Effect of MAPK inhibitors on the transcription of miR-155, miR-455, miR-146 and miR-125a.

<p>(A) BMDMs were treated for 1 h where indicated with 2 µM PD 184352, 5 µM SB203580 or 10 µM SP 600125. Cells were then stimulated with 100 ng/ml LPS for 1 h and total RNA isolated. The levels of pri-miR-155, pri-miR-455, pri-miR-146a, pri-miR-146b and pri-miR-125a were determined by Q-PCR. 18S levels were used as a loading control. Error bars represent the standard deviation of independent stimulations on cultures from 4 mice. (B) As (A) except that cells were pretreated for 1 h with 2 µM PD 184352 or 5 µM SB203580 were indicated and then stimulated with 10⁶ cells/ml heat killed <i>C. albicans</i> for 4 h. For comparison of LPS or <i>C. albicans</i> alone relative to inhibitor treated conditions, <i>p</i> values of less than 0.05 are indicated by * and less than 0.01 by ** (students <i>t</i>-test).</p

Regulation of miRNA by IL-10.

<p>A) BMDMs were stimulated with either 100 ng/ml IL-10, 100 ng/ml LPS or a combination of both IL-10 and LPS for 1 or 6 h. Total RNA was isolated, and the levels of pri-miR-155, pri-miR-455, pri-miR-146a, pri-miR-146b, pri-miR-125a were determined by Q-PCR. Error bars represent the standard deviation of independent cultures from 4 mice. B) BMDMs were cultured from either wild type (black bars) or IL-10 knockout (white bars) mice and stimulated with 100 ng/ml LPS for the indicated times. RNA was isolated and pri-miR-155 levels determined by Q-PCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype.</p

Primer sequences used for Q-PCR.

<p>Primer sequences used for Q-PCR.</p

Heat killed <i>Candida albicans</i> stimulates the transcription of miR-155, miR-455, miR-146 and miR-125a.

<p>BMDMs were isolated from C57/Bl6 mice. Cells were stimulated with 10⁶ cells/ml of heat killed <i>C. albicans</i> for the indicated times. Cells were then lysed and total RNA isolated as described in the methods. (A) The levels of miR-155, miR-455, miR-146a, miR-146b, miR-125a-5p (grey bars) and miR-125a-3p (black bars) were determined by a Taqman based Q-PCR (Applied Biosystems). 18S levels were used to normalise the amount of RNA in the reaction. (B) The levels of pri-miR-155, pri-miR-455, pri-miR-146a, pri-miR-146b and pri-miR-125a were determined by Q-PCR. 18S was used as a loading control. Error bars represent the standard deviation of independent stimulations cultures from 4 mice.</p

Heat killed <i>Candida albicans</i> stimulates ERK1/2, p38 and NFκB.

<p>(A) BMDMs were stimulated for the indicated times with 10⁶ cells/ml heat killed <i>C. albicans</i>. Cells were lsyed in SDS sample buffer and the levels of phospho ERK1/2, total ERK1/2, phospho p38, total p38, phospho JNK, phospho p105 and total IκB were determined by immunoblotting. (B) BMDMs were incubated with 2 µM PD184352 or 5 µM SB203580 as indicated for 60 min prior to stimulation with 10⁶ cells/ml heat killed <i>C. albicans</i> for 60 min. Cells were lsyed in SDS sample buffer and the levels of phospho ERK1/2, total ERK1/2, phospho p38, total p38 and phospho MK2 were determined by immunoblotting. (C) BMDMs were stimulated with 10⁶ cells/ml heat killed <i>C. albicans</i>. Total RNA was isolated and the levels of IκBα mRNA determined by Q-PCR. Error bars represent the standard deviation from 4 independent cultures. (D) Where indicated BMDMs were treated with 15 µM BMS 345541 or 10 µM BAY 65-1942 for 1 h. Cells were then stimulated with 10⁶ cells/ml heat killed <i>C. albicans</i> for 4 h, and IκB levels determined by Q-PCR. Error bars represent the standard deviation from 4 independent cultures.</p

miRNAs up-regulated by LPS treatment of BMDMs.

<p>Microarray analysis was carried out by LC Sciences on BMDMs either treated with heat killed <i>C. albicans</i> for 16 h or left untreated. Average fold induction and <i>p</i> values were obtained from analysis of duplicate experiments. The miRNAs that showed a fold change greater than 1.5 and <i>p</i> value of <0.01 are represented.</p

miR-146b, mir-155, miR-455 and miR-125a are located in annotated genes.

<p>(A) The locations of the miRNA are shown relative to the genes in which they are contained. The structure of the genomic loci are shown, with coding exons shown by filled boxes and non-coding exons by open boxes. miR-155 is located in the exon of the non-protein coding BIC gene, while miR-146b and miR-455 are encoded in the introns of Tmem180 and Col27a1 respectively. miR-125a is located 5′ to the Ncrna00085 transcript. ESTs suggest that a further exon (hatched box), containing miR-125a, exists upstream of Ncrna00085. (B) BMDMs were stimulated with 100 ng/ml of LPS (upper panel) or 10⁶ cells/ml heat killed <i>C. albicans</i> (CA, lower panel) for the indicated times. Cells were then lysed and total RNA isolated as described in the methods. The levels of mRNA for Col27a1 were determined by Q-PCR. Error bars represent the standard deviation of stimulations from 4 independent cultures. (C) As (B) except that the levels of Tmem180 using primers spanning exons 3 to 4 were measured.</p

Effect of IKKβ inhibitors on the transcription of miR-155, miR-455, miR-146 and miR-125a.

<p>(A) BMDMs were treated for 1 h where indicated with 15 µM BMS 345541 or 10 µM BAY 65-1942. Cells were then stimulated with 100 ng/ml LPS for 1 h and total RNA isolated. The levels of pri-miR-155, pri-miR-455, pri-miR-146a, pri-miR-146b and pri-miR-125a were determined by Q-PCR. 18S was used as a loading control. Error bars represent the standard deviation of independent stimulations cultures from 4 mice. (B) As (A) except that cells were stimulated with 10⁶ cells/ml heat killed <i>C. albicans</i> for 4 h. (C) Where indicated BMDMs were pretreated with 2 µM PD 184352, 10 µM BAY 65-1942 or 15 µM BMS 345541 as indicated. Cells were stimulated with 100 ng/ml LPS or 10⁶ cells/ml heat killed <i>C. albicans</i> for 30 min and the levels of phospho ERK1/2, total ERK1/2, phospho MK2 and phospho p105 were determined by immunoblotting. (D) As (C) except that total RNA was isolated and the levels of pri-miR-125a determined by Q-PCR. Error bars represent the standard deviation of independent stimulations cultures from 4 mice.</p

miRNAs up-regulated by heat killed <i>Candida albicans</i> treatment of BMDMs.

<p>Microarray analysis was carried out by LC Sciences on BMDMs either treated with heat killed <i>C. albicans</i> for 16 h or left untreated. Average fold induction and <i>p</i> values were obtained from analysis of triplicate experiments. The miRNAs that showed a fold change greater than 1.5 and <i>p</i> value of <0.01 are represented.</p

Expression analysis of Ins1/2; Gcg and MafA upon SB-747541A treatment.

<p>(A-C) Co-expression and quantification of Ins1/2 and Gcg expression domains by immunohistochemistry on pancreatic sections from E15.5 stage explants cultured for 4 days in DMSO or SB747541A. P-values = 4×10⁻⁴ for Ins1/2+;Gcg- and 0.02 for Gcg+;Ins1/2-, n≥5 pancreata each for DMSO and SB-747651A treatment and for each biological replicate, n = 3 biological replicates. (D-H) Expression and quantification of MafA positive cells in pancreatic explants treated with DMSO (D, F) and SB-747541A (E, G) for 4 days. Scale bar on all panels = 25μm. Values are averages of 2 independent experiments ± standard error unless otherwise stated. All P-values were calculated by Student’s t test, comparing DMSO and SB-747651A treated samples in 2 independent experiments, *≤ 0.05, **≤ 0.01, ***≤0.001, two-tailed.</p