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Image Search Results
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A, C,E) ChIP assays were carried out in HeLa cells stimulated with IFN-γ for 0–2 hrs. Cell lysates were immunoprecipitated (IP’d) with control antibody or with antibody to endogenous 19S ATPase S6a, Sug1, or S7 and associated DNA was isolated and analyzed by real-time PCR using primers and probe spanning the CIITApIV proximal promoter. Real time PCR values were normalized to the total amount of DNA in the reaction (Input). IP values are represented as ATPase binding to CIITApIV promoter DNA relative to unstimulated samples. (B,D,F) ChIP signal at the inactive gene CD4. The control IgG values were 0.004±0.001. Values for control IgG and either Sug1 IP, S7 IP or S6a IP represent the mean ± SEM of three biologically independent experiments * p<0.05.
Article Snippet:
Techniques: Immunoprecipitation, Control, Isolation, Real-time Polymerase Chain Reaction, Binding Assay
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A–B, D–E) HeLa cells were stimulated with IFN-γ as indicated and were harvested four hrs post treatment with 10 µM MG132 or 10 µM Lactacystin. mRNA was extracted and cDNA was generated using indicated reverse primers followed by amplification via real-time PCR. CIITA mRNA transcripts were obtained using primers and probes specific for CIITA exon IV and exon VII were normalized to GAPDH. (C) 18S rRNA transcripts for control and MG132 treated cells were obtained using primers and probe specific for 18S rRNA and were normalized to GAPDH. The 18 hr control sample was set to 100%. Data shown represents the mean ± SEM of three biologically independent experiments.
Article Snippet:
Techniques: Generated, Amplification, Real-time Polymerase Chain Reaction, Control
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: HeLa cells were transfected with HSE-Luciferase reporter, control siRNA, or S6a siRNA and were treated with MG132 six hrs prior to harvest. Cells were harvested following 48 hrs of incubation, lysed in cell lysis buffer, and analyzed by Luciferase assay. Luciferase readings obtained were normalized by Bradford assay. Data shown represents values obtained from three independent experiments. The negative control is a mixture of non-inducible reporter construct and constitutively expressing Renilla luciferase construct. The positive control is an inducible transcription factor-responsive construct expressing firefly luciferase, and a constitutively expressing Renilla luciferase construct.
Article Snippet:
Techniques: Transfection, Luciferase, Control, Incubation, Lysis, Bradford Assay, Negative Control, Construct, Expressing, Positive Control
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A–I) ChIP assays were carried out in HeLa cells stimulated with IFN-γ for 0–2 hrs. Cell lysates were IP’d with control antibody or with antibody to endogenous Sug1 (A and B), S7 (D and E), or S6a (G and H) and associated DNA was isolated and analyzed by real-time PCR using primers and probes spanning CIITApIV exon IV (A, C, E) and exon VII (B, D, F). Real time PCR IP values were normalized to the total amount of DNA (input); IP values are represented as ATPase binding to CIITApIV exon IV or exon VII DNA relative to unstimulated samples. (C,F,I) ChIP signal at the inactive gene CD4. The control IgG values were 0.005±0.001. Values for control and IP represent mean ± SEM of three biologically independent experiments. *p<0.05, **p<0.005. G. Mobility shift assay of Sug1 with a 90 nucleotide single stranded DNA on a native 8% polyacrylamide gel with a tris-borate magnesium running buffer; 0.7 µM DNA, 0.85 µM sug1, and 500 µM ATP. DNA was visualized with SYBER Green II dye.
Article Snippet:
Techniques: Control, Isolation, Real-time Polymerase Chain Reaction, Binding Assay, Mobility Shift
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A–C) HeLa cells were co-transfected with Myc tagged S6a, S7, or Sug1 and Flag tagged Hexim or HA tagged CDK9 as indicated. Cells were lysed and IP’d with Myc beads (lane 1) as a positive control, mouse isotype IgG (lane 2) as a negative control, flag beads (lane 3), and HA beads (lane 4). IP samples (top panel) and lysates (bottom panel) were IB’d for Myc, Flag, and HA as indicated. (D–E) HeLa cells were lysed and IP’d with either Hexim or CDK9 (lane 1) as a positive control, mouse isotype IgG (lane 2) as a negative control, or with S6a (lane 3), S7 (lane 4), and Sug1 (lane 5). IP samples (top panel) and lysates (bottom panel) were IB’d for Hexim or CDK9 as indicated. Results shown are indicative of data from three biologically independent experiments.
Article Snippet:
Techniques: Transfection, Positive Control, Negative Control
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A) HeLa cells were transfected with myc tagged S6a, S7 or Sug1 as indicated. Cells were lysed and IP’d with myc beads (first lane, top panels) as a positive control, with mouse isotype IgG (second lane, top panels) as a negative control, and with Ser5p-RNA Pol II antibody (third lane, top panels). IP’d samples (top panels) and lysates (middle and bottom panels) were IB for myc ATPases or for Ser5p-RNA pol II as indicated. (B) HeLa cells were lysed and IP’d with Ser5p-RNA Pol II (lane 1) as a positive control, mouse isotype IgG (lane 2) as a negative control, or with S6a (lane 3), S7 (lane 4), or Sug1 (lane 5). IP samples (top panel) and lysates (bottom panel) were IB’d Ser5p-RNA Pol II as indicated. Results shown are indicative of data from three biologically independent experiments.
Article Snippet:
Techniques: Transfection, Positive Control, Negative Control
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: A. HeLa cells were transfected with siRNA or were treated with proteasome inhibitors as indicated. Cells were harvested following 48hrs of siRNA incubation. Cell lysates were IB’d with Ser2p-RNA pol II antibody (top panels), Ser5p-RNA pol II (middle panels), or with RNA pol II antibody (bottom panels). Cells treated with proteasome inhibitors serve as a positive control for degradation dependent effects. Results shown are indicative of data from three biologically independent experiments. Sug1, S7, and S6a protein expression was effectively decreased using specific siRNA. Actin blots demonstrate loading and siRNA specificity controls .
Article Snippet:
Techniques: Transfection, Incubation, Positive Control, Expressing
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A,C,E) ChIP assays were carried out in HeLa cells transfected with ATPase specific or with control siRNA and stimulated with IFN-γ for 0–2 hrs. Cell lysates were crosslinked, sonicated, lysed, and IP’d with either antibody against endogenous RNA pol II or with control antibody (IgG). Associated DNA was analyzed via real-time PCR using primers and probe specific for the CIITApIV proximal promoter. Real time PCR IP values were normalized to total amount of reaction DNA (Input). The values for control IP and RNA Pol II IP represent the mean of three biologically independent experiments *p<0.05, **p<0.005, ***p<0.0005 versus control siRNA. (B, D, F) ChIP signal at the inactive gene CD4. Sug1, S7, and S6a protein expression was effectively decreased using ATPase specific siRNA .
Article Snippet:
Techniques: Transfection, Control, Sonication, Real-time Polymerase Chain Reaction, Expressing
Journal: PLoS ONE
Article Title: Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
doi: 10.1371/journal.pone.0091200
Figure Lengend Snippet: (A, C, E) ChIP assays were carried out in HeLa cells transfected with ATPase specific or with control siRNA and stimulated with IFN-γ for 0–2 hrs. Cell lysates were crosslinked, sonicated, lysed, and IP’d with either antibody against endogenous TBP or with control antibody (IgG). Associated DNA was analyzed via real-time PCR using primers and probe specific for the CIITApIV proximal promoter. Real time PCR IP values were normalized to total amount of reaction DNA (Input). The values for control IP and TBP IP are representative data *p<0.05, **p<0.005, ***p<0.0005 versus control siRNA. (B, D, F) ChIP signal at the inactive gene CD4. Sug1, S7, and S6a protein expression was effectively decreased using ATPase specific siRNA .
Article Snippet:
Techniques: Transfection, Control, Sonication, Real-time Polymerase Chain Reaction, Expressing
Journal: Cell & Bioscience
Article Title: The role of GLI1 for 5-Fu resistance in colorectal cancer
doi: 10.1186/s13578-017-0145-7
Figure Lengend Snippet: Characterization of 5-FU resistant LoVo-R cells. a Shows morphology of LoVo and LoVo-R cells. b Shows the IC50 dose of 5-FU calculated from measurement of cell viability in different concentrations of 5-FU (48 h). The X-axis is 5-FU concentration (μg/ml), and the Y-axis is O.D. values. Significant difference was indicated by *p < 0.05, **p < 0.005, or ***p < 0.0005
Article Snippet: The human
Techniques: Concentration Assay
Journal: Cell & Bioscience
Article Title: The role of GLI1 for 5-Fu resistance in colorectal cancer
doi: 10.1186/s13578-017-0145-7
Figure Lengend Snippet: Up-regulated of GLI1 signaling axis in LoVo-R (in comparison with LoVo) cells. After next generation sequencing, we performed ingenuity pathway analysis (IPA). a Shows up-regulation of GLI1 and its signaling molecules, and the up-regulated genes are in red . b Detection of GLI1 protein in LoVo and LoVo-R cells. β-actin was used as the internal control
Article Snippet: The human
Techniques: Comparison, Next-Generation Sequencing, Control
Journal: Cell & Bioscience
Article Title: The role of GLI1 for 5-Fu resistance in colorectal cancer
doi: 10.1186/s13578-017-0145-7
Figure Lengend Snippet: The effect of GLI1/2 knockdown on 5-FU response in LoVo-R cells. a Real-time PCR detection of GLI1 after GLI1 shRNA expression. b Detection of GLI1 protein by Western blotting. c The effect of GLI1 -ShRNAs (shown as shGLI1) on the IC50 of 5-FU (measured as shown in Fig. b). d Effects of GLI2 shRNAs (shown as shGLI2) on 5-FU response. Significant difference was indicated by *p < 0.05, **p < 0.005, or ***p < 0.0005
Article Snippet: The human
Techniques: Knockdown, Real-time Polymerase Chain Reaction, shRNA, Expressing, Western Blot
Journal: Cell & Bioscience
Article Title: The role of GLI1 for 5-Fu resistance in colorectal cancer
doi: 10.1186/s13578-017-0145-7
Figure Lengend Snippet: The effects of GLI1 knockdown on gene/protein expression. a Real-time analysis of GLI1, GLI2, Snai1 and Snai2 in GLI2 shRNAs-expressing LoVo-R cells. Significant difference was indicated by ***p < 0.0005. b Effects of Gli2 shRNAs on vimentin expression. c The effect of GLI1 knockdown on Sox2 and CD44 proteins
Article Snippet: The human
Techniques: Knockdown, Expressing
Journal: Cell & Bioscience
Article Title: The role of GLI1 for 5-Fu resistance in colorectal cancer
doi: 10.1186/s13578-017-0145-7
Figure Lengend Snippet: Cell invasiveness assay. Different cells were subjected to cell invasiveness as described in the methods, and invasive cells were visualized by blue in the staining ( a ). b Shows the summary from three independent experiments, with the value (number of invasive cells) from LoVo cells as 100
Article Snippet: The human
Techniques: Staining
Journal: Oncology Letters
Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1
doi: 10.3892/ol.2017.5709
Figure Lengend Snippet: miR-150 is downregulated in OS patient tissues and cell lines. (A) Relative expression levels of miR-150 in 40 OS patient tissue samples and adjacent normal tissue sample counterparts. The relative expression levels of miR-150 were determined using RT-qPCR analysis. (B) Relative expression levels of miR-150, determined using RT-qPCR analysis in U2OS, MG63 and SaOS2 OS cell lines and the normal hFOB1.19 human OS cell line. Student's t -test was used to analyze significant differences.*P<0.05. Data are presented as the mean + standard deviation. OS, osteosarcoma; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Article Snippet: The SaOS2, U2OS and
Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction
Journal: Oncology Letters
Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1
doi: 10.3892/ol.2017.5709
Figure Lengend Snippet: miR-150 overexpression is inversely correlated with OS cell growth. (A) U2OS, (B) MG63 and (C) SaOS2 OS cell lines were transfected with miR-150 and the numbers of cells were determined at different time points using a fluorescence-based CyQUANT cell proliferation assay kit. In all cases, Student's t -test was used to analyze significant differences, *P<0.05. Data are presented as the mean + standard deviation. OS, osteosarcoma; miR, microRNA; NC, negative control.
Article Snippet: The SaOS2, U2OS and
Techniques: Over Expression, Transfection, Fluorescence, CyQUANT Assay, Proliferation Assay, Standard Deviation, Negative Control
Journal: Oncology Letters
Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1
doi: 10.3892/ol.2017.5709
Figure Lengend Snippet: miR-150 overexpression is inversely correlated with OS cell migration and invasion. Transwell migration and invasion assays of (A) U2OS, (B) MG63 and (C) SaOS2 OS cell lines. The assays were performed following transfection of the cell lines with miR-150. Representative images of migrated and invaded cells on the membrane (magnification, ×100). OS, osteosarcoma; miR, microRNA; NC, negative control.
Article Snippet: The SaOS2, U2OS and
Techniques: Over Expression, Migration, Transfection, Membrane, Negative Control
Journal: Oncology Letters
Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1
doi: 10.3892/ol.2017.5709
Figure Lengend Snippet: miR-150 negatively regulates gene expression of ROCK1 in OS cell lines. (A) Reverse transcription-quantitative polymerase chain reaction analysis of mRNA expression of ROCK1 in U2OS, MG63 and SaOS2 OS cell lines overexpressing miR150. Student's t -test was used to analyze significant differences. *P<0.05. Data are presented as the mean + standard deviation. (B) Western blot analysis of protein expression of ROCK1 in U2OS, MG63 and SaOS2 OS cell lines overexpressing miR150. A corresponding NC was used in the absence of miR-150. β-actin protein was used as an additional control. miR, microRNA; ROCK1, Rho-associated kinase 1; NC, negative control.
Article Snippet: The SaOS2, U2OS and
Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Western Blot, Control, Negative Control
Journal: FEBS Open Bio
Article Title: Doxorubicin promotes breast cancer cell migration and invasion via DCAF13
doi: 10.1002/2211-5463.13330
Figure Lengend Snippet: DCAF13 promotes EMT in human breast cancer. (A) Hierarchical cluster analysis of differentially expressed genes (|logFC| > 1 and P adj < 0.001) between DCAF13_high and DCAF13_low breast cancer samples. (B) The volcano plot of differential gene expression between DCAF13_high and DCAF13_low breast cancer samples. The horizontal dashed line marks the threshold ( P adj < 0.05) for defining a gene as significantly upregulated or downregulated. The vertical dashed lines represent two‐fold differences in expression. Significantly differentially expressed genes are shown as red dots ( P adj < 0.05 and |log2FC| > 1). (C) GSEA analysis was performed to identify the pathways altered in DCAF13_high samples compared to DCAF13_low samples. (D) A quantitative PCR was carried out to verify DCAF13 knockdown efficiency in BT549 cells and MDA‐MB‐231 cells. Data are presented as the mean ± SD. ( n = 3 independent experiments). *** P < 0.001, unpaired t ‐test. (E,F) Western blot and immunofluorescence staining assays were carried out to examine the effect of DCAF13 knockdown on EMT marker gene expression. Scale bar = 50 μm.
Article Snippet:
Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Knockdown, Western Blot, Immunofluorescence, Staining, Marker
Journal: FEBS Open Bio
Article Title: Doxorubicin promotes breast cancer cell migration and invasion via DCAF13
doi: 10.1002/2211-5463.13330
Figure Lengend Snippet: Doxorubicin promotes breast cancer cell migration, invasion and EMT via DCAF13. (A) Doxorubicin treatment increased DCAF13 expression in breast cancer cells. BT549 cells and MDA‐MB‐231 cells were treated with doxorubicin and total RNA was extracted for real‐time PCR analysis. Three independent experiments were carried out and the results were quantified and represented as the mean ± SD. *** P < 0.001, unpaired t ‐test. (B,C) Doxorubicin promotes breast cancer cell migration via DCAF13. Wound healing assays were carried out to assess the effect of doxorubicin treatment on breast cancer cell migration and the involvement of DCAF13. Three independent experiments were carried out and the results were quantified and represented as the mean ± SD. ** P < 0.01, unpaired t ‐test. Scale bar = 100 μm. (D,E) Doxorubicin promotes breast cancer cell invasion via DCAF13. Transwell cell invasion assays were performed to assess the effect of doxorubicin treatment on breast cancer cell invasion and the involvement of DCAF13. Three independent experiments were carried out and the results were quantified and represented as the mean ± SD. ** P < 0.01, *** P < 0.001, unpaired t ‐test. Scale bar = 100 μm. (F) Doxorubicin promotes the EMT in breast cancer cells via DCAF13. A western blot assay was performed to examine the effect of doxorubicin treatment on EMT marker gene expression and the involvement of DCAF13.
Article Snippet:
Techniques: Migration, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, Gene Expression
Journal: Oncotarget
Article Title: Chronic inflammation contributes to the development of hepatocellular carcinoma by decreasing miR-122 levels
doi: 10.18632/oncotarget.7740
Figure Lengend Snippet: A. Analysis of miR-122 expression in liver biopsy specimens from CHB patients, HCC tumors, and healthy controls (HC) by real-time PCR. Results were normalized to a U6 endogenous control, RNU6B. miR-122 levels in HC were arbitrarily set to 1.0. B. Real-time PCR analysis of miR-122 levels in CHB patients with ALT below and above 40 U/L. C. Correlation analysis of miR-122 levels and serum ALT levels in CHB patients. D. Meta-analysis of published gene expression microarray data comparing 15 alcohol-induced hepatitis samples to seven healthy livers. miR-33a and miR-122 targets predicted by TargetScan (TS) and miR-122 targets validated by reporter assay (mirbase) were analyzed. The mean fold change in expression of miR-122 targets was compared to miR-33a targets or all genes; the number of genes in each group is shown. E. Huh-7 cells were treated with 1000 IU/ml IL-6, TNF-α, IL-1α, IL-1β, TGF-β, IFN-γ, IFNα, or PBS as a control for 12 h. miR-122 expression was detected by real-time PCR. F. miR-122 levels were analyzed after treatment with the indicated amounts of IL-6 (left) or TNF-α (right). G. Huh-7 cells were treated with 1000 IU/ml IL-6 or TNF-α for 12 h. The expression of pri-miR-122 was analyzed by real-time PCR. H. HepG2 cells were transfected with miR-122 mimic or control mimic (left) and Huh-7 cells were transfected with miR-122 inhibitor or control inhibitor (right). miR-122 levels were assessed 48 h after transfection by real-time PCR. Data are presented as the mean ± SD from three independent experiments. p <0.05 (*), p < 0.01 (**), and p <0.0001 (***), compared to controls.
Article Snippet: Human hepatoma cell lines Huh-7 and
Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Gene Expression, Microarray, Reporter Assay, Transfection