were performed 7 days after this treatment (Figure 3(a)). Alizarin complexone (A3882; Sigma-Aldrich) and calcein (C0875; Sigma-Aldrich) used to label newly formed bone were intraperitoneally injected at 100 mg/g of body weight on the day before rapid maxillary expansion (RME) and sacrificing, respectively. To inhibit the function of IP₃R, 2-aminoethyl diphenylborinate (2-APB; 3170846; Merck Millipore, Darmstadt, Germany) was applied at a concentration of 70 μM, 24 h after incubation.

Cells were pretreated with or without NMII kinase inhibitors, Y27632 (10 μM, EMD Millipore), ML-7 (10 μM, EMD Millipore) and PIK (100 μM) or Ca²⁺ inhibitors, 2APB (10 μM, EMD Millipore) and BAPTA (50 μM, EMD Millipore) for 1 h, and incubated with GC in the presence or absence of the inhibitors for 6 h. Cell were washed and fixed with 4% paraformaldehyde, permeabilized with 1% Triton X100, and stained with anti-E-cadherin (BD Bioscience), anti-pMLC (Cell Signaling Technology, Carlsbad, CA), anti-GC antibodies, and DAPI for nuclei. Cells were analyzed by CFM. Images were acquired as Z-series of 0.37 μm slices, and 3D composites obtained. Epithelial exfoliation will be quantified using xz images as described for the tissue explants. The distribution of E-cadherin and pMLC was quantitatively analyzed by measuring the FIR at the apical junctional to the cytoplasmic area (from xy images) or at the apical to lateral surface area (from xz images) in individual cells.

We used the drug 2-Aminoethoxy-diphenylborate (2-APB) (EMD Millipore, Billerica, MA, USA) that blocks IP₃ receptors. 2-APB was first dissolved at 100 mM in DMSO, and then diluted to 30 μM in buffer solution. Cells were allowed to preincubate in 2-APB for 10 minutes.
Ryanodine (20 μM), PA (50 μM), Nifedipine (10 μM).

HIV-1-infected T cells were preincubated with 100 μM 2-aminoethyl diphenylborate (2-APB) (Merck), 5 μM oligomycin (Sigma), 1 μM nocodazole (Sigma), 50 μM Mdivi (Sigma), 10 mM EGTA (Fluka), 10 μM BAPTA-AM (Life Technologies), or 0.5% dimethyl sulfoxide (DMSO) as a control for 30 min prior to mixing with target T cells. 2-APB was removed by washing after treatment of HIV-1-infected cells. Infected cells were then mixed with target cells and processed for immunofluorescence microscopy as described above. Cell-free virus production was quantified with an enzyme-linked immunosorbent assay (ELISA) to measure p24 (35 (link)). Viability and ATP concentration of the treated cells were measured by trypan blue exclusion (Life Technologies) and CellTiter-Glo (Promega), respectively.

All chemicals and solvents were used as supplied. SKF96365 (1), CAI (2), 2-APB (3), Pyr2 (4), Pyr3 (5), Synta-66 (6), RO2959 (7) AnCoA4 (10), leflunomide (15), Gd^3+, (18) and La³⁺ (19) were purchased from Sigma-Aldrich (UK). CM4620 (14) was purchased from MedChemExpress (Insight Biotechnology Ltd, UK). Teriflunomide (16) was purchased from APExBIO (UK). MRS1845 (11) was purchased from Santa Cruz Biotechnology (Insight Biotechnology Ltd, UK). Compounds that were not commercially available were synthesised. CM3457 (13) was synthesised by Dr Rajendra Gosain (unpublished). JPIII (17) was synthesised as previously reported [31 (link)]. The synthetic route for GSK7975A (9) and GSK5503A (8) largely followed the patent protocol and is summarised in S2 and S3 Figs, respectively [66 ]. Synthesis of the 7-azaindole series compound (12) (S4 Fig) loosely followed the patent protocol [67 ]. Detailed chemistry methods for compound syntheses can be found in S1 Appendix and NMR data for all compounds and intermediates synthesized can be found in S5–S17 Figs.

VSMC calcification was induced by incubation with a calcifying medium, which consisted of growth medium supplemented with 10 mM β-GP (Sigma-Aldrich). A high-magnesium medium was produced by adding MgSO₄, with a final Mg²⁺ concentration of 3 mM. 2-APB (Sigma-Aldrich), the TRPM7 inhibitor, was added to reach a final concentration of 10⁻⁴ M. Following incubation for 14 days, cells were washed twice with phosphate-buffered saline (PBS; Beijing Solarbio Science & Technology Company Co., Ltd., Beijing, China) and fixed with 95% ethanol. The cells were exposed to 0.2% Alizarin red (pH 8.3; Beijing Solarbio Science & Technology Company Co., Ltd.). Subsequent to washing with PBS, the cells were visualized and images were captured to record the incidence of induced calcification by an inverted phase contrast microscope (type LH50A; Olympus Corporation, Tokyo, Japan). The software used to capture images was NIS-Element F3.0 (Olympus Corporation). Subsequently, calcium deposited in the extracellular matrix was extracted with 0.6 M HCl for 24 h at 37°C. The calcium content in the supernatant was measured with the o-cresolphthalein complexone method using a Calcium Assay kit according to the manufacturer’s instructions (BioSino Biotechnology & Science, Inc., Beijing, China) and normalized relative to the protein concentration of the same culture.