Journal: Nucleic Acids Research

Article Title: Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF

doi: 10.1093/nar/gkp577

Figure Lengend Snippet: Translational repression of c/ebp Beta isoforms by miR-155. ( A ) Schematic overview of the c/ebp Beta transcript. The miR-155 target site is indicated in the 3′ UTR with the 8-nt seed match boxed. TAD, transactivation domain; bZIP, basic region leucine zipper domain; ATG, translational start site; LAP, liver-enriched transcriptional activation protein; LIP, liver-enriched transcriptional inhibitory protein. ( B ) Assessment of luciferase reporter activity in HeLa cells cotransfected with pre-miR-155 in combination with either the Renilla/firefly luciferase psiCHECK2 control, the psiCHECK2 luciferase construct (miR-155 sensor) harbouring the miR-155 perfect match target site, the psiCHECK2 construct harbouring the c/ebp Beta 3′ UTR, or the psiCHECK2 c/ebp Beta 3′ UTR Mut construct harbouring a mutated miR-155 target site. Values represent mean ± SD ( n = 3). The pre-miR-155 significantly represses the luciferase activity of the c/ebp Beta 3′ UTR sensor construct (*** indicates P < 0.001). ( C ) Western blot analysis of c/ebp Beta LAP*, LAP and LIP isoforms and Pu.1 in Raw264.7 cell lysates. Protein extracts from Raw264.7 cells cotransfected with 5 nM pre-miR-155 in combination with either LNA control (5 nM) or LNA-antimiR (5 nM) were subjected to western blot analysis. (Right) Quantification of the LIP and LAP protein bands shown on the western blot. Values represent mean ± SD ( n = 3). ( D ) Western blot analysis of the c/ebp Beta LAP and LIP isoforms and Pu.1 protein in THP-1 cell lysates. Protein extracts from THP-1 cells cotransfected with pre-miR-155 (5 nM) in combination with either LNA control (5 nM) or LNA-antimiR (5 nM) were subjected to western blot analysis. Data are representative of three independent experiments.

Article Snippet: A primary monoclonal mouse-anti-c/ebp Beta antibody (SC-7962, Santa Cruz Biotechnology), a primary rabbit-anti-PU.1 (#2258, Cell signaling Technology), a primary mouse-anti- Tubulin-alpha Ab-2 (MS-581-P, Neomarkers) and HRP-conjugated secondary rabbit anti-mouse (P0447, DAKO) or swine anti-rabbit antibodies (P0399, DAKO) were used according to the manufacturer's instructions.

Techniques: Activation Assay, Luciferase, Activity Assay, Construct, Western Blot

Journal: Nucleic Acids Research

Article Title: Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF

doi: 10.1093/nar/gkp577

Figure Lengend Snippet: Regulation of c/ebp Beta isoforms by endogenous miR-155 in Raw264.7 cells during LPS stimulation. Western blot analysis of c/ebp Beta LAP*, LAP and LIP isoforms in Raw264.7 cell lysates. Protein extracts from Raw264.7 cells either mock (control) or LNA-antimiR (5 nM) transfected before treatment with 100 ng/ml LPS for 18 h were subjected to western blot analysis. (Right) Quantification of the LIP, LAP and LAP* protein bands shown on the western blot. Values represent mean ± SD ( n = 3) (** indicates P < 0.01; * indicates P < 0.05). Data represent three independent experiments.

Article Snippet: A primary monoclonal mouse-anti-c/ebp Beta antibody (SC-7962, Santa Cruz Biotechnology), a primary rabbit-anti-PU.1 (#2258, Cell signaling Technology), a primary mouse-anti- Tubulin-alpha Ab-2 (MS-581-P, Neomarkers) and HRP-conjugated secondary rabbit anti-mouse (P0447, DAKO) or swine anti-rabbit antibodies (P0399, DAKO) were used according to the manufacturer's instructions.

Techniques: Western Blot, Transfection

Journal: Nucleic Acids Research

Article Title: Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF

doi: 10.1093/nar/gkp577

Figure Lengend Snippet: miR-155 regulates c/ebp Beta in the splenocytes of LPS-treated mice. ( A ) Fluorescence-microscopy analysis of LNA oligonucleotide uptake in mouse splenocytes sorted in either B-cell (CD19) or monocytic (CD11b) cell fractions. Mice were dosed intravenously with 25 mg/kg FAM-labeled LNA oligonucleotide for three consecutive days, and spleens were dissected the day after last dose. ( B ) Quantitative RT-PCR analysis of miR-155 expression in splenocytes of mice treated with LNA-antimiR or LNA control by three intravenous injections of 25 mg/kg over three consecutive days. Spleen samples were dissected 2 h after intraperitoneal injection of 0.5 mg/kg LPS. Values represent mean ± SD ( n = 5). The LNA-antimiR compound effectively antagonizes miR-155 expression in LPS-treated mice compared to saline treated controls (*** indicates P < 0.001). ( C ) Western blot analysis of the c/ebp Beta LAP and LIP isoforms in splenocytes of mice treated with saline ( n = 3), LNA-antimiR ( n = 5) or LNA control ( n = 5) by three intravenous injections of 25 mg/kg over three consecutive days. Spleen samples were dissected 24 h after intraperitoneal injection of 0.5 mg/kg LPS. (Right) Quantification of the LIP and LAP protein bands shown on the western blot. Systemically delivered LNA-antimiR compound derepresses the LIP isoform of c/ebp Beta compared to both saline- and LNA control-treated mice (** P < 0.01). Values represent mean ± SD.

Article Snippet: A primary monoclonal mouse-anti-c/ebp Beta antibody (SC-7962, Santa Cruz Biotechnology), a primary rabbit-anti-PU.1 (#2258, Cell signaling Technology), a primary mouse-anti- Tubulin-alpha Ab-2 (MS-581-P, Neomarkers) and HRP-conjugated secondary rabbit anti-mouse (P0447, DAKO) or swine anti-rabbit antibodies (P0399, DAKO) were used according to the manufacturer's instructions.

Techniques: Fluorescence, Microscopy, Labeling, Quantitative RT-PCR, Expressing, Injection, Western Blot