Figure 5.

Galectin-3 upregulates HEY-1 and induces NICD in hFOB cells. (A) Recombinant galectin-3 induced mRNA expression level of HEY-1. RNA samples were extracted at 1 week after addition of recombinant galectin-3 (1.6 μM) with or without inducers for osteoblast differentiation. mRNA level was determined by quantitative real-time PCR. Results are presented as fold changes compared to the vehicle control. White bars, culture with vehicle control. Black bars, with galectin-3. (B) Cancer-secreted galectin-3 induced mRNA expression level of HEY-1 in co-culture experiment. RNA samples were extracted at 6 days after co-culture with transfected cancer clones with or without secretion of galectin-3. Results are presented as fold changes compared to the empty vector cells. Data represent means ± SD. White bars, co-culture with empty vector cells. Black bars, co-culture with galectin-3 transfected stable clone. EV, empty vector cells. *P < .05 versus vehicle or empty vector cells. (C) Galectin-3 induced NICD in hFOB cells in a time-dependent manner. Recombinant galectin-3 was added to culture medium in 1.6 μM as final concentration, respectively. (D) Suppression study using lactose; 75 mM of lactose or sucrose was used as final concentration. Cell lysates were extracted 12 hours after treatment. (E) Suppression study using HL31 anti–galectin-3 rabbit polyclonal antibody; 15 μg/ml HL31 was used as final concentration. Cell lysates were extracted 12 hours after treatment. The same concentration of rabbit isotype IgG was used as control (data were not shown). (F) Immunofluorescence images of hFOB with or without galectin-3 stimulation. Recombinant galectin-3 was added to culture medium in 1.6 μM as final concentration. Cells were fixed 24 hours after treatment. Images were shown for NICD (TRITC, red) and 4′,6-diamidino-2-phenylindole (nuclear stain, blue). White bar indicates 10 μm as scale.