Figure S2.
Establishment of cell lines expressing ezrin, MA-ezrin, and iMC-linker, and the expression and localization confirmation of NFATc1, a master transcription factor, and TRAP, an osteoclast differentiation marker. (A) Representative western blot of parental cells and stable cell lines overexpressing HA-tagged ezrin, MA-ezrin (MA), and iMC-linker (iMC) without RANKL treatment, using anti-HA tag and anti–β-actin antibodies. (B) Representative western blot of parental cells and stable cell lines above without or with RANKL treatment for 36 h, using anti-NFATc1 and anti–β-actin antibodies. (C) Quantification of B. Mean ± SEM, n = 3 experiments. Note that overexpression of these exogenous genes does not inhibit the induction of NFATc1 expression induced by RANKL. P value obtained from one-way ANOVA with Tukey’s test. ns: not significant; *P < 0.05; **P < 0.01. (D) Confocal images of the four cell lines above without or with RANKL treatment for 36 h, stained with anti-NFATc1 antibody. Scale bar: 20 μm. (E) The nucleus-to-cytoplasm ratio of NFATc1 signal intensity in mononuclear cells. Solid and dotted lines in violin plots show median and quantiles. The total number of cells analyzed was as follows: n = 224 (parental, RANKL−), 197 (parental, RANKL+), 153 (ezrin, RANKL−), 200 (ezrin, RANKL+), 141 (MA, RANKL−), 117 (MA, RANKL+), 288 (iMC, RANKL−), and 304 (iMC, RANKL+). P value obtained from one-way ANOVA with Tukey’s test. ns: not significant; *P < 0.05; ****P < 0.0001. Note that the nucleus-to-cytoplasm ratios of these stable cell lines tend to be higher compared with parental cells; however, they do not inhibit the nuclear transport of NFATc1. (F) The four cell lines stained for TRAP activity. Multinucleated cells are surrounded by dotted lines. Note that all cell lines indicate the activity of TRAP after RANKL treatment for 84 h. Scale bar: 200 μm. Source data are available for this figure: SourceData FS2.

Establishment of cell lines expressing ezrin, MA-ezrin, and iMC-linker, and the expression and localization confirmation of NFATc1, a master transcription factor, and TRAP, an osteoclast differentiation marker. (A) Representative western blot of parental cells and stable cell lines overexpressing HA-tagged ezrin, MA-ezrin (MA), and iMC-linker (iMC) without RANKL treatment, using anti-HA tag and anti–β-actin antibodies. (B) Representative western blot of parental cells and stable cell lines above without or with RANKL treatment for 36 h, using anti-NFATc1 and anti–β-actin antibodies. (C) Quantification of B. Mean ± SEM, n = 3 experiments. Note that overexpression of these exogenous genes does not inhibit the induction of NFATc1 expression induced by RANKL. P value obtained from one-way ANOVA with Tukey’s test. ns: not significant; *P < 0.05; **P < 0.01. (D) Confocal images of the four cell lines above without or with RANKL treatment for 36 h, stained with anti-NFATc1 antibody. Scale bar: 20 μm. (E) The nucleus-to-cytoplasm ratio of NFATc1 signal intensity in mononuclear cells. Solid and dotted lines in violin plots show median and quantiles. The total number of cells analyzed was as follows: n = 224 (parental, RANKL−), 197 (parental, RANKL+), 153 (ezrin, RANKL−), 200 (ezrin, RANKL+), 141 (MA, RANKL−), 117 (MA, RANKL+), 288 (iMC, RANKL−), and 304 (iMC, RANKL+). P value obtained from one-way ANOVA with Tukey’s test. ns: not significant; *P < 0.05; ****P < 0.0001. Note that the nucleus-to-cytoplasm ratios of these stable cell lines tend to be higher compared with parental cells; however, they do not inhibit the nuclear transport of NFATc1. (F) The four cell lines stained for TRAP activity. Multinucleated cells are surrounded by dotted lines. Note that all cell lines indicate the activity of TRAP after RANKL treatment for 84 h. Scale bar: 200 μm. Source data are available for this figure: SourceData FS2.

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