Mowiol 4 88

The synthesis of the ligands and complexes was performed according to the protocol described by Sgarbossa and co-workers [18 (link)]. Complexes and ligands were dissolved in DMSO as a 10 mM stock solution and stored at room temperature. SB202190, AZD6244, and SP600125 were purchased from Selleck Chemical (Houston, TX); they were dissolved in DMSO as a 20 mM stock solution and stored at −20 °C. Vanadyl sulfate (VOSO₄), 4’,6-diamidino-2-phenylindole (DAPI), Mowiol^® 4-88, RNase, propidium iodide, EDTA, protease inhibitor cocktails, and monoclonal anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). VOSO₄ was freshly dissolved in medium. DMEM, DMEM-F12, RPMI 1640, and fetal bovine serum (FBS) were purchased from Aurogene (Rome, IT). EGF, insulin, hydrocortisone, penicillin/streptomycin antibiotic mixture, amphotericin B, and glutamine were purchased from Sigma-Aldrich (Milan, IT).

NIH/3T3 cells were cultivated until 70–90% confluency on microscopy coverslips. Cells were washed three times for 5 min with 2 ml PBS^{Ca2+ Mg2+} (Sigma-Aldrich). Cells were fixed for 10 min at room temperature in 4% paraformaldehyde. Cells were washed as described and permeabilized with 0.2% ice-cold TritonX-100 in PBS for 5 min. Cells were blocked in 2 ml 5% non-fat dried milk in PBS^{Ca2+ Mg2+} for 1h at room temperature. Primary antibody binding was performed overnight at 4°C with a concentration of 4 μg/ml antibody in PBS^{Ca2+ Mg2+}/5% non-fat dried milk powder. Secondary antibody binding was performed at room temperature for 2 h with a concentration of 0.5 μg/ml antibody in PBS^{Ca2+ Mg2+}/5% milk powder. Cells were stained with 1μg/ml DAPI in PBS^{Ca2+ Mg2+} for 3 min, washed again with PBS and mounted on microscopy slides using Mowiol^® 4–88 (Sigma-Aldrich).

To perform immunocytochemistry, fibers were fixed immediately with 4% paraformaldehyde (PFA) for 20 min. After the fixation method 0.1 M glycine in phosphate buffer saline (PBS) was used to neutralize excess formaldehyde. Fibers were permeabilized with 0.5% Triton-X (TritonX-100, Sigma) for 10 min and blocked with a serum-free protein blocking solution (Dako, Los Altos, CA) for 30 min. Slides were rinsed three times with PBST solution. Primary antibodies (anti-RyR1, anti-Septin7, and skeletal muscle-specific anti-α-actinin, see ‘Key resources table’) diluted in blocking solution were added to the fibers and slides were incubated overnight at 4°C in a humidity chamber. Samples were washed three times with PBST and incubated with fluorophore-conjugated secondary antibodies for 1 hr at room temperature. After washing three times, a drop of mounting medium was added to each slide (Mowiol 4-88, Sigma) and coverslips placed on the mounting medium. Images from Alexa Fluor 488, TRITC, and DAPI-labeled samples were acquired with an AiryScan 880 laser scanning confocal microscope (Zeiss, Oberkocken, Germany) equipped with a ×20 air and a ×40 oil objective. Excitation at 488, 543, and 405 nm wavelengths was used to detect fluorescence of the aforementioned secondary antibodies, respectively, while emission collected above 550 nm with a long-pass filter.

Larval brain dissections were performed in PBS and fixed in 4% formaldehyde, pH 7.0 for 20 min. Subsequently, brains were washed in PBS and mounted in Mowiol^® 4–88 (Sigma-Aldrich, 81381, St. Louis, MO, USA). Samples were dissected in the morning or in the afternoon and imaged in the afternoon of the same day or the following morning, respectively.

Parasites were seeded on poly L-lysine treated coverslips and fixed in 2.5% glutaraldehyde. Coverslips were mounted on glass slides using Mowiol 4–88 (Sigma-Aldrich). Images were acquired using an Axiophot microscope at 63x magnification and an Andor camera. Length and width of the parasite cell body, and flagellum length were measured for at least 200 promastigotes using the Image J Fidji software package (https://imagej.net/). The ratios flagellum over body length and body length over body width were determined for the 200 parasites and the Kruskal-Wallis test was used for statistical analysis. The experiment was performed in duplicate.

MG63 cells were seeded at 15 × 10³ cell/cm² on a glass coverslip in a 12 well-plate and incubated for 24 h. Then, cells were treated with the different compounds at their IC₅₀ values. After 24 h, cells were washed twice with PBS and fixed for 15 min in a 4% (w/v) paraformaldehyde solution (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) at room temperature. After three washes with PBS, cells were stained for 45 min at room temperature with FITC-phalloidin (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) and Hoechst 33342 (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany). FITC-phalloidin and Hoechst 33342 were solubilized in PBS at a final concentration of 1µg/mL. Finally, sample were washed and mounted with Mowiol^® 4-88 (product reference, 81381; Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) containing DABCO as antifading. Images were acquired with a Nikon eclipse 90i fluorescence microscopy (Nikon Corporation, Tokyo, Japan).

Live and whole-mount hybridized embryos were photographed on agarose-coated dishes using either an epifluorescence Leica MZ16 F stereomicroscope (1X Plan Apo objective, NA0.141) (Leica, Wetzlar, Germany) equipped with digital camera or an Axio Zoom.V16 fluorescence stereomicroscope (Zeiss). Phalloidin-stained embryos were acquired on an Axiovert 200M fluorescence microscope (Zeiss) equipped with ApoTome.2 to enhance resolution. Evaluation of ISV defects was carried out on developing vessels in the region of the trunk above the prolongation of the yolk, as indicated in Figure 4A.
Confocal analysis of filopodia was performed using a LSM510 laser scanning microscope (Zeiss). To this purpose, 26–28 hpf embryos were fixed overnight with a PBS-based solution containing 1% PFA, 0.1% glutaraldehyde and 3% sucrose and mounted on glass slides with Mowiol 4.88 (Sigma). For consistency reasons and in order to minimize stage-related discrepancies, filopodia evaluation was carried out on the ISV pair in which the first vessel had already reached the roof of the trunk and the adjacent vessel was still growing up.