Image analysis system

On the second day of culture, the cells in the chamber slide (BD Biosciences) were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100. After blocking, the cells were incubated with antibodies against CK7 (1:200; Proteintech, United States) and vimentin (1:200; Proteintech, United States) overnight at 4°C. After washing, Alexa Fluor 594 goat anti-mouse immunoglobulin G (red; 1:200; Proteintech) and Alexa Fluor 488 goat anti-rabbit immunoglobulin G (green; 1:200; Proteintech) were used as secondary antibodies and the cells were incubated in darkness for 1 h. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI) (1 mg/mL). Images were obtained with a microscope and camera connected to a computer with an image analysis system (Zeiss).

Primary bone marrow osteoclast precursors cells were isolated following the protocols described in our previous publications to assess the impact of miR-29a-AS and pre-miR-29a on the osteoclastogenic potentials of the harvested primary bone marrow osteoclast precursors [26 (link)]. Briefly, nucleated cells in bone marrow were isolated using RBC Lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) and incubated in α-minimum essential medium (MEM) with 10% FBS and 20 ng/mL macrophage -colony stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) for 24 h. The floating cells were collected upon incubation. Then, 10⁵/well macrophages (24-well plates) were incubated in osteoclastogenic medium comprising α-MEM, 10% FBS, 20 ng/mL M-CSF and 20 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) (R&D Systems, Minneapolis, MN, USA) for one week. F-actin ring formation in osteoclasts was probed employing F-actin antibody conjugated with Alexa Fluor^® 488 Phalloidin (Life Technologies, Grand Island, NY, USA) and 4′,6-diamidino-2-phenylindole (DAPI)-Fluoromount G (Southern Biotech, Birmingham, AL, USA). The total number of F-actin rings in 3 different fields (×100 magnification) per well and 3 wells per animals were counted employing a Zeiss inverted microscope and image-analysis software (Zeiss Image Analysis System, Oberkochen, Baden-Württemberg, Germany).

We assessed the impacts of miR-29a-AS and pre-miR-29a on the osteogenic potentials of the harvested primary bone marrow mesenchymal cells. Briefly, primary bone marrow mesenchymal cells harvested from femurs were mixed with red blood cell lysis buffer (11814389001; Sigma-Aldrich, St. Louis, MO, USA) to isolate mononuclear cells. Upon incubating mononuclear cells in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) overnight, adherent cells were collected and incubated in osteogenic medium (10⁵ cells/well, 24-well plates) (StemPro™ Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA) for 18 days. The extent of mineralization was assessed employing von Kossa Stain Kits (ab150687; Abcam, Cambridge, UK), following instructor’s protocol. Calcium in mass deposits would be stained with black color by the Kit, and the black color-stained area (mm²/filed) of von Kossa-stained mineralized matrices in each ×125 magnification field were measured applying light microscopy (Zeiss Image Analysis System, Oberkochen, Baden-Württemberg, Germany) [19 (link),21 (link)].