Di 8 anepps

Nonactin, KCl, HEPES, phosphate-buffered solution (PBS), EDTA, pentane, ethanol, calcein, triton X-100, sephadex G-50, DMSO, di-8-ANEPPS, PEG-8000, amphotericin B (AmB), and gramicidin A (GrA) were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK). KCl solutions (0.1 or 2.0 M) were buffered using 10 mM HEPES-KOH at pH 7.4. Lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (CHOL), were obtained from (Avanti Polar Lipids, Inc., USA). All experiments were performed at room temperature (25 °C).
The variations in the chemical structures of the tested chromone-containing allylmorpholines are presented in Table 1. A description of the synthesis of the compounds is given in [7 (link)]. Relationships between compound numbers used in this work and in the study by [7 (link)] are given in Supplementary Table S1. Molecular weights, the ionization constants (pKa), logarithms of the octanol/water partition coefficients (LogP), and molecular dipole moments (μ) of the tested compounds are presented in Supplementary Table S1.

All chemicals used were of reagent grade. Synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), and cholesterol (Chol) were obtained from Avanti^® Polar Lipids (Alabaster, Alabama, AL, USA). Nonactin, di-8-ANEPPS, KCl, HEPES, KOH, DMSO, gramicidin A (GrA), nystatin (Nys), sildenafil citrate, vardenafil hydrochloride, and tadalafil were purchased from Sigma-Aldrich^® (Merck KGaA, Darmstadt, Germany). The chemical structures of the tested PDE-5 inhibitors are presented in Table 1.
KCl solutions (0.1 M or 2.0 M) were buffered using 5 mM HEPES–KOH at pH 7.4. All experiments were performed at room temperature (25 ± 2 °C).

Experimental imaging of t-tubules was performed by staining live cells with di-4-ANEPPS (Sigma-Aldrich), di-8-ANEPPS (Sigma-Aldrich), RH237 (ThermoFisher), CellMask (ThermoFisher) or FM1–43FX (ThermoFisher). In fixed cells and tissue sections, t-tubules were stained with wheat germ agglutinin conjugated to Alexa Fluor (488, 546 or 633). Dyadic proteins were labelled with primary antibodies against Caveolin-3 (Cav-3; Abcam, ab2912) and bridging integrator 1 (Bin-1; Santa Cruz, Sc-23918), and secondary antibodies coupled to Alexa Fluor 488 or 546 (ThermoFisher). Presented images of dyadic proteins and t-tubules were obtained using an LSM800 Airyscan confocal microscope (Zeiss, Jena, Germany) using a 63× magnification oil immersion objective. To test the versatility of the plugin, analysis of images captured with other confocal microscopes (Zeiss LSM 510 and 710) was also performed.