Fitc phalloidin

Proximity ligation assay was performed using commercially available Duolink PLA kit (Sigma-Aldrich, Cat. DUO92101-1KT). After 16 h incubation of hTERT RPE-1 cells on micro-patterned lines, they were fixed with 4% PFA and permeabilized with 0.1%Triton-X. Proximity ligation assay was performed according to the manufacturer’s protocol. For this experiment, pairs of mouse anti-emerin (Leica Biosystems, NCL-EMERIN)/ rabbit anti-Lamin B1 (Abcam, ab16048) or mouse anti-emerin (Leica Biosystems, NCL-EMERIN)/rabbit anti-nesprin-1 (Sigma-Aldrich, HPA019113) antibodies were used. The nuclei were stained with DAPI and F-actin was visualized using FITC-phalloidin (Sigma-Aldrich, Cat. P5282). The images were projected and signals were quantified using the custom-built ImageJ macro using Triangle segmentation method. The DAPI signal was used to exclude the nuclear signal whereas the F-actin marked the area of the cell. For each cell, a number of signals in the cytoplasm was calculated.

After 48-hour incubation, samples were washed with phosphate buffer solution (PBS) twice1 (link). After fixed with 2.5% glutaraldehyde solution for 2 h, the samples were washed with PBS for three times, treated with 1% osmium tetroxide for 2 h, and then dehydrated in ethanol of ascending concentrations (30, 50, 70, 80, 90, 95, 100 (v/v)) for 5 min respectively. Subsequently, the samples were immersed in isoamyl acetate for 20 min, vacuum-dried at 40 °C for 4 h, and then coated with gold-palladium. Cells morphology on the dried scaffolds was observed using SEM2 . After fixed with Formalin solution (3.7% formaldehyde in PBS) for 15 min, the fixed samples were washed with PBS twice, and then stained with FITC-Phalloidin (Sigma, St. Louis, USA) for 40 min. Cell nuclei were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime Biotech, Jiangsu, China) solution for 10 min. The samples were observed under a confocal laser scanning microscope (CLSM, Nikon, Japan).

RAW 264.7 cells were purchased from the Cell Bank of the Chinese Academy of Medical Sciences. Bone marrow monocytes (BMMs) isolated from Cko and Ctrl mouse femurs and tibias were treated with 100 ng/ml receptor activator of nuclear factor-κB ligand (RANKL; R&D Systems) and 30 ng/ml mouse macrophage colony-stimulating factor (M-CSF; R&D Systems) for 6 days. All the serum- or supplement-containing media were replaced every 2 days. Cells were exposed to hypoxia by incubation under 2% O₂ and 5% CO₂ conditions with balanced N₂ in the Invivo2 Hypoxia Workstation (Rushkinn, Waltham, MA). BMMs were treated under hypoxic conditions for the last 48 h. The cells were stained with a TRAP staining kit, toluidine blue staining kit (Solarbio) and FITC phalloidin (Sigma-Aldrich). Osteoclasts were identified by TRAP staining, and TRAP-positive osteoclasts were counted in multiple sections. For the bone resorption assay, BMMs were seeded on bone slices in 24-well plates and cultured with osteoclastogenic medium for 14 days. The medium was aspirated, and sodium hypochlorite was used to bleach the samples three times. Those methods are the same as our previous study (Tian et al., 2020 ).

F-actin staining using FITC-phalloidin (Sigma-Aldrich, St. Louis, MO) was carried out using 1 × 10⁶ cells. The cells were pelleted, fixed, and permeabilized with CytoPerm/Cytofix buffer (BD Biosciences, San Jose, CA) for 20 min at room temperature and washed with cold Perm/Wash buffer (BD Biosciences, San Jose, CA) twice, followed by staining with 5 μl of 0.3 mM FITC-labeled phalloidin (Sigma-Aldrich, St. Louis, MO) for 30 min on ice in the dark. The cells were washed twice with cold Perm/Wash buffer, resuspended in 1% paraformaldehyde, and analyzed on a FACSCalibur (BD Biosciences, San Jose, CA).

The OBs were seeded on the surfaces of different samples and cultured in the incubator of 0.5, 1, and 4 h. Then, the OBs were washed three times with PBS, followed by fixation with 4% paraformaldehyde (PFA) and 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, United States) staining for 10 min. Confocal laser scanning microscopy (CLSM, C2 Plus, Nikon, Tokyo, Japan) was applied to capture the cell fluorescent images by randomly selecting six fields under × 10 magnification.
The seeded and fixed OBs for 24 h were stained with FITC-phalloidin (Sigma-Aldrich) and counterstained with DAPI, from which the fluorescent images were captured under × 20 magnification by CLSM.

As described previously, FITC-phalloidin staining was used for cardiomyocyte surface area determination [21 (link)]. After treatment, cells were washed with Phosphate Buffered Saline for three times. The cells were then fixed in 4% paraformaldehyde for 30 min, and permeabilized in in 0.1% Triton X-100 for 10 min. Subsequently, cells were blocked with 10% normal goat serum for 10 min, and stained by FITC-phalloidin (10 μg/ml, Sigma-Aldrich) for 30 min at 37°C. Stained cells were photographed under a fluorescence microscope and cell surface area was quantified using ImageJ software.