Figure S2.
(Related toFig. 2). (A and B) Depletion of meosin by siRNA in hOC. Western blot analysis of moesin and activated ERM (P-ERM) expression level in hOC after treatment on day 0 with nontargeting siRNA (siCTL) or siRNA targeting moesin (siM), on days 3, 6, and 10 of differentiation. (A) Representative experiment and (B) quantification of moesin (left) and P-ERM (right) expression level normalized to actin. Each circle represents a single donor, n = 7–8, SDs are shown. (C–E) Effect of LPC treatment on hOC fusion and activated ERM expression level (P-ERM). On day 6 of differentiation, hOC were treated with LPC (+LPC, 17 h), or treated and then washed (90 min) (+/− LPC) or no treated (control, CTL). (C and D) Microscopy analysis of hOC fusion. (C) Representative microscopy images: F-actin (phalloidin, white) and nuclei (DAPI, cyan). Scale bar, 50 µm. (D) Quantification of fusion index (left) and area occupied by multinucleated OC (right). Each circle represents a single donor, n =3–4. (E) Western blot analysis of P-ERM expression level in each condition, normalized to actin. Representative blot (left panel) and quantification (right panel). Each circle represents a single donor, n = 4. (F) P-ERM signal by western blot analysis in murine inflammatory osteoclasts (DC-OC) versus control osteoclasts (MN-OC). See material and methods. Representative blot of P-ERM expression level in each condition (upper panel) and quantification of P-ERM expression level, normalized to actin (lower panel). Each circle represents a single mouse, n = 4, SDs are shown. (G–J) Effect of HIV infection on activated ERM expression level (P-ERM) in hOC (G and H) and macrophages (I and J). (G and H) On day 6 of differentiation, hOC were infected with the viral strain NLAD8-VSVG (+HIV-1) or not (CTL) and analyzed 8 days after infection. (G) Quantification of the fusion index (each circle represents a single donor, n = 4) and (H) representative western blot analysis of P-ERM expression level (left), and quantification of P-ERM expression level, normalized to actin (right, each circle represents a single donor, n = 5, SD is shown). (I and J) On day 6 of differentiation, macrophages were infected with the viral strain NLAD8-VSVG (+HIV-1) or not (CTL) and analyzed 7 days after infection. (I) Quantification of the fusion index (each circle represents a single donor, n = 4) and (J) representative western blot analysis of P-ERM expression level (left) and quantification of P-ERM expression level, normalized to actin (right). Each circle represents a single donor, n = 6, SDs are shown. Predicted molecular weights are indicated on western blots. Statistical analyses: (D and E) one-way ANOVA and then Tukey multiple comparison tests. P value is indicated on the graphs, *P ≤ 0.05. Source data are available for this figure: SourceData FS2.

(Related to Fig. 2 ). (A and B) Depletion of meosin by siRNA in hOC. Western blot analysis of moesin and activated ERM (P-ERM) expression level in hOC after treatment on day 0 with nontargeting siRNA (siCTL) or siRNA targeting moesin (siM), on days 3, 6, and 10 of differentiation. (A) Representative experiment and (B) quantification of moesin (left) and P-ERM (right) expression level normalized to actin. Each circle represents a single donor, n = 7–8, SDs are shown. (C–E) Effect of LPC treatment on hOC fusion and activated ERM expression level (P-ERM). On day 6 of differentiation, hOC were treated with LPC (+LPC, 17 h), or treated and then washed (90 min) (+/− LPC) or no treated (control, CTL). (C and D) Microscopy analysis of hOC fusion. (C) Representative microscopy images: F-actin (phalloidin, white) and nuclei (DAPI, cyan). Scale bar, 50 µm. (D) Quantification of fusion index (left) and area occupied by multinucleated OC (right). Each circle represents a single donor, n =3–4. (E) Western blot analysis of P-ERM expression level in each condition, normalized to actin. Representative blot (left panel) and quantification (right panel). Each circle represents a single donor, n = 4. (F) P-ERM signal by western blot analysis in murine inflammatory osteoclasts (DC-OC) versus control osteoclasts (MN-OC). See material and methods. Representative blot of P-ERM expression level in each condition (upper panel) and quantification of P-ERM expression level, normalized to actin (lower panel). Each circle represents a single mouse, n = 4, SDs are shown. (G–J) Effect of HIV infection on activated ERM expression level (P-ERM) in hOC (G and H) and macrophages (I and J). (G and H) On day 6 of differentiation, hOC were infected with the viral strain NLAD8-VSVG (+HIV-1) or not (CTL) and analyzed 8 days after infection. (G) Quantification of the fusion index (each circle represents a single donor, n = 4) and (H) representative western blot analysis of P-ERM expression level (left), and quantification of P-ERM expression level, normalized to actin (right, each circle represents a single donor, n = 5, SD is shown). (I and J) On day 6 of differentiation, macrophages were infected with the viral strain NLAD8-VSVG (+HIV-1) or not (CTL) and analyzed 7 days after infection. (I) Quantification of the fusion index (each circle represents a single donor, n = 4) and (J) representative western blot analysis of P-ERM expression level (left) and quantification of P-ERM expression level, normalized to actin (right). Each circle represents a single donor, n = 6, SDs are shown. Predicted molecular weights are indicated on western blots. Statistical analyses: (D and E) one-way ANOVA and then Tukey multiple comparison tests. P value is indicated on the graphs, *P ≤ 0.05. Source data are available for this figure: SourceData FS2.

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