After incubation with blocking agent, cells were washed two times with ice-cold FACS buffer and further incubated on ice for 30 min in 100 l of PE-conjugated rat anti-mouse TLR4 (CD284) or with isotype-specific PE-conjugated rat anti-mouse IgG1 prepared in FACS buffer. (7, 8). Further support for its tumor suppressor function has come from the finding that SNPs G446A (W149X) and T442C (C148R) in the gene are associated with familial risk for chronic lymphocytic leukemia (CLL)6 and for breast, prostate, and colorectal cancers (9,C15). On the other hand, ectopic expression of ARL11 in lung carcinoma was reported to induce apoptosis, suggesting that ARL11 down-regulation promotes tumor cell survival (8). A high degree of conservation of homologs in metazoans such as zebrafish, transcripts are mostly abundant in lymphoid tissues (spleen, bone marrow, and lymph nodes), which is also supported by co-expression analysis from data mining approaches (8, 14). We also searched for transcript expression in different immune cell types compiled in the Immunological Genome Project (ImmGen) database (https://www.immgen.org/)7 (37) and found that transcripts of predominated in macrophages, followed by monocytes and neutrophils. This led us to investigate the function of this uncharacterized protein in macrophages. Here, we demonstrate that ARL11 expression is usually up-regulated upon lipopolysaccharide (LPS) stimulation in macrophages and regulates the pro-inflammatory macrophage effector functions. ARL11 was required for LPS- or pathogen-mediated activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPKs). Notably, ERK1/2 colocalized with ARL11 at the cortical actin structures, and the two proteins interacted with each other, dependent upon ERK1/2 phosphorylation status. Taken together, our findings reveal that ARL11 regulates activation of the ERK1/2 MAPK signaling pathway in response to LPS stimulation and thereby regulates multiple pro-inflammatory effector Betamethasone hydrochloride functions of macrophages. Results ARL11 is expressed in macrophages, and its expression is enhanced upon LPS stimulation Computational analysis of transcript levels in different immune cell types using the ImmGen database revealed that was predominantly expressed in macrophages, monocytes, and neutrophils. To test this, we verified ARL11 expression in cell lysates from primary bone marrowCderived mouse macrophages (BMDMs), mouse macrophage cell lines (RAW264.7 and J774 cells), and a human monocyte-derived macrophage cell line (phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells) using an anti-peptide antibody raised against the N-terminal 17 amino acids of ARL11, a region that is identical in both human and mouse ARL11 protein (Fig. 1siRNACtransfected cell lysates (Fig. 1, and and sequence (identical between human and mouse ARL11 protein) represents the peptide sequence against which ARL11 antibody was generated. indicate the specific band as labeled, and -tubulin was used as the loading control. and and and and and gene silencing alters cell proliferation, we decided the growth rate of control shRNAC and shRNACtransfected RAW264.7 cells using alamarBlue? dye reduction. No significant differences in the proliferation rate of ARL11-depleted RAW264.7 cells were observed as compared with the control cells (Fig. S2with expression inhibits phagocytosis, LPS-induced pro-inflammatory cytokine secretion, and nitric oxide production in macrophages. and #shRNACtransfected RAW264.7 cells stimulated with 1 g/ml LPS for 24 h. A typical multiple-pseudopodia formation was observed in the case of control cells, whereas these morphological changes were inhibited upon silencing. and bioparticles in ARL11-depleted macrophages. Control shRNAC and shRNACtransfected RAW264.7 cells untreated or treated with 1 g/ml LPS for 24 h were allowed to phagocytose Alexa Fluor 488Cconjugated bioparticles. After 30 min of uptake, the cells were washed and analyzed by flow cytometry. The histograms show the flow cytometry results of analyzing the macrophages for Alexa Fluor 488 signal (= 3; ***, < 0.001; ****, < 0.0001; Student's test). and and and siRNA. After 72 h of siRNA transfection, Betamethasone hydrochloride cells were stimulated with 100 ng/ml LPS for the indicated time periods, supernatants from the cultures were collected, and the concentration of IL-6 (= 3) (< 0.01; ***, < 0.001; ****, < 0.0001; Student's test). To determine ARL11's role in regulating macrophage effector functions, we first examined the phagocytic ability of control and ARL11-depleted cells. To this end, we analyzed phagocytosis of Alexa Fluor 488Cconjugated bioparticles in control and ARL11-depleted cells by flow cytometry. Notably, the Betamethasone hydrochloride phagocytic capacity of silencing led to significantly lower IL-6 and TNF production, as determined by quantitative RT-PCR and enzyme-linked immunoassay (Fig. 2, shRNA, 3-fold increase from 0 to 48 h). We noted that this impaired LPS-mediated effector responses in ARL11-depleted RAW264.7 cells were not due to reduced cell-surface levels of TLR4, suggesting that ARL11 acts downstream of the TLR4 pathway (Fig. S2and and and and and and (and in RAW264.7 Rabbit Polyclonal to RALY cells and upon silencing in PMA-differentiated THP-1 cells (Fig. S3, and and shRNA 1Ctransfected RAW264.7 cells were treated with 1 g/ml LPS for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total Betamethasone hydrochloride ERK1/2, p38, JNK1/2, and.