Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: The Cnot genes maintain self-renewal by repressing early trophectoderm (TE) transcription factors. (A): Cnot1, Cnot2, and Cnot3 knockdown did not immediately affect known self-renewal factors and pathways. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) in M15 medium. Cells were collected 48 hours after transfection, and total Stat3, Smad1, b-Catenin as well as phospho-Stat3, phospho-Smad1, phosphor-b-Catenin, Oct4, and Nanog levels were determined by Western blot. Starved: control-transfected ESCs cultured in serum-free and LIF-free medium for additional 4 hours. (B): Comparing gene expression changes caused by perturbations of known self-renewal factors: Cnot1, 2, and 3 silencing induced similar changes to those of Oct4 or Sox2 silencing. Pearson's correlation coefficients were calculated between microarray datasets and depicted in a heatmap. The self-renewal factors were clustered by unsupervised hierarchical clustering based on the correlation coefficients. Microarray datasets used for this plot are listed in Supporting Information Table 2. (C): Cnot2 or Cnot3 overexpression cannot rescue Oct4 or Sox2 silencing-induced differentiation. Oct4GiP cells and Oct4GiP cells overexpressing Cnot2 (Cnot2-Rescue, same as in Fig. 1C) or Cnot3 (Cnot3-Rescue, same as in Fig. 1C) were transfected with control, Oct4 (Oct4-KD), or Sox2 (Sox2-KD) siRNAs, and the % differentiation was determined by the Oct4GiP reporter assay. (D): Cnot1, Cnot2, and Cnot3 knockdown induced TE differentiation in the presence of sustained Oct4 expression. ZHBTc4 cells that constitu-tively express Oct4 at the normal level from a Tet-Off promoter were transfected with control or Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), Cnot3-siRNA2 (Cnot3-KD), and the expression of TE markers Cdx2 and Gata3 was determined by qRT-PCR after 4 days. (E): Cdx2 deletion partially rescued Cnot1, Cnot2, and Cnot3 silencing-induced differentiation. Oct4GiP (WT) or dKO23-5 (Cdx2-/- ) cells were transfected with Control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD), and the expression of lineage markers was determined by qRT-PCR 96-hour after transfection. Abbreviations: ESC, embryonic stem cell; KD, Knockdown; WT, wild type.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Transfection, Western Blot, Cell Culture, Expressing, Microarray, Over Expression, Reporter Assay, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Silencing Cnot1, Cnot2, or Cnot3 led to mouse embryonic stem cell (ESC) differentiation. (A): Silencing Cnot1, Cnot2, or Cnot3 resulted in ESC differentiation based on the Oct4GiP reporter assay. Oct4GiP ESCs were transfected with indicated siRNAs (two different siR NAs for each CCr4-Not complex gene) in M15 medium and cultured for 4 days. The percentage of differentiated cells (% differentiation) was determined by measuring the percentage of green fluorescent protein-negative cells by fluorescence-activated cell sorting (FACS) at the end of the culture. (B): Expression of siRNA-resistant Cnot2 or Cnot3 rescued the differentiation caused by Cnot2 or Cnot3 knockdown, respectively. Oct4GiP cells or Oct4GiP cells expressing siRNA-resistant Cnot2 (Cnot2-Rescue) or Cnot3 (Cnot3-Rescue) were transfected with Control, Cnot1-siRNA1, Cnot2-siRNA2, or Cnot3-siRNA2, and the percentage of differentiated cells was determined by the Oct4GiP reporter assays. Note that Cnot2-Rescue cells were not able to rescue the differentiation caused by Cnot1 or Cnot3 silencing, and Cnot3-Rescue cells were not able to rescue Cnot1 or Cnot2 silencing. ***, p < .001. (C): Silencing Cnot1, Cnot2, or Cnot3 resulted in morphological changes and loss of alkaline phosphatase (AP) staining in ESCs. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were stained with the AP staining kit and imaged 4 days after transfection. (D): Cnot1, Cnot2, or Cnot3 silencing led to downregulation of ESC marker and upregulation of differentiation markers. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were harvested for quantitative real-time PCR (qRT-PCR) analysis 4 days after transfection. ESC marker: Oct4; differentiation markers: Cdx2, Eomes, Gata3, Hand1, and Krt8. (E): Cnot1, Cnot2, or Cnot3 silencing reduced cell proliferation or viability in 2i medium. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) and cul tured in 2i medium. Cell numbers were counted by FACS 4 days after transfection and normalized to control-transfected cells. (F): Cnot1, Cnot2, or Cnot3 silencing led to differentiation in 2i medium. Oct4GiP cells were transfected with indicated siRNAs and cultured in 2i medium. Cells were harvested for qRT-PCR analysis 4 days after transfection. (G): Expression of C-terminally HA-tagged Cnot2 (Cnot2-HA) in E14Tg2a cells. Expression of the exogenous Cnot2-HA was detected in Western blot with the HA-antibody, and Ran was used as a loading control. Expression of total (endogenous and exogenous) Cnot2 was determined by qPCR in wild-type E14Tg2a (E14) and Cnot2-HA expressing cells. The expression of the Cnot2-HA was estimated to be ∼2-fold of the endogenous Cnot2 on the mRNA level. (H): Identification of Cnot1 and Cnot3 in Cnot2-HA immunoprecipitation. HA-pull-down was carried out in E14Tg2a cells expressing Cnot2-HA. The presence of Cnot1, Cnot2-HA, and Cnot3 in the total lysate and pull-down sample (HA-beads) were detected by Western blot. Note that Oct4 was not detected in the pull down sample. As a negative control, protein-A beads were used in an independent pull-down. Abbreviations: HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Reporter Assay, Transfection, Cell Culture, Fluorescence, FACS, Expressing, Staining, Marker, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Cnot1, Cnot2, and Cnot3 are required for human embryonic stem cell (ESC) self-renewal. (A): Cnot1, Cnot2, and Cnot3 were down-regulated during human ESC differentiation. H1 human ESCs were differentiated for 7 days using 100 ng/ml human recombinant BMP4. The expression levels of Cnot1, Cnot2, and Cnot3 as well as Oct4 and differentiation markers Cdx2 and Hand1 were determined by quantitative realtime PCR (qRT-PCR). (B): Silencing of Cnot1, Cnot2, or Cnot3 led to morphological changes of human ESCs. H1 cells were imaged 6 days after transfection. Phase-contrast images highlight the undifferentiated morphology of human ESCs in the lipids-only transfected cells (mock) versus the differentiated phenotype in the Cnot1, Cnot2, or Cnot3 siRNA transfected cells. (C): Silencing of the Cnot genes led to upregulation of the Cdx2 and Gata3 proteins. H1 cells were transfected with lipids-only (mock), Oct4, Cnot2, or Cnot3 siRNAs. Cells were fixed and stained for Cdx2 or Gata3 expression by immunofluorescence staining 6 days after transfection. (D): Silencing of the Cnot genes led to downregulation of the ESC marker and upregulation of the extraembryonic markers. H1 cells were harvested 6 days after transfection and marker expression was determined by qRT-PCR. Abbreviations: BMP, bone morphogenetic protein; DAPI, 4′-6-diamidino-2-phenylindole.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence, Marker

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Hela, HepG2, and Huh7 cells were seeded in a 12-well plate for overnight, then left untreated or treated with human IFN-α (1,000 IU/ml) for 18 or 36 h. The untreated cells (control) were harvested together with the cells treated by IFN-α for 36 h. ISG20 expression was detected by Western blot. Hela cells transfected with plasmid F-ISG20 expressing the FLAG-tagged ISG20 were used as positive control for ISG20 Western blot (lane 2). β-actin expression was presented as loading control. (B) PHHs and HepG2 cells were cultured in 12-well-plate and treated with type I IFN-α (1,000 IU/ml), type II IFN-γ (100 ng/ml) or type III IFN-λ (100 ng/ml), or left untreated (control) for 2 days. The levels of ISG20 expression were determined by Western blot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Northern Blot, Hybridization, Over Expression, Western Blot, Bioprocessing, FLAG-tag, Expressing, Control

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepDES19 cells were transfected with 100 nM of control siRNA (Lane 1–3) or ISG20 siRNA (siISG20) (lane 4–6) twice with a 24 h interval after tet being withdrawn. Culture medium was replaced 12 h after the 2nd siRNA transfection, and cells were either left untreated as controls (lane 1 &4) or treated with 100 IU/ml (lanes 2 & 5) or 1,000 IU/ml (lanes 3 & 6) of IFN-α. Cells were harvested 5 days after 2nd transfection. Viral total RNA (top panel), encapsidated pgRNA (upper middle panel), and core DNA (lower middle panel) were subjected to Northern and Southern analyses, respectively. ISG20 protein expression was revealed by Western blot, and β-actin served as loading control (bottom panels). The relative levels of viral nucleic acids and ISG20 expression in the siISG20 transfected or IFN-α treated samples (lanes 2–6) are expressed as the percentage of the control sample (lane 1). The data presented here are representative of two independent experiments.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2-NTCP12 cells stably transduced by control lentiviral shRNA (shcontrol) or ISG20 lentiviral shRNA (shISG20) were spinoculated with HBV at 100 vge/cell. 16 h later, the infected cells were mock treated or treated with 1,000 IU/ml of IFN-α for 6 days, and the cells were subjected to the following analyses: (A) The expression of ISG20 was analyzed by Western blot. (B) HBV infectivity was assessed by HBcAg immunofluorescence, and the percentage of HBcAg-positive cells were calculated from multiple microscopic field of view (mean±SD, n = 5). Nuclei were stained with DAPI. (C) HBV total RNA were quantified by qPCR and the relative expression levels to β-actin mRNA were plotted as fold change to control samples (HBV infected shcontrol cells without IFN-α treatment) (mean±SD, n = 3).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Stable Transfection, Control, shRNA, Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20 D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected in situ by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Hybridization, Enzyme Immunoassay, In Situ, Southern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20 D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20 D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20 D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See for more experimental details.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Control, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells in 6-well-plate were co-transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol in the absence of HA-ISG20 D94G (lane 2) or increased amount of HA-ISG20 D94G (1 μg, 2 μg, 4 μg; lanes 3–5). The total amount of transfected DNA was kept constant (6 μg/well) by adding control vector plasmid (lanes 2–4). 5 days later, total cellular HBV RNA and protein (FLAG-Pol and HA-ISG20 D94G ) were determined by Northern and Western blot, respectively, as input controls (top panels). Immunoprecipitation was performed by using antibodies against HA or FLAG epitopes, followed by Northern blot analysis of HBV RNA (bottom panels).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Control, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32 P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustrations of the wildtype HBV ε RNA and mutants. The substructural domains of ε, including the lower stem, bulge, upper stem, and apical loop, are marked on the full-length form. Shorter versions of ε include the upper stem loop (US+L), lower stem with wildtype or mutant bulge sequence as loop (LS+B, LS+Bm), and LS+B with bottom 4 base-pairs removed from the lower stem (LSΔ4+B). These RNA fragments were chemically synthesized and 5’ end radiolabeled for ISG20 EMSA. (B) EMSA of ISG20 binding with full-length (FL) ε, US+L, and LS+B. (C) EMSA of ISG20 binding with LS+B, LS+Bm, and LS+B. (D) HepG2 cells were transfected with plasmid pMS transcribing the 2.1kb HBV RNA, or pMSΔ4bp transcribing the 2.1kb HBV with 4 nucleotides removed from the bottom right arm of the lower stem of ε, in the absence or presence of F-ISG20. HBV RNA and FLAG-tagged ISG20 were analyzed by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Mutagenesis, Sequencing, Synthesized, Binding Assay, Transfection, Plasmid Preparation, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Control, Plasmid Preparation, Construct, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: 0.1 µg of 5’-radiolabeled synthetic RNA substrates, specifically (A) the intact ε, upper stem-loop region (US+L), and lower stem with bulge serving as loop (LS+B); and (B) the intact ε, single-stranded left arm portion of ε, and 30-mer poly(rA), were incubated with the indicated amount of RNase A or purified His-ISG20 in nuclease reaction buffer for 15 min, then the reactions were terminated and the mixtures were fractionated through 10% TBE-Urea denaturing polyacrylamide gel, and the dried gel was subjected to autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Incubation, Purification, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: The major viral intermediates and products at each major HBV replication steps are illustrated. cccDNA (or other HBV transcription template)-derived 3.5kb RNA (including precore mRNA and pgRNA) and other shorter subgenomic RNA species (2.4/2.1kb surface mRNA and 0.7kb X mRNA) are aligned to show the location of ε on different RNA species. The black circle dots indicate the 5’ cap of mRNA, the zigzag lines represent the polyA tails. ISG20 is shown as a rectangle box and its targeting sites on HBV RNA are indicated by arrowheads. As a consequence of ISG20-mediated HBV RNA degradation, the illustrations and labels of viral proteins/antigens, pgRNA encapsidation and DNA replication are shown in gray color schemes. The solid gray triangles indicate capsid proteins, viral polymerase is shown in an oval shape before ε binding and then a gray circle dot in the nucleocapsids after pgRNA encapsidation.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Derivative Assay, Binding Assay

Journal: Aging Cell

Article Title: The variant senescence‐associated secretory phenotype induced by centrosome amplification constitutes a pathway that activates hypoxia‐inducible factor ‐1α

doi: 10.1111/acel.13766

Figure Lengend Snippet: Centrosome amplification triggers a variant senescence‐associated secretory phenotype (SASP). (a) Experimental scheme for the matrigel‐coated transwell invasion experiment. Transwell inserts physically separate mCherry MDA‐MB468 cells (top chamber) from the indicated MCF10A cells (bottom chamber). Paracrine invasion is scored from the number of mCherry MDA‐MB468 cells that migrate to the bottom chamber. (b, c) MCF10A cells with centrosome amplification promote the invasion of MDA‐MB468 mCherry cells. Representative images (b) and fold increase (c) in mCherry MDA‐MB468 cells that crossed the matrigel‐coated transwell upon co‐culturing with the indicated MCF10A cells. (d) Centrosome amplification alters expression of genes related to cell motility and senescence. Ingenuity Pathway Analysis (IPA) of gene expression changes in MCF10A cells with centrosome amplification relative to controls revealing the top pathways altered by centrosome amplification. *Hepatic Fibrosis/ Hepatic Stellate Cell Activation is a senescence‐regulated process (Krizhanovsky et al., ). (e–h) Induction of senescence‐related gene expression in cells with centrosome amplification. Gene set enrichment analysis (GSEA) revealed strong enrichment of genes upregulated (e, g) or downregulated (f, h) with senescence in MCF10A cells with centrosome amplification (e, f) and RPE‐1 cells with centrosome amplification (g, h) relative to controls. NES: normalised enrichment score; FDR: false discovery rate. (i, j) Induction of secreted protein expression in cells with centrosome amplification. Gene set enrichment analysis (GSEA) revealed enrichment of genes annotated to the extracellular region in MCF10A cells with centrosome amplification (i) or RPE‐1 cells with centrosome amplification (j). NES: normalised enrichment score; FDR: false discovery rate. (k, l) Induction of senescence‐related gene expression in cells with centrosome amplification. Gene set enrichment analysis (GSEA) revealed strong enrichment of genes upregulated (k) or downregulated (l) with senescence in RPE‐1 tetraploids relative to “evolved tetraploids.” NES: normalised enrichment score; FDR: false discovery rate. (m, n) Proliferation arrest after centrosome amplification. Representative images (m) and quantification (n) of cells that cycled through S‐phase (24 hr. EdU‐label, red) and DAPI (blue) in MCF10A cells with (PLK4) and without (608) centrosome amplification. (o) Centrosome amplification induces retinoblastoma protein phosphorylation. Rb, Phospho Rb S780 and GAPDH (loading control) immunoblots of lysates from MCF10A cells with and without centrosome amplification. (p, q) Centrosome amplification increases cell size. Suspended (trypsinised) (p) and adherent (q) cell size measured for MCF10A cells with or without centrosome amplification. (r, s) Increased SA‐β‐Gal staining in cells with centrosome amplification. Representative images (r) and quantification (s) of SA‐β‐Gal (blue) staining of MCF10A cells with and without centrosome amplification or positive control with doxorubicin treatment. (t, u) Centrosome amplification does not induce DNA damage. Representative images (t) and quantification (u) of γ H2AX foci (green) in MCF10A cells with (PLK4) and without (608) centrosome amplification as compared to DNA damage from doxorubicin treatment. Cells in S‐phase are labelled with Edu (red) pulse and excluded from the quantification. (v, w, x) Centrosome amplification does not induce paracrine invasion. (v) Experimental scheme for the paracrine senescence assay. Transwell inserts ensure physical separation between MCF10A PLK4(top), 608(top) or wild‐type cells (bottom). Representative images (w) and quantification (x) of SA‐β‐Gal (blue) staining of MCF10A wild‐type cells that were co‐cultured with PLK4 or 608 or doxorubicin treated cells. (y, z) RNAi‐mediated knockdown of p53 or LATS2 releases MCF10A cells with centrosome amplification from proliferation arrest. (y) Quantification of control, p53 knockdown and LATS2 knockdown MCF10A cells with (PLK4) and without (608) centrosome amplification, which cycled through S‐phase (24 hr EdU‐label, red). (z) p53, LATS2 and GAPDH (loading control) immunoblots of lysates from control 608, p53 KD 608, LATS2 KD 608, control PLK4, p53 KD PLK4 and LATS2 KD PLK4 cells. Scale bar, 50 μm. All data are means ± SEM from n = 3 independent experiments, ** p < 0.01, **** p < 0.0001; analysed with Student's t test except m, which was with one‐way ANOVA, Tukey's multiple comparison test. Scale bars, 50 μm.

Article Snippet: The ANGPTL4 mouse antibody mAb11F6C4, used at 40 μg/ml for blocking experiments, was a gift from Tan Nguan Soon, Andrew (Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore). siRNA against CYBA p22phox (Santa Cruz), smartpool siRNA against LATS2 and p53 (Dharmacon).

Techniques: Amplification, Variant Assay, Expressing, Gene Expression, Activation Assay, Phospho-proteomics, Control, Western Blot, Staining, Positive Control, Cell Culture, Knockdown, Comparison

Journal: Aging Cell

Article Title: The variant senescence‐associated secretory phenotype induced by centrosome amplification constitutes a pathway that activates hypoxia‐inducible factor ‐1α

doi: 10.1111/acel.13766

Figure Lengend Snippet: Centrosome amplification‐induced ROS. (a, b) RNAi‐mediated knockdown of p53 or LATS2 does not affect nuclear HIF‐1α accumulation after centrosome amplification. Shown are representative images (a) and quantification (b) of nuclear HIF‐1α levels in the indicated cells. (c) Small molecule Rac‐1 inhibition prevents the accumulation of nuclear HIF‐1α after centrosome amplification. The indicated MCF10A cells were treated with 50 μm NSC23766 or vehicle and HIF‐1α nuclear accumulation was measured. (d, e) CRISPR‐mediated gene disruption of TRIO blocks nuclear HIF‐1α accumulation after centrosome amplification. Gene targeting of a pool of cells was performed in the indicated MCF10A cells prior to the initiation of centrosome amplification. Shown are representative image (d) and quantification (e) of nuclear HIF‐1α levels in the indicated cells. (f) siRNA knockdown of p22 phox inhibits nuclear HIF‐1α accumulation after centrosome amplification. (g) Trio is required for the upregulation of HIF‐1α‐induced genes in MCF10A cells with centrosome amplification. Shown is a GSEA plot comparing cells with centrosome amplification with or without TRIO gene disruption. (h) The induction of ANGPTL4 by centrosome amplification requires Trio and ROS. Shown is the fold induction of ANGPTL4 from RNA‐Seq after centrosome amplification in MCF10A cells after the indicated treatments. (i) Accumulation of superoxide after centrosome amplification. Superoxide levels were measured by dihydroethidium labelling in MCF10A cells with and without centrosome amplification. Pyocyanin treatment is the positive control. (j) Conversion of superoxide into hydrogen peroxide further induces nuclear HIF‐1α accumulation in cells with centrosome amplification. Shown are the fold changes in nuclear HIF‐1α in the indicated MCF10A cells after treatment with TEMPOL. (k, l) Catalase blocks the nuclear accumulation of HIF‐1α in cells with centrosome amplification. Representative images (k) and quantification (l) of HIF‐1α in the indicated MCF10A cells with and without catalase medium addition. (m) GSEA showing that catalase treatment prevents the upregulation of the custom HIF‐1α signature up gene set in MCF10A cells. All data are means ± SEM from n = 3 independent experiments, * p < 0.05, ** p < 0:01, *** p < 0:001, **** p < 0:0001; analysed with one‐way ANOVA, Tukey's multiple comparison test. Scale bars, 50 μm. (n) Model for centrosome amplification‐induced SASP.

Article Snippet: The ANGPTL4 mouse antibody mAb11F6C4, used at 40 μg/ml for blocking experiments, was a gift from Tan Nguan Soon, Andrew (Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore). siRNA against CYBA p22phox (Santa Cruz), smartpool siRNA against LATS2 and p53 (Dharmacon).

Techniques: Amplification, Knockdown, Inhibition, CRISPR, Disruption, RNA Sequencing, Positive Control, Comparison

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 3. Caveolin-1 interacts with ICAM-1 primarily through filamin A. HeLa cells treated with siRNA for filamin A, filamin B, caveolin-1 (Cav1), or control (ctrl) were used in a pull-down (PD) assay using the biotinylated pep- tide encoding the intracellular domain of ICAM-1. Cell lysates and isolated proteins were analyzed by Western blot for caveolin-1, filamin A, filamin B, and tubulin (protein loading control).

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Control, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 6. Filamin mediates lateral membrane mobility of ICAM-1. FRAP analysis in combination with confocal imaging was used to study membrane dynamics of GFP-CAAX as a control (A) and ICAM-1-GFP (B) in HeLa cells trans- fected with control (gray line) or filamin B (dark line) siRNA. Western blot in A shows expression analysis of filamin B. Tubulin is included as a loading con- trol. Dashed lines indicate pre-bleaching intensity set at 100%. A, the fluores- cence recovery of GFP-CAAX was not affected by reduced expression of fil- amin B. Graph is representative of four independent experiments. B, images show ICAM-GFP distribution in control siRNA or filamin B siRNA-treated cells. Bar, 20 m. In control siRNA-treated cells, ICAM-1-GFP recovered to 70% within 2.5 min (gray lines), whereas filamin B-deficient cells showed recovery to maximal 40% (dark lines). The graph is representative of four independent experiments. C, mobile fractions of both experiments are calculated and show that filamin B siRNA significantly reduces the mobile fraction of ICAM- 1-GFP but not GFP-CAAX. Data are mean S.E. of four independent experi- ments;*,p0.01.D,measurementoftheslope(K)oftherecoverydepictedin A and B shows that reduction of filamin B did not affect the speed of motility ofICAM-1-GFPorofGFP-CAAXintheplasmamembrane.DataaremeanS.E. of four independent experiments.

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Membrane, Imaging, Control, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 7. Filamin B mediates recruitment of ICAM-1. A, ICAM-1 antibody-coated beads were incubated with ICAM-1-GFP expressing HeLa cells and the recruitment of ICAM-1-GFP to the beads was recorded for 20 min using time lapse confocal imaging. Time in minutes is shown above the images. Cells were treated with control siRNA or filamin B siRNA, as indicated on the left. Still images in A show ICAM-1-GFP recruitment around ICAM-1 antibody-coated beads in siRNA control cells after 4 to 8 min (open arrowheads in upper panels), whereas ICAM-1-GFP recruitment occurs not before 20 min in cells with reduced filamin B expression (lower images). Bar, 10 m. Lower panels show line-scan of the fluorescent intensity of ICAM-1-GFP surrounding the bead, indicated by the white dashed bar. B, quantification of the number of cup structures, formed around adherent beads on cells treated or not (ctrl) with siRNA to filamin B. *, p 0.001.

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Incubation, Expressing, Imaging, Control

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 8. Filamin B regulates ICAM-1-mediated adhesion and transmi- gration under static and flow conditions. A, reduced filamin B levels reduced the adhesion of ICAM-1 antibody-coated beads to ICAM-1-GFP expressing HeLa cells. Beads were counted per field of view, 20 fields per experiment were counted and three independent experiments have been carried out. HeLa cells were either not treated, or transfected with ICAM-1- GFP and treated with control siRNA (ctrl) or filamin B siRNA as indicated. Data are mean S.E. of a representative experiment performed in quadruple. *, p 0.05. B, differentiated HL60 cells were allowed to migrate across a HeLa ICAM-1-GFP monolayer in a Transwell system to 50 ng/ml SDF-1. Percentage of migration was determined as described under “Experimental Procedures.” HeLa cells were either not treated or transfected with ICAM-1-GFP and treated with control siRNA (ctrl) or filamin B siRNA as indicated. Data are mean S.E. of a representative experiment performed in quadruple. *, p 0.05. C, differentiated HL60 cells were perfused over monolayers of TNF-- treated primary human endothelial cells. Migration was visualized by adhe- sive cells crossing the monolayer thereby changing from a bright to a dim appearance (arrowheads). D, differentiated HL60 cells were flown over mono- layers of TNF--treated primary human endothelial cells. Left panel shows quantification of leukocyte migration across endothelial monolayers trans- fected with filamin B siRNA (si-filamin B), compared with migration across siRNA control treated endothelial cells (ctrl). Right panel shows quantification of adhesion of HL60 cells to endothelial cells treated with siRNA to filamin B. Data represent averages of duplicates from one of two independent experiments.

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Expressing, Transfection, Control, Migration

Journal: Endocrinology

Article Title: Stathmin, a microtubule regulatory protein, is associated with hypoxia-inducible factor-1alpha levels in human endometrial and endothelial cells.

doi: 10.1210/en.2008-1333

Figure Lengend Snippet: FIG. 2. Effects of stathmin suppression on hypoxia-induced HIF-1 expression in EM-1 and HUVEC. EM-1 and HUVEC were treated with control () or stathmin () siRNA and cultured under normoxic and hypoxic conditions for 5 h. Cell lysates were subjected to immunoblot analysis for HIF-1, HIF-1, and stathmin. The same blot was probed, stripped, and reprobed with each antibody. -Actin level was used as a loading control. Representative data from three independent experiments are shown.

Article Snippet: Treatment with small interfering RNA (siRNA) Cells at approximately 60% confluency in 12-well culture plates were transfected with the nontargeting control siRNA (50 nM, Allstar negative control; QIAGEN, Mississauga, Ontario, Canada) or stathmin siRNA (50 nM, sc-36127; Santa Cruz Biotechnology, Santa Cruz, CA) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Cell Culture, Western Blot

Journal: Endocrinology

Article Title: Stathmin, a microtubule regulatory protein, is associated with hypoxia-inducible factor-1alpha levels in human endometrial and endothelial cells.

doi: 10.1210/en.2008-1333

Figure Lengend Snippet: FIG. 1. Effects of stathmin suppression on hypoxia-induced HIF-1 expression in ESC. Primary cultured ESC and immortalized ESC line (EtsT) were treated with nontargeting control () or stathmin () siRNA and then cultured under normoxic and hypoxic conditions for 5 h. A, Each cell lysate was subjected to immunoblot analysis for HIF-1, HIF-1, and stathmin. The same blot was probed, stripped, and reprobed with each antibody. The level of -actin was used as a loading control. Representative data from three independent experiments are shown. B, Immunofluorescence of stathmin (upper panels) and HIF-1 (lower panels) in ESC. C, ESC were lysed with MSB as described in Materials and Methods. The amount of -tubulin in the soluble (S) and polymerized (P) fractions was determined by immunoblot analysis. The membrane was reprobed with antibodies against stathmin and -actin.

Article Snippet: Treatment with small interfering RNA (siRNA) Cells at approximately 60% confluency in 12-well culture plates were transfected with the nontargeting control siRNA (50 nM, Allstar negative control; QIAGEN, Mississauga, Ontario, Canada) or stathmin siRNA (50 nM, sc-36127; Santa Cruz Biotechnology, Santa Cruz, CA) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Control, Western Blot, Immunofluorescence, Membrane

Journal: Endocrinology

Article Title: Stathmin, a microtubule regulatory protein, is associated with hypoxia-inducible factor-1alpha levels in human endometrial and endothelial cells.

doi: 10.1210/en.2008-1333

Figure Lengend Snippet: FIG. 5. Effects of stathmin knockdown on the Akt signaling pathway in ESC, EtsT, EM-1, and HUVEC. ESC, EtsT, EM-1, and HUVEC were treated with control () and stathmin () siRNA and then cells were cultured under normoxic and hypoxic conditions for 5 h. The cell lysates collected from each cell type were subjected to immunoblot analysis for phospho-Akt (p-Akt; ser-473). The same blot was probed, stripped, and reprobed with anti-Akt antibody. Representative data from three independent experiments are shown.

Article Snippet: Treatment with small interfering RNA (siRNA) Cells at approximately 60% confluency in 12-well culture plates were transfected with the nontargeting control siRNA (50 nM, Allstar negative control; QIAGEN, Mississauga, Ontario, Canada) or stathmin siRNA (50 nM, sc-36127; Santa Cruz Biotechnology, Santa Cruz, CA) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Knockdown, Control, Cell Culture, Western Blot

Journal: Endocrinology

Article Title: Stathmin, a microtubule regulatory protein, is associated with hypoxia-inducible factor-1alpha levels in human endometrial and endothelial cells.

doi: 10.1210/en.2008-1333

Figure Lengend Snippet: FIG. 3. Effect of stathmin suppression on the expression of HIF-1 target genes in ESC, EM-1, and HUVEC. ESC, EM-1, and HUVEC were treated with control () and stathmin () siRNA and then cultured under hypoxia for 5 h. Total RNA was extracted and subjected to semiquantitative RT-PCR to analyze stathmin, VEGF, and HIF-1 mRNA expression. The primers for VEGF can detect the splicing variants of VEGF. Representative data from three independent experiments are shown.

Article Snippet: Treatment with small interfering RNA (siRNA) Cells at approximately 60% confluency in 12-well culture plates were transfected with the nontargeting control siRNA (50 nM, Allstar negative control; QIAGEN, Mississauga, Ontario, Canada) or stathmin siRNA (50 nM, sc-36127; Santa Cruz Biotechnology, Santa Cruz, CA) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Stathmin, a microtubule regulatory protein, is associated with hypoxia-inducible factor-1alpha levels in human endometrial and endothelial cells.

doi: 10.1210/en.2008-1333

Figure Lengend Snippet: FIG. 6. Proposed mechanism of stathmin-mediated inhibition of HIF-1 and its target genes in human uterine cells. Under hypoxic conditions, HIF-1 protein that has escaped from ubiquitin-mediated proteasomal degradation translocates from the cytoplasm to the nucleus and heterodimerizes with HIF-1. The HIF-1 complex binds the hypoxia response element (HRE) and up-regulates its target genes, VEGF. The PI3K/Akt signaling pathway is needed for hypoxia-induced HIF- 1 protein accumulation because a PI3K inhibitor, wortmannin, represses HIF-1 levels. Knockdown of stathmin decreased the level of hypoxia-induced HIF-1 protein and VEGF expression via repressing the PI3K/Akt signaling pathway.

Article Snippet: Treatment with small interfering RNA (siRNA) Cells at approximately 60% confluency in 12-well culture plates were transfected with the nontargeting control siRNA (50 nM, Allstar negative control; QIAGEN, Mississauga, Ontario, Canada) or stathmin siRNA (50 nM, sc-36127; Santa Cruz Biotechnology, Santa Cruz, CA) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Inhibition, Ubiquitin Proteomics, Knockdown, Expressing

Journal: Cell death discovery

Article Title: Squaramides enhance NLRP3 inflammasome activation by lowering intracellular potassium.

doi: 10.1038/s41420-023-01756-9

Figure Lengend Snippet: Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), TGN46 (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.

Article Snippet: Coverslips were incubated with sheep anti-human TGN46 (Biorad, AHP500G) and rabbit anti-human Golgin97 (Abcam, ab84340) in PBS-Triton for 1 h at RT.

Techniques: Activation Assay, Control, Western Blot, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Western Blot, Produced, Expressing, Immunofluorescence, Cell Culture, Clinical Proteomics, Membrane, Fluorescence, SDS Page, Molecular Weight, Activation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) The remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells 48 h after serum withdrawal. When the data were plotted as the percentage of the initial cell viability without serum withdrawal, it was shown that PAC1-CHO had remaining cell viability (57.34±5.91%) that was significantly higher than that of M-PAC1-CHO (36.96±6.85%) or pcDNA-CHO (37.89±7.11%) (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). (B) The intracellular caspase3 activities after serum withdrawal. The reactions of pcDNA-CHO were considered not result from PAC1 because pcDNA-CHO did not express PAC1 or PACAP; therefore, all the data were plotted as fold changes in pcDNA-CHO. As shown, PAC1-CHO had significantly lower caspase3 activity than M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO), whereas there was no significant difference between M-PAC1-CHO and pcDNA-CHO. (C) The intracellular Bcl-2 levels after serum withdrawal. After the data were plotted as the fold changes of pcDNA-CHO, it was shown that PAC1-CHO had significantly higher Bcl-2 level about 2 folds of that in M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments. (D) The detection of β-catenin, cyclin D1 and c-myc levels in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells by western blotting. The western blotting results and the statistical analysis showed that the levels of β-catenin, cyclin D1 and c-myc (tow targets of β-catenin) in PAC1-CHO cells were significantly higher than those in M-PAC1-CHO or pcDNA-CHO cells (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). These findings indicated that overexpression of wild type PAC1 endowed CHO with anti-apoptotic activities against serum withdrawal, suggesting that PAC1 had ligand independent basal activity, while M-PAC1 did not. And Wnt/β-catenin signals were involved in the anti-apoptotic activity of PAC1-CHO. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Activity Assay, Western Blot, Over Expression

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Knockdown of endogenous PACAP and PAC1 with shRNA in Neuro2a. Western blotting assays showed that shRNA against PACAP significantly diminished the expression of endogenous PACAP in neuro2a/PACAP - , and further transfection with shRNA plasmids against PAC1 (+) to neuro2a/PACAP - cells decreased the PAC1 levels significantly, while control plasmids (-) did not interfere with expression of PAC1. The knockdown of PACAP and PAC1 in neuro2a produced a chance for the detection of the correlation of PAC1 down-regulation with its ligand independent basal activity. (B) The remaining cell viabilities of nero2a/PACAP - transfected with PAC1 shRNA plasmids (+) or control plasmid (-). After the data were plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that down-regulation of PAC1 with PAC1 shRNA plasmids (+) decreased the remaining cell viabilities to almost a half of the remaining cell viabilities transfected with control plasmids (-) 48 h after serum withdrawal (*, P<0.01, shRNA + vs. shRNA-). (C) Western blotting of β-catenin, cyclin D1 and c-myc in the nero2a/PACAP - cells transfected with PAC1 shRNA plasmids (+) or control plasmids (-). After the relative protein levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that PAC1 shRNA plasmids (+) significantly decreased the levels of β-catenin, cyclin D1 and c-myc compared with control plasmids (+)(*, P<0.01, shRNA+ vs. shRNA-). These data suggested that down-regulation of PAC1 in the natural cells such neuro2a with high expression of PAC1 inhibited the anti-apoptotic activities in the ligand free condition. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Knockdown, shRNA, Western Blot, Expressing, Transfection, Control, Produced, Activity Assay, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: Expression of RANKL in osteoblasts and osteoclastogenesis in co-cultures of bone marrow macrophages and calvarial osteoblasts induced by OSM are dependent on Shc1 and STAT3. Osteoblasts in which the Shc1 gene (A) or the Stat3 gene (D) was knocked-down by siRNA were treated with OSM at 100 ng/mL for 24 h before analysis of Tnfsf11 gene expression. Osteoblasts transfected with scrambled RNA (siSCR) were similarly treated and analyzed. Osteoblasts in which Shc1 (B,C) or Stat3 (E,F) was knocked down (or siSCR as control) were exposed to OSM (100 ng/mL) or vehicle and co-cultured with BMMs for 3 days before TRAP staining and counting of TRAP + MuOCL (A,C,D,F) . Values represent means for four wells and SEM is shown as vertical bars. Significant differences are indicated by horizontal lines where ** P < 0.01; *** P < 0.001; analyzed by two-way ANOVA followed by Tukey post hoc-test . The difference in OSM-induced response with and without silencing analyzed by two-way ANOVA was statistically significant [interaction P -value in A ( P < 0.0001), C ( P < 0.005), D ( P < 0.0001) and F ( P < 0.0001)]. Scale bar in (B,E) is 50 μm.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Expressing, Gene Expression, Transfection, Control, Cell Culture, Staining

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: OSM and, to a lesser extent, LIF stimulate bone resorption and RANKL production in calvarial bones, calvarial osteoblasts and stromal cells. (A) Mouse calvarial bones were cultured in the absence or the presence of LIF or OSM (both 0.1–100 ng/mL) and bone resorption was assessed by 45 Ca release after a 5-day culture period. (B) RT-qPCR was performed using mRNA extracted from calvarial bones treated with either LIF or OSM (both at 100 ng/mL) for 24 h to assess the expression of Tnfsf11 . (C) Protein expression of RANKL after 48 h was also analyzed in calvarial bone treated with LIF or OSM (both at 100 ng/L). (D) Mouse calvarial osteoblasts were incubated in the absence (Co) or the presence of LIF (100 ng/mL) or OSM (100 ng/mL) for 48 h and expression of Tnfsf11 was analyzed by semi-quantitative RT-PCR. (E) The expression of Tnfsf11 mRNA in calvarial osteoblasts stimulated by LIF and OSM a different concentrations (0.1-100 ng/mL) was performed using quantitative RT-PCR. (F) The mRNA expression of Osmr and Lifr in osteoblasts was compared at three different time points. (G) The receptor components Il6st, Lifr and Osmr are expressed in ST-2 stromal cells as assessed by RT-PCR. (H) The mRNA expression of Tnfsf11 in ST-2 cells cultured without (Co) or with LIF or OSM (both at 100 ng/mL) for 48 h was analyzed. Values represent means for six bones (calvarial bones) or four wells (cell culture experiments) and SEM is shown as vertical bars. * , ** , and *** , indicate significant difference compared to untreated (Co) cells, * P < 0.05, ** P < 0.01, and *** P < 0.001, respectively. Statistical significance was determined by ANOVA using Levene's homogeneity test followed by Dunnett's T3 post-hoc tests vs. Co. In (F) , Tukey post-hoc test was used to compare all groups and no statistical difference was observed.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: OSM, but not LIF, stimulates osteoclastogenesis in bone marrow cell cultures. Mouse bone marrow cells (BMC) were cultured in the absence (Co) or the presence of LIF, or OSM (both at 100 ng/mL), PTH or 1,25(OH) 2 -vitamin D3 (D3; both at 10 −8 M) for 7 days before staining (A) and counting (B) of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts (TRAP + MuOCL). Quantitative real-time PCR analysis of mRNA expression of cathepsin K ( Ctsk , C ), TRAP ( Acp5 , D ), calcitonin receptor ( Calcr , E ) RANKL ( Tnfsf11 , F ) and OPG ( Tnfsf11b , G ) in BMC cultured without (Co) or with LIF or OSM (both at 100 ng/mL) for 7 days. Values represent means for four wells and SEM is shown as vertical bars. *** , indicates significant difference compared to untreated (Co) cells, *** P < 0.001. Statistical significance was determined by ANOVA using Levene's homogeneity test followed by Dunnett's 2-sided (B–F) or Dunnett's T3 (G) post-hoc test.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: Lack of effect by OSM and LIF on osteoclast formation in bone marrow macrophage cultures. (A,B) BMMs were cultured in M-CSF (30 ng/mL) or differentiated to osteoclasts by addition of M-CSF + RANKL (30 and 4 ng/mL, respectively) in the presence or absence of LIF or OSM (both at 100 ng/mL) for 3 days before staining for tartarate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts (TRAP + MuOCL), which then were counted. No osteoclasts were formed in the absence of RANKL. (C) Quantitative PCR analyses of Il6st, Lifr , and Acp5 in BMM cultured for 72 h with M-CSF (30 ng/ml) or M-CSF+RANKL (30 and 4 ng/ml, respectively). Values represent means for four wells and SEM is shown as vertical bars. In (C) *** , indicates significant difference compared to cells treated with M-CSF (M), P < 0.001. In (B) Statistical significance was determined one-way ANOVA followed by Tukey's post-hoc test and in (C) , Student's t -test was used for comparison between M and M + R groups for each gene.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction, Comparison