Slfn4 over-expression caused splenomegaly.

<p>(A) Macroscopic appearance of spleens from <i>Slfn4</i> over-expressing mice (right) and from MacBlue littermate controls (left). (B) Organ weights are expressed as percentage of body weight. Data are combined from four independent experiments and are displayed as mean + SEM.</p

Putative regulatory elements within the <i>Slfn4</i> promoter.

<p>The <i>Slfn4</i> proximal promoter is a TATA-containing promoter with putative binding sites for STAT1, IRF family members, PU.1 and AP1. The TSS, designated +1, is marked with an arrow.</p

Increased macrophages and granulocytes in spleen from MacBlue/UAS-<i>Slfn4</i>-V5 mice.

<p>Splenocytes from MacBlue littermate controls (A) and MacBlue/UAS-<i>Slfn4</i>-V5 mice (B) were stained for the cell surface markers F4/80 (macrophage), Ly-6G (granulocyte), B220 (CD45R, B cell), and CD3ε (T cell). The samples were analysed by FACS and quadrants were set based upon isotype control profiles. Data are representative of two independent experiments.</p

Induction of <i>Slfn</i> expression by LPS in BMM, and in the CIA mouse model.

<p>(A) BMM were stimulated with LPS over a time course (0h no treatment control, 2h, 4h, 8h, 24h), RNA was extracted and reverse transcribed, and the expression of <i>Slfn1</i>, <i>Slfn2</i>, <i>Slfn4</i>, <i>Slfn5</i>, <i>Slfn8</i>, and <i>Slfn9</i> was determined using quantitative real-time PCR. Data (mean + SEM) are combined from 3 independent experiments. * <i>P</i>&lt;0.05 compared to control; ** <i>P</i>&lt;0.01 compared to control; *** <i>P</i>&lt;0.001 compared to control. (B) RNA from whole joints (disease affected, score 3; or unaffected, score 0) from mice with CIA was used to synthesise cDNA. mRNA expression of <i>Slfn1</i>, <i>Slfn2</i>, <i>Slfn4</i>, <i>Slfn5</i>, <i>Slfn8</i>, <i>Slfn9</i>, <i>csf1r</i>, and <i>Emr1</i> was determined using real-time PCR. The data, displayed as fold induction relative to control (score 0), are combined from two independent experiments (mean + range).</p

Increased neutrophils and macrophages in the livers and spleens of MacBlue/UAS-<i>Slfn4</i>-V5 mice.

<p>(A) Hematoxylin and eosin staining was performed on liver paraffin sections. Arrowheads indicate aggregates of neutrophils in the liver. Liver sections from MacBlue/UAS-<i>Slfn4</i>-V5 and MacBlue littermate controls were also stained for F4/80 (brown). Arrows indicate F4/80 expressing cells surrounding aggregates of neutrophils. (B) Hematoxylin and eosin staining was also performed on spleen paraffin sections. Arrows indicate clusters of megakaryocytes. Spleen sections from MacBlue/UAS-<i>Slfn4</i>-V5 and MacBlue littermate controls were also stained for F4/80 (brown). Sections were examined using an Olympus BX-51 microscope with a DP-70 digital camera and DP controller imaging software (Olympus). RP, red pulp; WP, white pulp.</p

Decreased monocyte and elicited macrophage numbers in Slfn4 over-expressing mice.

<p>BM, bone marrow; PBL, peripheral blood leukocytes; TEPM, thioglycollate-elicited peritoneal macrophages. FACS data are combined from at least three independent experiments. Data represent mean ± SEM. For BM and PBL, data were collected from two independent transgenic lines and littermate controls. For TEPM, data were generated from the transgenic line with the highest transgene expression (F4) and littermate controls.</p><p>*<i>P</i>&lt;0.05;</p><p>**<i>P</i>&lt;0.01.</p

<i>Slfn4</i> mRNA is induced via autocrine type I IFN and is down-regulated during macrophage differentiation.

<p>(A) BMM were stimulated with LPS (10 ng/ml), dsRNA Poly(I∶C) (30 µg/ml), Imiquimod (15 µg/ml), Zymosan (150 µg/ml), synthetic phosphorothioate CpG oligonucleotide 1668S (0.3 µM) and non-stimulatory control oligonucleotide 1668S-GC (0.3 µM), Pam3Cys (15 ng/ml), or IFN-γ (500 pg/ml) over a 24h time course. The data, displayed as fold induction relative to control (0h), are combined from two independent experiments (mean + range). (B) BMM from IFNAR-1^−/− and wild type mice were stimulated with LPS over a time course. The data are combined from two independent experiments (mean + range). C) HMDM were stimulated with LPS over a time course (0h no treatment control, 2h, 6h, 24h). The data are combined from eight independent donors (mean + SEM). (D, E) Bone marrow progenitors were differentiated into BMM in the presence of CSF-1 over a 6-day time course. Data are combined from three independent experiments (mean + SEM). <i>Slfn4</i> (A–D) and <i>csf1r</i> (E) mRNA expression was determined using quantitative real-time PCR. * <i>P</i>&lt;0.05 compared to 0h or day 0 control; ** <i>P</i>&lt;0.01 compared to 0h or day 0 control; *** <i>P</i>&lt;0.001 compared to 0h or day 0 control.</p