Deletion of ERK1 impairs osteoclast formation without any effects on osteoblasts.

<p>WT and ERK1^−/− mouse femur sections were stained with Trichrome Goldner-Masson stain (A) and revealed for TRAP activity (B). Ob.S/BS and Oc.S/BS parameters were calculated from Goldner trichrome and TRAP activity sections, respectively. Data are the means±S.E.M. (n  = 5 per group). Data represent the mean±SEM, the Mann-Whitney test was used to calculate the <i>P</i> value.</p

ERK1^−/− mice have a reduced fraction of bone marrow monocytes.

<p>(A) Representative FACS plot of BM monocytes defined as Gr1⁺CD115⁺ cells. (B) Proportion of monocytes as described above in the total BM in WT (n = 11) and ERK1^−/− (n = 12). Data represent the mean±SEM. The <i>t</i>-test was used to calculate the <i>P</i> value.</p

ERK1^−/− microenvironment induces a defect in WT HSC activity.

<p>(A) Scheme of transplantation assay. (B) Analysis of WT donor cells engraftment in WT (n = 10) and ERK1^−/− mice (n = 10) primary recipient 24 weeks after transplantation. (C) Analysis of WT donor cells engraftment in WT (n = 9 per group) and ERK1^−/− mice (n = 16) secondary recipient 24 weeks after transplantation. In panels B and C, total donor cells are shown as a percentage of live cells. Individual lineages are shown as a percentage of donor-derived cells. Data represent the mean±SEM, the <i>t</i>-test was used to calculate the <i>P</i> value.</p

ERK1^−/− microenvironment alters the lodging and the homing efficiencies of BM cells.

<p>Lodging of the bone marrow cells into a non irradiated host were quantified 3 h (A) and 24 h (B) after injection. Homing of the bone marrow cells into a lethally irradiated host were quantified 3 h (C) and 24 h (D) after injection. Results are presented as scatter plots showing the percentage of recovered CFSE⁺ cells in the BM 3 and 24 hours after transplantation. The non parametric Mann-Whiney test was used to calculate the <i>P</i> value. Horizontal bars show the mean values.</p

ERK1 deficiency impairs osteoclastogenesis.

<p>(A) Total number of osteoclasts per well following 5 days of culture under osteoclastogenic condition (n = 3) (B) Representative picture of TRAP-positive multinucleated osteoclasts generated from BMNCs of WT and ERK1^−/− mice. (C) Relative expression of cathepsin K (CTSK), calcitonin receptor (CaR), and receptor activator of nuclear factor <i>kappa</i> B (RANK) in WT and ERK1^−/− osteoclasts. (D) Representative pictures of the bone resorption pits formed by WT and ERK1^−/− derived osteoclasts. (E) Bar graph representing the quantification of the resorptive area per dentin slice for WT and ERK1^−/− osteoclasts (n = 3). Data represent the mean± SEM. The <i>t</i>-test was used to calculate the <i>P</i> value.</p

ERK1 loss alters the bone architecture.

<p>Cortical and trabecular parameters in WT and ERK1^−/− mice (n = 5 for each group). (A) midshaft diaphysis cortical thickness, (B) bone volume/tissue volume (% BV/TV), (C) trabecular thickness (Tb.Th), (D) trabecular number (Tb.N), (E) trabecular separation (Tb.S). The non parametric Mann-Whiney test was used to calculate the <i>P</i> value. Horizontal bars show the mean values.</p

IFITM3 is a <i>bona fide</i> virion-associated protein.

<p>Virion particles produced as described above were then analyzed by immuno-gold electron microscopy. Briefly, unfixed viral preparations purified by ultracentrifugation and produced in the presence or absence of IFITM3 were incubated with anti-Flag antibodies, followed by incubation with a gold-conjugated secondary antibody (arrows). Representative pictures are shown here. The graph displays the number of gold particles counted on a per virion basis.</p

CCR5 usage relieves the negative effects of IFITM3 on HIV-1 replication and on its ability to decrease the virion particles infectivity.

<p>A) Human CCR5 was introduced in the IFITM3-stable SupT1 cells used before, by retroviral-mediated gene transduction and cells were challenged with the indicated viruses. HIV spreading was assessed by exo-RT activity over time (day 0 through 7). The panels and the histogram overlay present the patterns of expression obtained for IFITM3 and CCR5 following WB and flow cytometry analyses. The graph presents normalized data obtained in 2 to 3 independent experiments. B) Virions obtained at late times after infection were harvested, normalized and used to infect HeLaP5 cells that contain a β-galactosidase reporter gene under the control of the HIV-1 LTR.</p

Comparison between the antiviral effect of IFITMs reported in the literature for the different viruses and mediated by the pool of IFITM proteins in target cells, with the negative imprinting of the virion particles infectivity reported in this study.

<p>Given their high identity, the antiviral effects of IFITM2 and 3 are presented together, separately from those of IFITM1. Variations in the magnitude of the antiviral effects reported in the different studies have not been taken into account here, as they are likely influenced by the specific experimental conditions used, so that the effects of IFITMs on viral infectivity are presented as negative, absent (none), or controversial, even when a single conflicting report exists. When data in the literature was not directly comparable to ours (i.e. the same virus was not used), data was compared to its closest relative, marked in <i>italicus</i>. The effects of the expression of IFITMs in target cells against AAV and MeV were measured in this study and are presented in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.s008" target="_blank">S6 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.g007" target="_blank">Fig 7C</a>, respectively.</p

CD45 depletion excludes a potentially confounding role of exosome-incorporated IFITMs on virion infectivity.

<p>SupT1 cells stably expressing the different IFITMs were infected with HIV-1 and VSV. At a late time after infection of the cell culture, supernatants containing newly-produced virions were harvested and divided in two fractions that were either incubated with CD45-conjugated microbeads or left untreated. After the microbeads removal, virion particles were purified by ultracentrifugation, normalized and then used for WB and infectivity analyses. The WB panels present typical results obtained, while the graph presents averages and SEM obtained in 3 independent experiments. No statistically significant differences were observed between depleted and non-depleted fractions, after a Student t test.</p