c-Myc activation is not associated with degree of RHPS4 sensitivity.

<p>(A) c-Myc transcription factor assay. Jurkat cell nuclear extracts show activation and specificity of c-Myc proportional to concentration of extract analyzed and in the presence of wild-type or mutant competitor. (B–C) No significant difference in c-Myc activation was observed between untreated PFSK-1 or C6 cells and RHPS4-treated cells. Asterisk denotes significant reduction in c-Myc levels when either PFSK-1 or C6 untreated cells were exposed to a wild-type oligonucleotide competitor (p≤0.05). (D–E) c-Myc quantitative reverse transcriptase PCR. No difference in PFSK-1 or C6 c-Myc gene expression was observed between representative RHPS4-treated cells and untreated cells.</p

Acute RHPS4 exposure is associated with telomerase inhibition in brain tumor cells <i>in vitro</i>.

<p>(A) TRAP assay using ethanol-precipitated telomere extended DNA products after 30 minutes extension in non-drug treated brain tumor cells. High levels of telomerase activity are observed in each cell line. (B–D) TRAP assays in RHPS4-treated brain tumor lysates reveals complete telomerase inhibition in all cell lines at each drug concentration. 0.1 µg of total protein lysate was loaded per well in each TRAP assay. <i>CHAPS, CHAPS buffer only no lysate control; TS, telomere substrate internal control 61-bp oligonucleotide.</i></p

Acute RHPS4 exposure inhibits proliferation of high grade brain tumor cells <i>in vitro</i>.

<p>Proliferation of tumor cells was impaired in malignant brain tumor cells after acute 72 hours exposure to RHPS4. (A–C) PFSK-1, DAOY, U87 and (E) Res196 cells exhibited IC₅₀ values of 2.7, 2.2, 1.1, and 1.6 µM respectively when 0.5–5.0 µM RHPS4 was used, representing a significant inhibition of cell proliferation (p≤0.05 for each drug concentration versus untreated). (D, F–G) Within this concentration range, KNS42, C6 and GB-1 cells were resistant to RHPS4. (H–I) At higher concentrations of RHPS4 exposure C6 and GB-1 cells exhibited IC₅₀ values of 26 µM and 32 µM respectively, representing a significant inhibition of cell proliferation (p≤0.05 for each drug concentration versus untreated). Error bars indicate standard error from three independent experiments. (J–M) Light microscopy of PFSK-1, DAOY, C6 and GB-1 cells showing a marked reduction in cellular density after RHPS4 exposure. <i>Magnifications, x20; Scale bar = 25 </i><i>µm</i>.</p

Acute RHPS4 exposure alters cell cycle dynamics of brain tumor cells <i>in vitro</i>.

<p>PFSK-1 cells exhibited a dose-dependent increase in the proportion of cells in G1-phase. In contrast DAOY, C6 and GB-1 cells exhibited a dose-dependent increase in the proportion of cells in S-phase. PFSK-1 further shows a moderate accompanying increase of sub-G0/1 cells at the higher RHPS4 concentration (5 µM). Percentages are the mean from three independent experiments. Asterisk denotes a significant difference relative to untreated cells.</p

RHPS4 sensitivity in normal neural and endothelial cells <i>in vitro</i> and <i>ex vivo</i>.

<p>(A) C17.2 cerebellar progenitor cells and (B) HBMEC endothelial cells are sensitive to RHPS4 with an IC₅₀ of 15 µM and 5 µM respectively. (C) Primary rat ependymal <i>ex vivo</i> cultures exhibited functional impairment of ependymal CBF after 3 µM or 30 µM RHPS4 exposure (p≤0.01). (D) A significant reduction in cilia tip distance was observed after either 3 µM or 30 µM RHPS4 exposure (p≤0.01). Error bars represent standard error of the mean from four separate rat brains/experiments.</p

Telomere length measurement and RHPS4-mediated inhibition of Taq polymerase <i>in vitro</i>.

<p>(A) Mean TRF lengths for PFSK-1/DAOY cells (3.8 kb/7.8 kb) and C6/GB-1 (7.5 kb/3.9 kb) cells were determined prior to RHPS4 exposure. (B) Quantitative TRAP assay showing telomerase-mediated telomere extension after 30 minutes (standard TRAP assay) or 5 minutes extension time in non-drug exposed cells. (C) PCR gel showing telomere extension products after 5 minutes extension time in non-drug exposed cells. <i>1, no lysate control; 2–5, PFSK-1, DAOY, C6 and GB-1</i>. (D) PFSK-1 and DAOY showed low levels of telomerase activity at low RHPS4 concentrations and complete absence of activity at high RHPS4 concentrations, when RHPS4 was added pre-telomere extension. C6 and GB-1 showed complete absence of telomerase activity at both RHPS4 concentrations analyzed. (E) Telomerase activity was absent in all cell lines and at both RHPS4 concentrations when RHPS4 was added post-telomere extension. <i>CHAPS, CHAPS buffer only no lysate control; IC, internal control 61-bp telomere substrate oligonucleotide</i>.</p

data_sheet_1_Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages.PDF

<p>GPR84 is a member of the metabolic G protein-coupled receptor family, and its expression has been described predominantly in immune cells. GPR84 activation is involved in the inflammatory response, but the mechanisms by which it modulates inflammation have been incompletely described. In this study, we investigated GPR84 expression, activation, and function in macrophages to establish the role of the receptor during the inflammatory response. We observed that GPR84 expression in murine tissues is increased by endotoxemia, hyperglycemia, and hypercholesterolemia. Ex vivo studies revealed that GPR84 mRNA expression is increased by LPS and other pro-inflammatory molecules in different murine and human macrophage populations. Likewise, high glucose concentrations and the presence of oxidized LDL increased GPR84 expression in macrophages. Activation of the GPR84 receptor with a selective agonist, 6-(octylamino) pyrimidine-2,4(1H,3H)-dione (6-n-octylaminouracil, 6-OAU), enhanced the expression of phosphorylated Akt, p-ERK, and p65 nuclear translocation under inflammatory conditions and elevated the expression levels of the inflammatory mediators TNFα, IL-6, IL-12B, CCL2, CCL5, and CXCL1. In addition, GPR84 activation triggered increased bacterial adhesion and phagocytosis in macrophages. The enhanced inflammatory response mediated by 6-OAU was not observed in GPR84^−/− cells nor in macrophages treated with a selective GPR84 antagonist. Collectively, our results reveal that GPR84 functions as an enhancer of inflammatory signaling in macrophages once inflammation is established. Therefore, molecules that antagonize the GPR84 receptor may be potential therapeutic tools in inflammatory and metabolic diseases.</p

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type– and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.</p