Phalloidin staining

Podosomes were visualized by phalloidin staining (Sigma-Aldrich) following manufacturer’s instructions. After removing unbound phalloidin conjugates, cells were labeled with 1 μg/ml DAPI (Sigma-Aldrich). Microscopic imaging was performed using a fluorescence microscope (Zeiss Axio Observer D1). For mitochondrial analysis, live cells were labeled with 200 nM MitoTracker Orange (ThermoFisher) according to manufacturer’s recommendations. After staining, cells were fixed in 4% formaldehyde, labeled with 1 μg/ml DAPI and subjected to z-stack confocal microscopy analyses (Zeiss LSM 710 on an inverted Axio Observer.Z1 stand). Images were processed using Fiji/Image J software (Schindelin et al., 2012 (link)).

ASCs-T2DM were inoculated in a 6-well plate at a density of 2.5 × 10⁵ and divided into four groups with three multiple wells. Then, medium containing different concentrations of CGRP (10⁻⁷ mol/L, 10⁻⁸ mol/L, 10⁻⁹ mol/L, and 0 mol/L) was added and changed until the cell confluence reached about 50%. Whereafter, they were fixed with 4% paraformaldehyde (Solaibio, China) and then membrane was ruptured with 0.5% Triton X-100 (Sigma, USA). After washing, 1% BSA (Solaibio, China) was added to block the antigen. The cells were covered with phalloidin staining (Sigma, USA) and incubated at room temperature for 30 minutes. Finally, they were dyed with DAPI (Solaibio, China) and observed under a fluorescence microscope (Olympus, Japan).
ASCs-T2DM were inoculated into 96-well plates at a density of 3000/well. Each group consisted of 3 multiple holes and 3 control holes. At the same time of each day, the mixture with rate 10 : 1 of CGRP (10⁻⁷ mol/L, 10⁻⁸ mol/L, 10⁻⁹ mol/L, and 0 mol/L) and CCK-8 (EnoGene, China) was added to the test group to incubate for 3 hours in CO₂ incubators and then determine the absorbance value at 450 nm wavelength by a spectrophotometer (BioTek, USA). The average value was calculated, and the growth curve was drawn.