Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-3

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p>ed serially by site-directed deletion mutagenesis and designated as DS1, DS3, and DS2, respectively. HEK 293 cells were either untransfected (UN) or transfected with individual variants or vector control (VC) plasmid, as indicated at the top. The cells were divided into two groups for Western blotting or RT-PCR analyses 24 h after transfection. . Each lysate (30 μg) was resolved on a 10% SDS-polyacrylamide gel, immunoblotted with HA-specific antibody, and reprobed with anti-β-Actin antibody as a loading control. . Total RNA was extracted from each transfectant and reverse transcribed. PCR reactions using P3 primers, as indicated at the bottom of , were utilized and the resultant amplicons were resolved on 1% agarose gels. A β-Actin transcript and its protein were used as loading controls, as indicated on the right

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-6

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p> not for 96 h. The cell counts for each transfectants were quantified by a standard MTT assay. The results are presented as the means ± SEM of three independent experiments. ** Indicates that the DM3 variant rescued the cells from TGFβ-induced growth suppression (< 0.01). . Transfectants (2 × 10cells) expressing DM3, DM4, or vector control (VC) were seeded and stimulated with (+) or without (-) 5 ng/mL of TGFβ1 for 72 h. The cells were collected and stained with Annexin V and propidium iodide for detection of apoptotic cells. The percentage of the apoptotic cells in the total cell population was determined by flow cytometry. Data are presented as the fold increases in cell apoptosis compared with those of corresponding untreated cells

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-1

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p>the top. The riboprobes were synthesized by T7 RNA polymerase using T7 promoter-containing DNA fragment of individual variant as a template. The predicted protected fragments are 263 bp for D1 (DM1), 192 bp for D2 (DM2), 151 bp for D3 (DM3), and 90 bp for D4 (DM4). the mixed riboprobes (M) were incubated with (+) or without (-) RNase A/T1, respectively. the total RNA (10 μg aliquots) was hybridized with individual purified antisense riboprobes and the protected fragments were resolved on a 6% acrylamide/8 M urea sequencing gel. * Indicates an unidentified transcript variant in CA1109 tumor cells protected by the anti-DM3 riboprobe

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-7

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p>ted PCR reactions amplified coding sequences using two pairs of primers, P1 and P2, as indicated at the top of . The resultant amplicons were resolved in a 1% agarose gel followed by gel purification, gene cloning, and direct sequencing. The nucleotide sequences flanking the truncated region(s) in each variant are marked at the top of . . DM1 and DM2 are in-frame variants lacking nucleotides 16–75 and 15–80, respectively, located in the signal sequence of the gene. DM3 and DM4 represent cytoplasmic variants lacking transmembrane and partial intracellular domains, as indicated

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-4

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p>sate system. S-labeled proteins were resolved on a 10% SDS-polyacrylamide gel and visualized by autoradiography. The control plasmid (C) encoded for luciferase polypeptide (61 KDa). . Cells were transfected with a plasmid encoding for wild-type TβRI (WT) or the DM1 variant. After 24 h transfection, the transfectants were treated with 50 μg/mL cycloheximide (CHX) for the designed time courses, as indicated at the top. WT (15 μg) and DM1 (150 μg) transfectant lysates were analyzed by Western blotting using an anti-HA antibody. . Transfectants were treated simultaneously with CHX and 5 μM, 20 μM MG132, or DMSO (vehicle control) for 30 min in . Transfectants treated with CHX and 20 μM MG132 or 10 μM lactacystin (Lata) for 60 min were shown in . Protein levels were determined by Western blotting of WT, DM1, and DM2 transfectant lysates using an anti-HA antibody. The same blot was reprobed with β-actin antibody as a control

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-5

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p>he bottom, 1 μg of p3TP-Lux, and 0.05 μg of pRL-TK plasmid which was used to correct the differences in transfection efficiency between experiments. After transfection for 24 h, the cells were treated with TGFβ1 (5 ng/mL) for 24 h and luciferase activities in each lysates were determined using a dual-luciferase reporter assay system (Promega). Data are the mean ± SEM luciferase activities of four independent experiments normalized to individual protein level. . R1B cells were transfected with each variant gene as indicated. The cell growth rates of the transfectants in the presence or absence of TGFβ1 were determined using standard MTT assays as described in the "Methods" section. Data are presented as the mean ± SEM of ODabsorbance values of three independent experiments

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells"</p><p>http://www.biomedcentral.com/1471-2199/8/72</p><p>BMC Molecular Biology 2007;8():72-72.</p><p>Published online 17 Aug 2007</p><p>PMCID:PMC1995218.</p><p></p>ted PCR reactions amplified coding sequences using two pairs of primers, P1 and P2, as indicated at the top of . The resultant amplicons were resolved in a 1% agarose gel followed by gel purification, gene cloning, and direct sequencing. The nucleotide sequences flanking the truncated region(s) in each variant are marked at the top of . . DM1 and DM2 are in-frame variants lacking nucleotides 16–75 and 15–80, respectively, located in the signal sequence of the gene. DM3 and DM4 represent cytoplasmic variants lacking transmembrane and partial intracellular domains, as indicated

The functional influence of the RNA transcript complementary to pri-miR-122 for the expression levels of pri-miR-122 and miR-122.

<p>(<b>A</b>) RNA extracted from Huh-7 cells infected with various lenti-ADARs was prepared for assessment of the levels of <b>endogenous</b> miR-122, with that of si-GFP infected cells (set as 1). (<b>B</b>) RNA extracted from HepG2 cells transfected with either the wild-type or the mutant pri-miR-122 containing A-to-G at position −7 was processed for qRT–PCR. (<b>C</b>) The protein lysates extracted from Huh-7 (left) and HepG2 (right) cells infected with various lentiviruses were processed for the Western blotting analysis. (<b>D</b>) RNA extracted from Huh-7 cells infected with either lenti-si-GFP or lenti-si-AS-122, which targets the RNA transcript complementary to pri-miR-122, was processed for qRT–PCR for the antisense transcript (comp-AS-RNA) (left), the pri-miR-122 (middle), and miR-122 (right). (<b>E</b>) The HepG2 cells transfected with the control vector or the plasmid construct expressing comp-AS-RNA were processed for qRT–PCR, for examining the levels comp-AS-RNA (left), pri-miR-122 (middle), and miR-122 (right). The results were shown as the average of three experiments (mean ± SD), and the <i>P</i> values were calculated by <i>t</i> test (*<i>P</i><0.05; ***<i>P</i><0.001).</p

Antisense transcripts exist for pri-miR-214 and pri-miR-122, with A-to-I/(G) RNA editing at specific nucleotide residues.

<p>Schematic illustration of the gene structures for the RNA transcripts complementary to (<b>A</b>) pri-miR-214 and (<b>B</b>) pri-miR-122. (<b>C</b>) The representative results for sequencing of the strand-specific RT–PCR products from HCC with putative editing events in the sense (<b>S</b>) and antisense (<b>AS</b>) transcripts of pri-miR-214 and pri-miR-122, focusing on the regions covering the sites with potential editing events with the editing marked with red asterisks.</p

Specific editing of the RNA transcripts related with pri-miR-214 and pri-miR-122 in Huh-7 cells infected with lenti-ADAR2 lentiviruses.

<p>(<b>A</b>) The cells were uninfected (mock), infected with lenti-sh-luc (negative control), or infected with the lenti-ADAR1S, ADAR1L, and ADAR2 individually. The cell lysates were processed for Western blotting by probing with Abs as indicated at the bottom of each panel. The HRM results for miR-214 (<b>B</b>) and miR-122 (<b>C</b>). The raw data for the relative signals are shown in the upper panel; and the difference of the relative signal by comparing with the “no editing” standard are shown in the lower panel. The stem loop structures for pre-miR-214 (<b>D</b>) and pre-miR-122 (<b>E</b>) are illustrated schematically, with the nucleotides corresponding to mature miRs labeled with green. The positions of the nucleotides changed by overexpression of ADAR2 are marked in red, with the numbers showing their positions relative to the first nucleotide of mature miRs (as position no. 1). The summary results of the nucleotide changes in precursors of these two miRNAs are shown at the right panel, by sequencing of 100 clones from TA cloning of the RT–PCR products amplified by the RNA from lenti-ADAR2 infected cells.</p