Fig 1: HA suppresses osteoclastogenic gene expression in RAW264.7 cells. Real-time PCR shows: (a) relative NF??BmRNA expression; (b) relative NFATc1 mRNA expression; (c) relative MMP9 mRNA expression; (d) relative Cathepsin K mRNA expression in RAW 264.7 cells treated with RANKL (50 ng/ml), HA (1×) and the combination of HA and RANKL, control = PBS treated for 4 indicated days. *,#Significant differences versus control by t test. Data are means ± standard error with triplicates. HA, hippuric acid; MMP9, matrix metallopeptidase 9; mRNA, messenger RNA; NF-?B, nuclear factor kappa-light-chain-enhancer of activated B cells; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; RANKL, receptor activator of nuclear factor ?-? ligand
Fig 2: HA and 3‐3‐PPA inhibit RANKL‐induced osteoclastogenic signaling. (a) Western blot shows RANKL activated NFATc1, cFos, MMP9 and Cathepsin K protein expression after RAW264.7 cells were treated for 3 days. Both 1× HA and 10× 3‐3‐PPA inhibited those RANKL‐induced protein expression. MMP9‐1 and MMP9‐2 are same membrane but different exposure time. α‐tubulin is a protein loading control. (b) Western blot analysis shows β‐catenin and GSK3beta expression in RAW264.7 cells after 4 days treatment with either 1× HA, 10× 3‐3‐PPA, RANKL or their combinations, β‐actin is a protein loading control. (c) real‐time PCR shows mRNA expression of β‐catenin and downstream genes Cmyc and Traf6 in RAW264.7 cells after 4 days treatment with either 1× HA, 10× 3‐3‐PPA, RANKL or their combinations, GAPDH was used as a housekeeping gene. *p < .05 versus control by t test. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; HA, hippuric acid; MMP9, matrix metallopeptidase 9; mRNA, messenger RNA; RANKL, receptor activator of nuclear factor κ‐Β ligand
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