Journal: BioMed Research International

Article Title: Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

doi: 10.1155/2013/637850

Figure Lengend Snippet: Sequences of siRNA used in this report. The double-stranded sequences of siRNA and their locations in the segments and genes of FLuc and RLuc are illustrated. The lower strands represent the antisense strands. The numbering starts from nucleotide A of the start codon.

Article Snippet: The siRNAs were purified by HPLC, and their molecular weights were verified by LC/Mass. siRNA negative control (siNC) used as a negative control siRNA was purchased from MDBio, Inc.

Techniques:

Journal: BioMed Research International

Article Title: Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

doi: 10.1155/2013/637850

Figure Lengend Snippet: Fold repression caused by (a) siFLuc-I and (b) siFLuc-II on the reporter vectors pFluc and pRLuc-f. For transfection, the H1299 cells were grown in 6 cm Petri dishes at 70% confluence. One microgram (1 μ g) pSEAP2-Control, 1 μ g pFLuc or pRLuc-f, and siNC or siFLuc (20 nM final concentration) were mixed and transfected into the cells (see for details). Measurement of fold repression is mentioned in . The data presented are the mean ± SD of triplicates. P values are also indicated.

Article Snippet: The siRNAs were purified by HPLC, and their molecular weights were verified by LC/Mass. siRNA negative control (siNC) used as a negative control siRNA was purchased from MDBio, Inc.

Techniques: Transfection, Control, Concentration Assay

Journal: BioMed Research International

Article Title: Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

doi: 10.1155/2013/637850

Figure Lengend Snippet: Fold repression caused by (a) siRLuc-I and (b) siRLuc-II on the reporter vectors pRluc and pFLuc-r. For transfection, the H1299 cells were grown in 6 cm Petri dishes at 70% confluence. One microgram (1 μ g) pSEAP2-Control, 1 μ g pRLuc or pFLuc-r, and siNC or siRLuc (20 nM final concentration) were mixed and transfected into the cells. Measurement of fold repression is mentioned in . The data presented are the mean ± SD of triplicates. P values are also indicated.

Article Snippet: The siRNAs were purified by HPLC, and their molecular weights were verified by LC/Mass. siRNA negative control (siNC) used as a negative control siRNA was purchased from MDBio, Inc.

Techniques: Transfection, Control, Concentration Assay

Journal: BioMed Research International

Article Title: Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

doi: 10.1155/2013/637850

Figure Lengend Snippet: Secondary structures and thermodynamic stabilities of the two mRNA segments. The mRNA segments of the (a) Fluc gene (91 nt to 454 nt) and (b) RLuc gene (334 nt to 715 nt) were analyzed using an RNAfold program ( http://bibiserv.techfak.uni-bielefeld.de/rnafold/ ). The 2D structures and the entropy assigned to each nucleotide are shown. The siRNA binding sites are indicated by heavy lines.

Article Snippet: The siRNAs were purified by HPLC, and their molecular weights were verified by LC/Mass. siRNA negative control (siNC) used as a negative control siRNA was purchased from MDBio, Inc.

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability

doi: 10.1093/nar/gku978

Figure Lengend Snippet: USP20 promotes CHK1 activation in response to replication stress. (A and B) Depletion of USP20 delayed HU-induced CHK1 activation. 293T cells were transfected with a control siRNA (siCTR) or a USP20-specific siRNA (si1-USP20), cells were treated with HU 2 days after transfection. Total cell lysates were harvested at different time points after HU treatment and used for immunoblotting with antibodies as indicated. The efficiency of USP20 knockdown was determined by immunoblotting with antibodies against USP20 and β-actin (A). (C) Expression of siRNA-resistant form of USP20 rescued USP20-depletion-induced delay of CHK1 activation in response to HU treatment. 293T cells were transfected with siCTR or si1-USP20. USP20-depleted cells were introduced 48 h after the first transfection with FLAG vector or FLAG-USP20res, the transfectants were treated 2 days later with HU at different time points. Total cell lysates were harvested and used for immunoblotting with antibodies as indicated. (D) Overexpression of the catalytically inactive mutant USP20(C154S) attenuated HU-induced CHK1 activation. 293T cells were transfected with FLAG-USP20 or FLAG-USP20(C154S). Cells were treated 2 days later with HU at different time points. Total cell lysates were harvested and used for immunoblotting with antibodies as indicated.

Article Snippet: The siRNA oligonucleotide duplexes against USP20 (si1-USP20 sequence: CCATAGGAGAGGTGACCAA; si2-USP20 sequence: GGACAATGATGCTCACCTA), HERC2 (si1-HERC2 sequence: GCGGAAGCCTCATTAGAAA; si2-HERC2 sequence: GAGCTGATTTCTTGAGTAA) and the non-target siRNA control (siCTR) were purchased from Guangzhou RiboBio.

Techniques: Activation Assay, Transfection, Control, Western Blot, Knockdown, Expressing, Plasmid Preparation, Over Expression, Mutagenesis

Journal: Nucleic Acids Research

Article Title: HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability

doi: 10.1093/nar/gku978

Figure Lengend Snippet: USP20 promotes CLASPIN stability through deubiquitination. (A and B) USP20 interacted with CLASPIN. Total cell lysates were extracted from 293T cells (A) or 293T cells co-transfected with MYC-USP20 and FLAG-CLASPIN (B), and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. (C) Schematic structure of CLASPIN fragments. (D) The N terminus of CLASPIN (1–330 AAs) mediated the interaction with USP20. Total cell lysates extracted from 293T cells co-transfected with MYC-USP20 and FLAG-CLASPIN or its fragments were subjected to immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with an anti-MYC antibody. (E and F) Inhibition of USP20 expression decreased CLASPIN protein levels and shortened CLASPIN half-life. The siCTR- or si-USP20-transfected 293T cells were treated with CHX at different time points. Total cell lysates were harvested and used for immunoblotting with antibodies as indicated in (E), and quantification of the CLAPSIN/β-ACTIN ratio was plotted in (F). (G) USP20 deubiquitinated K48-linked polyubiquitination of CLASPIN in vivo . 293T cells were co-transfected with the expression constructs as indicated, total cell lysates were harvested 2 days later and subjected to immunoprecipitation followed by immunoblotting with antibodies as indicated. (H) USP20 depletion-induced delay of CHK1 activation in response to HU treatment was reversed by overexpression of CLASPIN. USP20-depleted 293T cells were transfected with FLAG-VEC or wild-type FLAG-CLASPIN and treated 2 days later with HU at different time points. Total cell lysates were extracted and used for immunoblotting with antibodies as indicated.

Article Snippet: The siRNA oligonucleotide duplexes against USP20 (si1-USP20 sequence: CCATAGGAGAGGTGACCAA; si2-USP20 sequence: GGACAATGATGCTCACCTA), HERC2 (si1-HERC2 sequence: GCGGAAGCCTCATTAGAAA; si2-HERC2 sequence: GAGCTGATTTCTTGAGTAA) and the non-target siRNA control (siCTR) were purchased from Guangzhou RiboBio.

Techniques: Transfection, Immunoprecipitation, Western Blot, Inhibition, Expressing, In Vivo, Construct, Activation Assay, Over Expression

Journal: Nucleic Acids Research

Article Title: HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability

doi: 10.1093/nar/gku978

Figure Lengend Snippet: HERC2 promotes USP20 ubiquitination and subsequent degradation. (A) USP20 interacted with HERC2. Total cell lysates were extracted from 293T cells and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. (B) Both USP20 and CLASPIN protein levels increased in response to HU treatment. 293T cells were treated with HU at different time points as indicated, total cell lysates were extracted and subjected to immunoblotting with antibodies as indicated. (C) Inhibition of HERC2 expression increased protein levels of USP20 and CLASPIN. 293T cells were transfected with siCTR or HERC2-specific siRNA (si1-HERC2 and si2-HERC2). Total cell lysates were harvested 2 days later and subjected to immunoblotting with antibodies as indicated. (D) Inhibition of HERC2 expression decreased the ubiquitination levels of FLAG-USP20. HERC2-depleted 293T cells were co-transfected with expression constructs of FLAG-USP20 and HA-UB. Total cell lysates were harvested 2 days later and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. (E) Depletion of HERC2 promotes HU-induced CHK1 activation. HERC2-depleted 293T cells were treated with HU at different time points as indicated. Total cell lysates were harvested and subjected to immunoblotting with antibodies as indicated. (F) Inhibition of HERC2 expression prolonged the half-life of USP20 and CLASPIN. Mocked- or HERC2-depleted 293T cells were treated with CHX at different time points as indicated, total cell lysates were harvested and subjected to immunoblotting with antibodies as indicated.

Article Snippet: The siRNA oligonucleotide duplexes against USP20 (si1-USP20 sequence: CCATAGGAGAGGTGACCAA; si2-USP20 sequence: GGACAATGATGCTCACCTA), HERC2 (si1-HERC2 sequence: GCGGAAGCCTCATTAGAAA; si2-HERC2 sequence: GAGCTGATTTCTTGAGTAA) and the non-target siRNA control (siCTR) were purchased from Guangzhou RiboBio.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Inhibition, Expressing, Transfection, Construct, Activation Assay

Journal: Nucleic Acids Research

Article Title: HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability

doi: 10.1093/nar/gku978

Figure Lengend Snippet: USP20 promotes genome stability and suppresses xenograft tumor growth. (A) Depletion of USP20 promotes cell proliferation. A549 cells were transfected with siCTR, si1-USP20 or si2-USP20, transfectants were split 2 days later into a 96-well plate (5000 cells per well) and treated 16 h later with different doses of HU for 72 h. Cell proliferation was determined by the MTT assay. The USP20 knockdown efficiency is shown in Supplementary Figure S10. (B and C) Depletion of USP20 compromised UV-induced intra-S-phase checkpoint. Mock- or USP20-depleted A549 cells described in panel (A) were UV irradiated with a dose of 20 J/m 2 , pulse-labeled 40 min later with BrdU for 20 min, fixed with 75% ethanol and stained with an anti-BrdU antibody and DAPI (B). BrdU-positive cells in randomly selected fields (more than 100 cells per treatment) were quantified in (C). Three independent experiments were performed. (D–F) USP20 depletion resulted in chromosome aberrations. A549 cells with mocked- or shRNA-mediated USP20 depletion (D) were subjected to a mitotic spread assay. Representative metaphase spreads were shown in (E) and the arrows pointed to the chromosome aberrations. A histogram summarizing percentages of spreads containing aberrant chromosomal structures was shown in (D). More than 80 spreads were randomly selected for analysis. Three independent experiments were performed. (G and H) USP20 suppressed xenograft tumor growth. Note that 5 × 10 6 mock-depleted (shCTR) or USP20-depleted (sh-USP20) HCT116 cells were injected subcutaneously into the right lower flanks of BALB/c nude mice. The tumor volume was measured starting from day 9 after injection and ending at day 51 at a frequency of twice a week. Tumor volumes were shown as mean ± SEM, and the Student's t -test was performed. Tumor images were shown in (G), while quantification results were shown in (H). * P < 0.05.

Article Snippet: The siRNA oligonucleotide duplexes against USP20 (si1-USP20 sequence: CCATAGGAGAGGTGACCAA; si2-USP20 sequence: GGACAATGATGCTCACCTA), HERC2 (si1-HERC2 sequence: GCGGAAGCCTCATTAGAAA; si2-HERC2 sequence: GAGCTGATTTCTTGAGTAA) and the non-target siRNA control (siCTR) were purchased from Guangzhou RiboBio.

Techniques: Transfection, MTT Assay, Knockdown, Irradiation, Labeling, Staining, shRNA, Injection