Alexa fluor 488

GBC-SD and NOZ cells were seeded in 12 well-plates, which were previously laid with sterile cover glasses, and incubated for 24h before being stained. 4% paraformaldehyde was used for cell fixation which were then permeabilized in 0.2% Triton X-100 at room temperature. 0.1% bovine serum albumin (BSA) was then used to block cells prior to an overnight incubation with anti-RACGAP1 (1/400), anti-LIG3 (1/200) and anti-γH2A.X (1/200) at 4°C. This was then followed by an incubation at 37 °C with a Alexa Fluor 488 or Alexa Fluor 594 conjugated secondary antibody (Yeasen Biotech, Shanghai, China). DAPI was then used to counterstain the cells before a Leica microscope was used to image the cells.

MC3T3-E1 cells were seeded on coverslips in 24-well plates in DMEM for 12 h and then cultured in osteogenic differentiation medium with different interventions. Cells were seeded in 24-well plates at a density of 2 × 10⁴ per well. After osteogenic differentiation, all cells on coverslips were fixed with 4% paraformaldehyde for 20 min, followed by infiltration with 0.2% Triton X-100 (Beyotime) for 20 min. Blocking solution was then used to seal the cells for 60 min. Further, the primary antibodies were added and incubated at 4 °C for 12 h, including anti-OCN (1:200), anti-Gli1 (1:500) and anti-Runx2 (1:200) antibodies. Then, washing the cells 3 times by PBS and incubated in the secondary fluorescent antibody (Alexa Fluor^® 488) and TRITC Phalloidin (Yeasen) for 1 h. Coverslips were placed with fluorescence anti-fading solution containing DAPI (Beyotime) on microscope slides. The images were obtained by a fluorescence microscope (Zeiss). Nuclei were stained with DAPI (blue), Gli1, Runx2 and OCN-positive cells are depicted in green. The actin cytoskeleton was stained with phalloidin in red. Cell nuclei were identified using DAPI⁺ regions and then colocalized pixel-wise with green regions to assess the nucleus translocation level of Gli1. A ratio of integrated density to the area (IntDen/Area) was expressed as average fluorescence intensity using ImageJ.

Human RCECs were cultured on chamber slides in 4-well plates to approximately 90% confluence in serum-free medium overnight before experiments. The cells were then treated with PDGF-C (125 ng/mL, Sino Biological Inc., Beijing, China) or VEGF (10 ng/mL, Sino Biological Inc., Beijing, China) for 1 hr. The slides were then washed, fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. The slides were blocked with 1% bovine serum albumin and 5% goat serum in PBS at room temperature for 1 hr. After incubation with rat anti-JAM-C (CRAM-18 F26) and rabbit anti-ZO1 (ab59720) (Abcam, Shanghai, China) antibodies and secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594, Yeasen, Shanghai, China). The slides were washed and mounted in medium (DAPI Fluoromount G; SouthernBiotech, Birmingham, AL). A negative control staining followed the same protocol except that the anti-JAM-C or anti-ZO1 antibodies. Fluorescence images were captured using a laser scanning microscope (Leica Microsystems, Wetzlar, Germany).