Fig 2: Ezrin is the only ERM family protein whose expression is reduced by RANKL treatment, and its expression is regulated at the transcriptional level. (A) Western blot analysis of cell lysates without or treated with RANKL for 60 h using anti-ezrin, anti-radixin, anti-moesin, and anti–β-actin antibodies. (B–D) Quantification of endogenous ezrin (B), radixin (C), and moesin (D) based on (A). Mean ± SEM, n = 6 experiments. P value obtained from two-tailed Student’s t test. ns: not significant; ****P < 0.0001. (E) Western blot analysis of cell lysates without or treated with RANKL and the proteasome inhibitor MG132 for 84 h using anti-ezrin and anti–β-actin antibodies. (F) Quantification of endogenous ezrin based on E. The values of each band were measured and normalized to cells not treated with RANKL and MG132. Mean ± SEM, n = 6 experiments. P value obtained from one-way ANOVA with Tukey’s test. ns: not significant; *P < 0.05; **P < 0.01. (G–J) qPCR analysis of the relative expression of Nfatc1 (G), Ezr (H), Rdx (I), and Msn (J). mRNA was extracted after 60 h of RANKL treatment and normalized to the expression of Gapdh mRNA. Mean ± SEM, n = 4 experiments. P value obtained from two-tailed Student’s t test. ns: not significant; *P < 0.05; ***P < 0.001. (K) Western blot analysis of Blimp1 knockdown cell lysates without or treated with RANKL for 84 h using anti-ezrin and anti–β-actin antibodies. (L) Quantification of endogenous ezrin based on K. The values of each band were measured and normalized to cells not treated with RANKL of si-Control group. Mean ± SEM, n = 7 experiments. P value obtained from one-way ANOVA with Tukey’s test. ns: not significant; *P < 0.05; ****P < 0.0001. (M) Representative western blot of siRNA knockdown cells with RANKL treatments for 70 h, using anti-ezrin, anti-radixin, anti-moesin, and anti–β-actin antibodies. (N–P) qPCR analysis of the relative expression of Fnbp1 (N) and Baiap2l1 (72 h, O; 84 h, P) mRNAs, normalized by that of Gapdh mRNA. Source data are available for this figure: SourceData FS3.

Fig 3: U46619 promotes Moesin-dependent migration of Hs578T cells.(A) Immunoblot of Hs578T stably expressing nontarget (NT) shRNA or shRNA targeting EZR, RDX, or MSN. (B) mRNA expression of EZR, RDX, and MSN in the Hs578T cell line. Data are from RNA-seq of 1,019 human cancer cell lines from the Cancer Cell Line Encyclopedia https://www.ebi.ac.uk/gxa/home (Papatheodorou et al, 2020). (C, D) Immunoblot of Hs578T cells treated with 100 nM U46619 for the indicated time in hours (C). (D) p-ERM over ERM signals was quantified and normalized to the vehicle (D). (E, F) Immunoblot of Hs578T cells knocked out for ROSA26 (control) or MSN using two independent sgRNAs and treated with 100 nM U46619 for 5 min (E). (F) p-ERM over actin signals was quantified and normalized to ROSA26 + vehicle (F). (G) Quantifications of cell velocity during 2D cell migration of Hs578T cells stably expressing nontarget shRNA (NT) or an shRNA targeting MSN and treated with vehicle or 10 nM U46619 for 6 h. Quantifications are represented as superplots and are presented as the mean ± s.d. of three independent experiments. Immunoblots (A, C, E) are representative of three independent experiments. Velocity quantifications (G) are presented as superplots. The speed of individual cells is shown as small dots, color-coded by the independent experiment. The mean ± s.d. of each biological replicate (n = 3 independent experiments) is shown as larger dots. (F) Dots represent the mean of independent experiments (F). P-values were calculated using Holm–Sidak’s multiple comparisons test with a single pooled variance **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.