Malachite green

For ultrastructural analysis, cells were fixed for 1 h at 22 °C in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences), and 0.05% malachite green (Sigma-Aldrich) in 100 mM sodium cacodylate buffer (pH 7.2). malachite green was incorporated into the fixative for stabilization of lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and were post-fixed for 1 h in 1% osmium tetroxide (Polysciences). Samples were then rinsed extensively in distilled water before en bloc staining for 1 h with 1% aqueous uranyl acetate (Ted Pella). Following several rinses in distilled water, the samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections 95 nm in thickness were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems), then stained with uranyl acetate and lead citrate and viewed on a Tecnai G2 Spirit BioTWIN transmission electron microscope (FEI) at 60 kV.

To remove ATP, actin monomers in G-buffer were incubated with Bio-Rad AG1-X2 100–200 mesh anion exchange resin (Dowex) (Bio-Rad, 1401241) rotating at 4°C for 5 min, followed by low-speed centrifugation to remove resin. Actin filaments (20 μM) were polymerized in 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM Hepes pH 7.4 for 1 hr at 23°C. Drp1 was diluted in 150 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM Hepes pH 7.4, then centrifuged to remove aggregates as described above.
Drp1 (1.3 μM) was mixed with actin filaments (1 μM), and the final ionic strength was adjusted to the equivalent of 75 mM KCl using 4 M KCl stock. Samples were incubated at 37°C for 5 min. At this point, GTP was added to a final concentration of 250 μM to start reactions at 37°C. Reactions were quenched at various time points by mixing 20 μL of sample with 5 μL of 125 mM EDTA in a clear flat-bottomed 96-well plate (Greiner). Six time points were acquired for each condition. Inorganic phosphate was determined by addition of 150 μl of Malachite green solution (1 mM Malachite green (Sigma Aldrich, 2290105–100 g), 10 mM ammonium molybdate ([Sigma Aldrich, A7302–100 g] in 1N HCl) to 25 μl quenched reactions. Absorbance at 650 nm was measured with a 96-well fluorescence plate reader (TECAN Infinite M1000, Mannedorf, Switzerland). GTP hydrolysis rates were determined by plotting phosphate concentration as a function of time.

Dorsal skin samples were collected from 7 months old WT and Sc65KO animals (n = 4) and fixed overnight at 4°C in 2.5% glutaraldehyde (Electron Microscopy Sciences (EMS), Hatfield, PA, USA), 0.05% malachite green (Sigma Aldrich) in 0.1M sodium cacodylate buffer, pH 7.2 (EMS). After washing with 0.1M sodium cacodylate buffer, the samples were post-fixed for 2 hrs with 1% osmium tetroxide (EMS), 0.8% potassium hexaferrocyanide (Sigma Aldrich) for 2 hours and 1% tannic acid (EMS) for 20 min. The samples were rinsed with molecular grade water and stained with 0.5% uranyl acetate (EMS) for 1 hour then dehydrated with a graded alcohol series and propylene oxide before embedding in Araldite/Embed 812 (EMS). Sections (50nm) were cut on a Leica UC7 ultra-microtome and collected on formvar carbon coated slot grids and post-stained with uranyl acetate and lead citrate. Imaging was done using a Technai F20 (FEI, Netherlands) at 80kV. Analyses of collagen fibril diameter (>200 fibrils/mouse) and of collagen inter-fibril space were performed with the Leica Application Suite v 3.0 image analysis software (Leica Microsystems, Milan, Italy) on 5 sections for each mouse (n = 3) at the magnification 19000X.

Three sets of Gap-LAMP primers were prepared in separate reaction tubes. Gap-LAMP reaction was performed in a total volume of 25 μL. The reaction was containing 0.2 μM for each outer primer (B3 and F3), 0.8 μM for each inner primer (BIP and FIP), 1 M betaine (Sigma-Aldrich, St. Louis, MO), 1× isothermal amplification buffer (20 mM Tris-HCl, 50 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄ and 0.1% Tween 20), 2 mM MgSO₄ (New England Biolabs, Ipswich, MA) and 0.9 mM dNTP for SEA primer set or 1.1 mM dNTP for normal and THAI primer sets. The reaction mixture was pre-heated at 95°C for 15 min and cooled on ice. Then, 8 U Bst 2.0 WarmStart DNA Polymerase (New England Biolabs) and 0.16 μg/μL malachite green (Sigma-Aldrich) were added and followed by incubating at 64°C for 60 min. After the incubation step, reaction tubes were placed under visible light for 15 min. Positive reaction with amplified DNA products showed light blue color solution. In contrast, the negative reaction without amplification showed a colorless solution.

CuCl₂·2H₂O, 1,3,5-tricarboxylic acid benzene (H₃BTC), were obtained from Alfa-Aeser Co. Malachite green (MG) and lead nitrate were supplied from Sigma-Aldrich chemicals Co. Sulfamic acid (SA), N,N-dimethylformamide “DMF”, ethanol, ethyl acetoacetate, benzaldehyde, urea resorcinol, and lead nitrate are all with analytical grades and were used without any further purification.

TBPH containing 40 wt.% in water was purchased from Acros and concentrated through rotary evaporation under 70 bar, 40 °C to get higher concentration (50 wt.%). Microcrystalline cellulose (MCC) with the size 20 µm used in this study was purchased from Sigma Aldrich. Propylene carbonate (3, 99.7% purity), vinyl ethylene carbonate (VEC, 99% purity), ethylene carbonate (EC, 98% purity), and butylene carbonate (BC, 98% purity) were obtained from Sigma Aldrich. Dimethyl sulfoxide was supplied from Sigma Aldrich. PEG with molecular weight in the range as follows: PEG 950–1050, PEG 35,000, PEO 200,000, PEO 400,000, PEO 1,000,000 were provided from Sigma Aldrich. Methylene blue, malachite green, cresol red, thymol blue, eosin Y disodium salt and methylene orange were obtained from Sigma Aldrich.