Fig 1: EN’s role in osteoclast differentiation and function. Raw264.7 cells were pre-treated with EN (10 and 20 μg/mL) for 1 h, followed by RANKL (40 ng/mL). (A) The area of resorption pits formed by mature osteoclast cells was evaluated using a bone resorption assay and visualized under a light microscope at 40× magnification (Scale bar. 100 μm). (B) The pit area measurements, quantified in pixels, are depicted in a bar graph. (C) The formation of actin rings in mature osteoclast cells was observed using a LSM 700 laser scanning confocal microscope, highlighting F-actin staining (upper line) and DAPI staining (bottom line) at 100× magnification (Scale bar = 200 μm). Data represent mean ± SEM (n ≥ 3). One-way analysis of variance (ANOVA) was utilized, followed by the Bonferroni post hoc test for pairwise comparisons. *** p < 0.001; n.s. = not significant. DMSO: dimethyl sulfoxide.
Fig 2: EN modulates MAPKs signaling. (A–C) Western blot analysis was used to determine the phosphorylation level of MAPKs in Raw264.7 cells pre-treated with EN (10 and 20 μg/mL) for 1 h, followed by RANKL (40 ng/mL) for 10 min. Protein expression levels were normalized to the expression levels of ERK, JNK, and p38, respectively. Data represent mean ± SEM (n ≥ 3). One-way analysis of variance (ANOVA) was utilized, followed by the Bonferroni post hoc test for pairwise comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. = not significant. DMSO: dimethyl sulfoxide.
Fig 3: EN attenuates RANKL-induced osteoclast differentiation. Raw264.7 cells were pre-treated with EN (10 and 20 μg/mL) for 1 h, followed by RANKL (40 ng/mL) for 4 days. (A) EN’s inhibitory effect on osteoclast differentiation in Raw264.7 cells via TRAP staining shown in dark purple was observed at 40× magnification under a light microscope (Scale bar. 400 μm). (B–D) Counts of TRAP-positive cells, multinuclear cells (≥3 nuclei), and larger multinuclear cells (≥10 nuclei), presented as percentages. Data are shown as mean ± SEM (n ≥ 3). One-way analysis of variance (ANOVA) was utilized, followed by the Bonferroni post hoc test for pairwise comparisons. ** p < 0.01; *** p < 0.001; n.s. = not significant. DMSO: dimethyl sulfoxide.
Fig 4: Impact of EN on osteoclast-specific gene expression. (A) The mRNA expression levels of osteoclast-specific genes were analyzed by RT-PCR in Raw264.7 cells treated with RANKL (40 ng/mL) with or without EN (10 and 20 μg/mL) for 2 days. (B–F) The RT-PCR results, normalized to GAPDH, for TRAP, CTSK, DC-STAMP, OSCAR and NFATc1 are presented in a bar graph. The quantitative data are expressed as mean ± SEM (n ≥ 3). One-way analysis of variance (ANOVA) was utilized, followed by the Bonferroni post hoc test for pairwise comparisons. ** p < 0.01; *** p < 0.001; n.s. = not significant. DMSO: dimethyl sulfoxide.
Fig 5: EN’s effects on NFATc1 and c-fos post RANKL treatment. (A) Western blot analysis was conducted to evaluate the expression levels of NFATc1 and c-fos in Raw264.7 cells 24 h post-treatment with RANKL (40 ng/mL). (B,C) The findings are displayed in a bar graph format. Data represent mean ± SEM (n ≥ 3). One-way analysis of variance (ANOVA) was utilized, followed by the Bonferroni post hoc test for pairwise comparisons. *** p < 0.001; n.s. = not significant. DMSO: dimethyl sulfoxide.
Supplier Page from Enzo Life Sciences, Inc. for Fc (human):RANKL (soluble) (mouse), (recombinant)