Structured Review

Promega 293t cells
miR-342 targets BIRC6 and down-modulates Apollon/BRUCE protein in HCC1937 cells A. Schematic representation of the interaction of miR-342 at nucleotides 204-210 of the wild-type (wt) and mutated (mut) BIRC6 3′ UTR cloned into pGL3 promoter vectors (pLuc–BIRC6 and pLuc-MUT-BIRC6). B. Quantification of relative luciferase activity (RLU) in <t>293T</t> cells upon transfection with pre-miR-342 or scramble. Data are given as the ratio between luciferase activity detected in pre-miR-342 vs scramble-transfected cells and represent mean ± SD from at least three independent determinations. *P
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1) Product Images from "miR-342 overexpression results in a synthetic lethal phenotype in BRCA1-mutant HCC1937 breast cancer cells"

Article Title: miR-342 overexpression results in a synthetic lethal phenotype in BRCA1-mutant HCC1937 breast cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.7617

miR-342 targets BIRC6 and down-modulates Apollon/BRUCE protein in HCC1937 cells A. Schematic representation of the interaction of miR-342 at nucleotides 204-210 of the wild-type (wt) and mutated (mut) BIRC6 3′ UTR cloned into pGL3 promoter vectors (pLuc–BIRC6 and pLuc-MUT-BIRC6). B. Quantification of relative luciferase activity (RLU) in 293T cells upon transfection with pre-miR-342 or scramble. Data are given as the ratio between luciferase activity detected in pre-miR-342 vs scramble-transfected cells and represent mean ± SD from at least three independent determinations. *P
Figure Legend Snippet: miR-342 targets BIRC6 and down-modulates Apollon/BRUCE protein in HCC1937 cells A. Schematic representation of the interaction of miR-342 at nucleotides 204-210 of the wild-type (wt) and mutated (mut) BIRC6 3′ UTR cloned into pGL3 promoter vectors (pLuc–BIRC6 and pLuc-MUT-BIRC6). B. Quantification of relative luciferase activity (RLU) in 293T cells upon transfection with pre-miR-342 or scramble. Data are given as the ratio between luciferase activity detected in pre-miR-342 vs scramble-transfected cells and represent mean ± SD from at least three independent determinations. *P

Techniques Used: Clone Assay, Luciferase, Activity Assay, Transfection

2) Product Images from "Herpes Simplex Virus 1 Tegument Protein US11 Downmodulates the RLR Signaling Pathway via Direct Interaction with RIG-I and MDA-5"

Article Title: Herpes Simplex Virus 1 Tegument Protein US11 Downmodulates the RLR Signaling Pathway via Direct Interaction with RIG-I and MDA-5

Journal: Journal of Virology

doi: 10.1128/JVI.06713-11

US11 inhibits SeV-mediated activation of the IFN-β and NF-κB promoter activities. (A, C, and D) HEK 293T cells were transfected with 500 ng of IFN-β promoter reporter plasmid p125-luc (A), NF-κB-Luc (C), or (pRDIII-I)4-Luc (D), together with Renilla luciferase plasmid pRL-TK (50 ng) and pCMV-HA empty vector or plasmids encoding the indicated viral proteins (1,000 ng). At 24 h after transfection, cells were left untreated or infected with 100 HAU ml −1 SeV as indicated, and luciferase activity was measured 16 h postinfection. (B) As in panel A, except an increased amount of US11-HA expression plasmid, as indicated, was used. The expression of US11 was analyzed by Western blotting using anti-HA and anti-β-actin (as a control) monoclonal antibodies. Data are expressed as relative luciferase activities with standard deviations for three independent experiments performed in duplicate. (E) HEK293T cells were transfected with pCMV-HA empty vector or US11-HA expression plasmid. At 24 h posttransfection, cells were mock infected or infected with 100 HAU ml −1 SeV for 16 h before RT-PCR was performed using GAPDH and IFN-β primers. (F) Medium from infected cells in panel E was isolated and analyzed by ELISA for IFN-β secretion as described in Materials and Methods. The data represent means + standard deviations for three replicates.
Figure Legend Snippet: US11 inhibits SeV-mediated activation of the IFN-β and NF-κB promoter activities. (A, C, and D) HEK 293T cells were transfected with 500 ng of IFN-β promoter reporter plasmid p125-luc (A), NF-κB-Luc (C), or (pRDIII-I)4-Luc (D), together with Renilla luciferase plasmid pRL-TK (50 ng) and pCMV-HA empty vector or plasmids encoding the indicated viral proteins (1,000 ng). At 24 h after transfection, cells were left untreated or infected with 100 HAU ml −1 SeV as indicated, and luciferase activity was measured 16 h postinfection. (B) As in panel A, except an increased amount of US11-HA expression plasmid, as indicated, was used. The expression of US11 was analyzed by Western blotting using anti-HA and anti-β-actin (as a control) monoclonal antibodies. Data are expressed as relative luciferase activities with standard deviations for three independent experiments performed in duplicate. (E) HEK293T cells were transfected with pCMV-HA empty vector or US11-HA expression plasmid. At 24 h posttransfection, cells were mock infected or infected with 100 HAU ml −1 SeV for 16 h before RT-PCR was performed using GAPDH and IFN-β primers. (F) Medium from infected cells in panel E was isolated and analyzed by ELISA for IFN-β secretion as described in Materials and Methods. The data represent means + standard deviations for three replicates.

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Activity Assay, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay

US11 impedes the activation of IRF3 downstream of the RLR signaling pathway. (A) US11 blocks IRF3 nuclear translocation induced by SeV infection. HeLa cells were transfected with the US11-HA expression plasmid or pCMV-HA vector. Twenty-four hours posttransfection, cells were then mock infected or infected with 100 HAU ml −1 SeV for 8 h. Cells were stained with mouse anti-HA MAb and rabbit anti-IRF3 antibody. FITC-conjugated goat anti-mouse (green) and TRITC-conjugated goat anti-rabbit (red) were used as the secondary antibody. Cell nuclei (blue) were stained with Hoechst 33258. The images were obtained by fluorescence microscopy using a ×40 objective. (B) Cells expressing US11-HA or vector from panel A were scored for nuclear translocation of IRF3. At least 100 cells were counted for each sample. Data shown are from one representative experiment of at least three. (C and D) US11 inhibits SeV-induced IRF3 phosphorylation and dimerization. HEK293T cells were transfected with the US11-HA expression plasmid. Twenty-four hours posttransfection, cells were then mock infected or infected with 100 HAU ml −1 SeV for 8 h. Protein extracts were subjected to SDS-PAGE (C) or native PAGE (D) for subsequent analysis with anti-phospho-IRF3 (Ser396) (C) or anti-IRF3 (D) antibody. IRF3 and actin as a loading control were also detected.
Figure Legend Snippet: US11 impedes the activation of IRF3 downstream of the RLR signaling pathway. (A) US11 blocks IRF3 nuclear translocation induced by SeV infection. HeLa cells were transfected with the US11-HA expression plasmid or pCMV-HA vector. Twenty-four hours posttransfection, cells were then mock infected or infected with 100 HAU ml −1 SeV for 8 h. Cells were stained with mouse anti-HA MAb and rabbit anti-IRF3 antibody. FITC-conjugated goat anti-mouse (green) and TRITC-conjugated goat anti-rabbit (red) were used as the secondary antibody. Cell nuclei (blue) were stained with Hoechst 33258. The images were obtained by fluorescence microscopy using a ×40 objective. (B) Cells expressing US11-HA or vector from panel A were scored for nuclear translocation of IRF3. At least 100 cells were counted for each sample. Data shown are from one representative experiment of at least three. (C and D) US11 inhibits SeV-induced IRF3 phosphorylation and dimerization. HEK293T cells were transfected with the US11-HA expression plasmid. Twenty-four hours posttransfection, cells were then mock infected or infected with 100 HAU ml −1 SeV for 8 h. Protein extracts were subjected to SDS-PAGE (C) or native PAGE (D) for subsequent analysis with anti-phospho-IRF3 (Ser396) (C) or anti-IRF3 (D) antibody. IRF3 and actin as a loading control were also detected.

Techniques Used: Activation Assay, Translocation Assay, Infection, Transfection, Expressing, Plasmid Preparation, Staining, Fluorescence, Microscopy, SDS Page, Clear Native PAGE

US11 blocks the formation of complex between RIG-I and MAVS or between MDA-5 and MAVS. (A and B) HEK293T cells (∼5 × 10 6 ) were cotransfected with 10 μg of plasmid pEF-Flag-RIG-I (A) or pEF-Flag-MDA-5 (B) or 10 μg of plasmid pMyc-MAVS and 10 μg of plasmid US11-HA or transfected with empty vector. At 36 h posttransfection, cells were lysed and the clarified supernatants were subjected to immunoprecipitation assays using anti-Myc MAb (IP: Myc) or nonspecific mouse monoclonal antibody (IgG1). US11, MAVS, or RIG-I or MDA-5 was detected by Western blotting using anti-HA, anti-Myc, or anti-Flag MAbs, respectively. (C and D) HEK293T cells were cotransfected with plasmids pMyc-MAVS and pEF-Flag-RIG-I (C) or pEF-Flag-MDA-5 (D), respectively. Twenty hours after transfection, cells were infected with either WT or US11 mutant HSV-1 at an MOI of 10 for another 16 h. The cells were subsequently lysed and subjected to immunoprecipitation assays as described for panel A. Additionally, rabbit anti-US11 pAb was used for detection of US11 expression after either WT or US11 mutant HSV-1 infection.
Figure Legend Snippet: US11 blocks the formation of complex between RIG-I and MAVS or between MDA-5 and MAVS. (A and B) HEK293T cells (∼5 × 10 6 ) were cotransfected with 10 μg of plasmid pEF-Flag-RIG-I (A) or pEF-Flag-MDA-5 (B) or 10 μg of plasmid pMyc-MAVS and 10 μg of plasmid US11-HA or transfected with empty vector. At 36 h posttransfection, cells were lysed and the clarified supernatants were subjected to immunoprecipitation assays using anti-Myc MAb (IP: Myc) or nonspecific mouse monoclonal antibody (IgG1). US11, MAVS, or RIG-I or MDA-5 was detected by Western blotting using anti-HA, anti-Myc, or anti-Flag MAbs, respectively. (C and D) HEK293T cells were cotransfected with plasmids pMyc-MAVS and pEF-Flag-RIG-I (C) or pEF-Flag-MDA-5 (D), respectively. Twenty hours after transfection, cells were infected with either WT or US11 mutant HSV-1 at an MOI of 10 for another 16 h. The cells were subsequently lysed and subjected to immunoprecipitation assays as described for panel A. Additionally, rabbit anti-US11 pAb was used for detection of US11 expression after either WT or US11 mutant HSV-1 infection.

Techniques Used: Multiple Displacement Amplification, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Infection, Mutagenesis, Expressing

3) Product Images from "GDF11 Modulates Ca2+-Dependent Smad2/3 Signaling to Prevent Cardiomyocyte Hypertrophy"

Article Title: GDF11 Modulates Ca2+-Dependent Smad2/3 Signaling to Prevent Cardiomyocyte Hypertrophy

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19051508

Effects of intracellular Ca 2+ and the PLC/IP 3 R pathway on nuclear translocation and SBE-luc activity in cultured cardiomyocytes. ( A ) Cells were pretreated with BAPTA-AM (100 µM), U-73122 (50 µM), U-73343 (50 µM), or Xestospongin C (50 µM) and then stimulated with GDF11 (10 nM) for 1 h; ( B ) Cardiomyocytes were co-transfected with SBE-luc and Renilla luciferase plasmids; pretreated with U-73122 (50 µM), U-73343 (50 µM), or Xestospongin C (50 µM); and stimulated with GDF11 (10 nM) for 24 h. Values are expressed as mean ± SEM of triplicates from three independent experiments. * p
Figure Legend Snippet: Effects of intracellular Ca 2+ and the PLC/IP 3 R pathway on nuclear translocation and SBE-luc activity in cultured cardiomyocytes. ( A ) Cells were pretreated with BAPTA-AM (100 µM), U-73122 (50 µM), U-73343 (50 µM), or Xestospongin C (50 µM) and then stimulated with GDF11 (10 nM) for 1 h; ( B ) Cardiomyocytes were co-transfected with SBE-luc and Renilla luciferase plasmids; pretreated with U-73122 (50 µM), U-73343 (50 µM), or Xestospongin C (50 µM); and stimulated with GDF11 (10 nM) for 24 h. Values are expressed as mean ± SEM of triplicates from three independent experiments. * p

Techniques Used: Planar Chromatography, Translocation Assay, Activity Assay, Cell Culture, Transfection, Luciferase

Effects of GDF11 on Smad2/3 phosphorylation, nuclear translocation, and SBE-Luc activity in cultured cardiomyocytes. ( A ) Cells were treated with GDF11 (10 nM) for different times (0–60 min) for Western blot analysis. The figure shows representative Western blots, and the graph shows relative phospho-Smad2/3 levels with respect to total Smad2/3 ratio; ( B ) Cells were stimulated with GDF11 (10 nM) for 60 min and then subjected to immunofluorescent staining with an anti-Smad2/3 antibody (green); nuclei were stained with Hoescht 33342 dye (blue); and sarcomeres were stained with phalloidin–rhodamine (red). The figure shows representative images for control and stimulated conditions; ( C ) Cells were treated with GDF11 for 1 h at different concentrations (1 pM to 100 nM) for immunocytochemistry experiments. Quantification of Smad2/3 staining is shown as the nuclear-to-cytoplasmic fluorescence ratio; ( D ) Cells were cotransfected with the plasmids SBE-Luc and Renilla-luciferase and stimulated with GDF11 for 24 h at different concentrations (1 pM to 100 nM). Smad2/3 activity was expressed as the SBE-Luc to Renilla luciferase ratio. Values are expressed as mean ± SEM of triplicates from three independent experiments. ** p
Figure Legend Snippet: Effects of GDF11 on Smad2/3 phosphorylation, nuclear translocation, and SBE-Luc activity in cultured cardiomyocytes. ( A ) Cells were treated with GDF11 (10 nM) for different times (0–60 min) for Western blot analysis. The figure shows representative Western blots, and the graph shows relative phospho-Smad2/3 levels with respect to total Smad2/3 ratio; ( B ) Cells were stimulated with GDF11 (10 nM) for 60 min and then subjected to immunofluorescent staining with an anti-Smad2/3 antibody (green); nuclei were stained with Hoescht 33342 dye (blue); and sarcomeres were stained with phalloidin–rhodamine (red). The figure shows representative images for control and stimulated conditions; ( C ) Cells were treated with GDF11 for 1 h at different concentrations (1 pM to 100 nM) for immunocytochemistry experiments. Quantification of Smad2/3 staining is shown as the nuclear-to-cytoplasmic fluorescence ratio; ( D ) Cells were cotransfected with the plasmids SBE-Luc and Renilla-luciferase and stimulated with GDF11 for 24 h at different concentrations (1 pM to 100 nM). Smad2/3 activity was expressed as the SBE-Luc to Renilla luciferase ratio. Values are expressed as mean ± SEM of triplicates from three independent experiments. ** p

Techniques Used: Translocation Assay, Activity Assay, Cell Culture, Western Blot, Staining, Immunocytochemistry, Fluorescence, Luciferase

4) Product Images from "Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression"

Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

Journal: Oncotarget

doi: 10.18632/oncotarget.15499

miR-520c regulatory region is hypermethylated in CRC cell lines and treatment with 5-Aza induces the expression of miR-520c-3p and downregulates S100A4 expression ( A , B ) SW480, SW620 and HCT116 cells were treated with 5-Aza (2 mM) for 3 days. qRT-PCR and Western blots were performed to analyze the impact of 5-Aza treatment on miR-520c-3p and S100A4 expression. RNUB6, RPII and β-actin served as internal controls. ( C ) HCT116 and SW620 cells transfected with the wild type S100A4-3′-UTR were treated with 5-Aza (2 μM) for 24 h and the luciferase activity was measured. Renilla luciferase activity was used for normalization. Percentage luciferase activity was significantly reduced after 5-Aza. ( *p
Figure Legend Snippet: miR-520c regulatory region is hypermethylated in CRC cell lines and treatment with 5-Aza induces the expression of miR-520c-3p and downregulates S100A4 expression ( A , B ) SW480, SW620 and HCT116 cells were treated with 5-Aza (2 mM) for 3 days. qRT-PCR and Western blots were performed to analyze the impact of 5-Aza treatment on miR-520c-3p and S100A4 expression. RNUB6, RPII and β-actin served as internal controls. ( C ) HCT116 and SW620 cells transfected with the wild type S100A4-3′-UTR were treated with 5-Aza (2 μM) for 24 h and the luciferase activity was measured. Renilla luciferase activity was used for normalization. Percentage luciferase activity was significantly reduced after 5-Aza. ( *p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Activity Assay

5) Product Images from "Overexpression of miR-125a in Myelodysplastic Syndrome CD34+ Cells Modulates NF-?B Activation and Enhances Erythroid Differentiation Arrest"

Article Title: Overexpression of miR-125a in Myelodysplastic Syndrome CD34+ Cells Modulates NF-?B Activation and Enhances Erythroid Differentiation Arrest

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093404

Effect of TLR stimulation and miR-125a inhibition on NF-κB activity in KG1 cells. NF-κB activation was measured after 24 hours from nucleofection of KG1 cells with the luciferase vectors and treatment with ASO. Results are expressed as relative to cells transfected with mock and represent mean ± SEM of n = 4 independent experiments. Method disclosure: technical problems regarding the endogenous Renilla control were experienced during these luciferase assays; only one experiment out of four efficiently expressed the Renilla luciferase and could be properly normalized. Because normalized results were almost identical to non-normalized data, we conducted a joint statistical analysis of the four experiments. Statistical significance: ***P
Figure Legend Snippet: Effect of TLR stimulation and miR-125a inhibition on NF-κB activity in KG1 cells. NF-κB activation was measured after 24 hours from nucleofection of KG1 cells with the luciferase vectors and treatment with ASO. Results are expressed as relative to cells transfected with mock and represent mean ± SEM of n = 4 independent experiments. Method disclosure: technical problems regarding the endogenous Renilla control were experienced during these luciferase assays; only one experiment out of four efficiently expressed the Renilla luciferase and could be properly normalized. Because normalized results were almost identical to non-normalized data, we conducted a joint statistical analysis of the four experiments. Statistical significance: ***P

Techniques Used: Inhibition, Activity Assay, Activation Assay, Luciferase, Allele-specific Oligonucleotide, Transfection

6) Product Images from "The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer"

Article Title: The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0535-8

Influence of hsa-miR-12528 on migratory activity in vitro and in vivo. Post transfection, the delayed cell motility was captured in a time-dependent manner in scratched A549 cells ( a ). Cell migration or invasion was measured after 48 h post transfection to allow for the permeabilization of the Trans-well membrane ( b ). Proteolytic enzyme activity of the secreted MMP-2 and -9 was analysed in the cultured soup for 48 h post transfection ( c ). The lung metastatic models that were injected with the miRNA mimic-transfected Fluc-stable A549 cells into the tail vein were grown for 5 weeks ( n = 8 mice/group). After 5 weeks, the expressed Fluc signals visually show the degree of metastatic spread to lung, and Fluc signal intensity was quantified by ROI gates ( d ). Nodules generated on the lung surface (left) and histological staining (H E) of lung tissues (right) visually show a distinct difference between each group ( e ) (bars, mean ± S.E.M.; * p
Figure Legend Snippet: Influence of hsa-miR-12528 on migratory activity in vitro and in vivo. Post transfection, the delayed cell motility was captured in a time-dependent manner in scratched A549 cells ( a ). Cell migration or invasion was measured after 48 h post transfection to allow for the permeabilization of the Trans-well membrane ( b ). Proteolytic enzyme activity of the secreted MMP-2 and -9 was analysed in the cultured soup for 48 h post transfection ( c ). The lung metastatic models that were injected with the miRNA mimic-transfected Fluc-stable A549 cells into the tail vein were grown for 5 weeks ( n = 8 mice/group). After 5 weeks, the expressed Fluc signals visually show the degree of metastatic spread to lung, and Fluc signal intensity was quantified by ROI gates ( d ). Nodules generated on the lung surface (left) and histological staining (H E) of lung tissues (right) visually show a distinct difference between each group ( e ) (bars, mean ± S.E.M.; * p

Techniques Used: Activity Assay, In Vitro, In Vivo, Transfection, Migration, Cell Culture, Injection, Mouse Assay, Generated, Staining

Signalling alteration of IGF-1R networks in the absence and presence of the hsa-miR-12528. The dynamic signalling graphics model based on the presence and absence of miR-12528. The upregulated IGF-1R in NSCLC mediates the interaction of ligands such as IGF-1. Upon internalization between the extracellular ligand and receptor, IGF-1R networks mediate the hyperphosphorylation of the Akt/mTOR, which subsequently leads to the development or progression of NSCLC. In the presence of miR-12528, blocking IGF-1R translation leads to a reduction of the interaction between IGF-1R and its ligand via the lack of the ligand-binding regions, and induces hypo-phosphorylation. Consequently, miR-12528 can negatively regulate NSCLC progression by modulating the cell cycle and apoptotic programmed cell death
Figure Legend Snippet: Signalling alteration of IGF-1R networks in the absence and presence of the hsa-miR-12528. The dynamic signalling graphics model based on the presence and absence of miR-12528. The upregulated IGF-1R in NSCLC mediates the interaction of ligands such as IGF-1. Upon internalization between the extracellular ligand and receptor, IGF-1R networks mediate the hyperphosphorylation of the Akt/mTOR, which subsequently leads to the development or progression of NSCLC. In the presence of miR-12528, blocking IGF-1R translation leads to a reduction of the interaction between IGF-1R and its ligand via the lack of the ligand-binding regions, and induces hypo-phosphorylation. Consequently, miR-12528 can negatively regulate NSCLC progression by modulating the cell cycle and apoptotic programmed cell death

Techniques Used: Blocking Assay, Ligand Binding Assay

Basic information, expression profiling and influence on the novel hsa-miR-12528 in lung cancer. Identification and cloning of the miR-12528 from lung cancer cells. The assumed secondary folding structure of miR-12528. Human genomic sequences were found using the RNAfold web-tool. The marked location is a mature miR-12528 sequence. The miR-12528 is located on chromosome 19p.13.3 (649215-649234) ( a ). Maturation of miR-12528 is dependent on the Dicer pathway, as shown via Dicer -knockdown and an miScript miRNA assay ( b ). Mature miR-12528 is conserved at a high rate in other species. These results were determined using the NCBI BLAST tool ( c ). The miR-12528 expression was assessed in both 7 NSCLC cell lines ( d ) and 20 pairs of NSCLC patient tissues ( e ) compared with normal WI-38 and BEAS-2B cells, and matched normal tissues. When classified histologically, cases 1–10 are adenocarcinoma type and cases 11–20 are squamous cell carcinoma type ( e ). The proliferative activity in 7 NSCLC cells was assessed using a XTT reagent 48 h post transfection with 100 nM mimics ( f ). The miR-12528 expression levels were observed using qRT-PCR between absence and presence of FBS stimulation ( g ) and between diploid normal WI-38 and immortalised WI-38 VA13 ( h ). These expression profiling data were normalised to RNU6B and performed in triplicate using an miScript assay on independent samples (bars, mean ± S.E.M.; * p
Figure Legend Snippet: Basic information, expression profiling and influence on the novel hsa-miR-12528 in lung cancer. Identification and cloning of the miR-12528 from lung cancer cells. The assumed secondary folding structure of miR-12528. Human genomic sequences were found using the RNAfold web-tool. The marked location is a mature miR-12528 sequence. The miR-12528 is located on chromosome 19p.13.3 (649215-649234) ( a ). Maturation of miR-12528 is dependent on the Dicer pathway, as shown via Dicer -knockdown and an miScript miRNA assay ( b ). Mature miR-12528 is conserved at a high rate in other species. These results were determined using the NCBI BLAST tool ( c ). The miR-12528 expression was assessed in both 7 NSCLC cell lines ( d ) and 20 pairs of NSCLC patient tissues ( e ) compared with normal WI-38 and BEAS-2B cells, and matched normal tissues. When classified histologically, cases 1–10 are adenocarcinoma type and cases 11–20 are squamous cell carcinoma type ( e ). The proliferative activity in 7 NSCLC cells was assessed using a XTT reagent 48 h post transfection with 100 nM mimics ( f ). The miR-12528 expression levels were observed using qRT-PCR between absence and presence of FBS stimulation ( g ) and between diploid normal WI-38 and immortalised WI-38 VA13 ( h ). These expression profiling data were normalised to RNU6B and performed in triplicate using an miScript assay on independent samples (bars, mean ± S.E.M.; * p

Techniques Used: Expressing, Clone Assay, Genomic Sequencing, Sequencing, Activity Assay, Transfection, Quantitative RT-PCR

Relationship between the IGF-1R gene and hsa-miR-12528 in A549 cells. The target validation of miR-12528. The pGL3-plasmid was recombined by inserting the 3′-UTR of IGF-1R. The binding sequence of miR-12528 is marked in yellow fills (top; wild type, WT), and was replaced with a mismatched sequence using site-directed mutagenesis (bottom; mutant, MT) ( a ). Luciferase activity of WT and MT was analysed by co-transfecting the recombinant pGL3 construction, pRL-TK and miRNA mimics, and normalised to pRL-TK; Renilla luciferase ( b ). The expression levels between mRNA and protein of IGF-1R gene were assessed using quantitative RT-PCR and western blot analysis and normalised to 18S rRNA or GAPDH. Western blot analysis was semi-quantified using NIH ImageJ programme ( c ). The signalling alteration of the IGF-1R-downstream pathway was analysed using a western blot analysis 48 h post transfection (mimic conc.; 50 nM and *; 100 nM) ( d ) (bars, mean ± S.E.M; * p
Figure Legend Snippet: Relationship between the IGF-1R gene and hsa-miR-12528 in A549 cells. The target validation of miR-12528. The pGL3-plasmid was recombined by inserting the 3′-UTR of IGF-1R. The binding sequence of miR-12528 is marked in yellow fills (top; wild type, WT), and was replaced with a mismatched sequence using site-directed mutagenesis (bottom; mutant, MT) ( a ). Luciferase activity of WT and MT was analysed by co-transfecting the recombinant pGL3 construction, pRL-TK and miRNA mimics, and normalised to pRL-TK; Renilla luciferase ( b ). The expression levels between mRNA and protein of IGF-1R gene were assessed using quantitative RT-PCR and western blot analysis and normalised to 18S rRNA or GAPDH. Western blot analysis was semi-quantified using NIH ImageJ programme ( c ). The signalling alteration of the IGF-1R-downstream pathway was analysed using a western blot analysis 48 h post transfection (mimic conc.; 50 nM and *; 100 nM) ( d ) (bars, mean ± S.E.M; * p

Techniques Used: Plasmid Preparation, Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Recombinant, Expressing, Quantitative RT-PCR, Western Blot, Transfection

The capacity of hsa-miR-12528 during tumourigenesis in an in vivo model. Influence of miR-12528 during tumourigenesis in vivo. A549 cells were subcutaneously injected in equal amounts into both flanks in an individual mouse. After stabilisation for 5 weeks, the left tumours were only injected with PBS, whereas the right tumours were injected with either NC, miR-12528 or ASO-12528 mimics twice weekly. After a total of 4 weeks from the time of mimic injection, 7 mice per group were sacrificed (a total of 21 mice) and the excised tumour mass was imaged and measured ( a ). The rations of tumour volumes indicate that the right tumours injected with NC, miR-12528 or ASO-12528 mimics were normalised to a left tumour (MOCK) within each individual mouse ( b ). The tumour sections were expressed with H E staining, Ki67-IHC and TUNEL assay. The H E staining result shows the histological morphology of tumour tissues. Positive results for Ki67 and TUNEL, markers of proliferation and apoptosis, were indicated by brown nuclear staining ( c )
Figure Legend Snippet: The capacity of hsa-miR-12528 during tumourigenesis in an in vivo model. Influence of miR-12528 during tumourigenesis in vivo. A549 cells were subcutaneously injected in equal amounts into both flanks in an individual mouse. After stabilisation for 5 weeks, the left tumours were only injected with PBS, whereas the right tumours were injected with either NC, miR-12528 or ASO-12528 mimics twice weekly. After a total of 4 weeks from the time of mimic injection, 7 mice per group were sacrificed (a total of 21 mice) and the excised tumour mass was imaged and measured ( a ). The rations of tumour volumes indicate that the right tumours injected with NC, miR-12528 or ASO-12528 mimics were normalised to a left tumour (MOCK) within each individual mouse ( b ). The tumour sections were expressed with H E staining, Ki67-IHC and TUNEL assay. The H E staining result shows the histological morphology of tumour tissues. Positive results for Ki67 and TUNEL, markers of proliferation and apoptosis, were indicated by brown nuclear staining ( c )

Techniques Used: In Vivo, Injection, Allele-specific Oligonucleotide, Mouse Assay, Staining, Immunohistochemistry, TUNEL Assay

The anticancer effect of hsa-miR-12528 in cell cycle and apoptosis pathway. The signalling alteration and effect for miR-12528 on cell cycle and apoptosis pathway. Forty-eight hours post transfection, Cdk-2, Cdk-4, Rb and pRb proteins in cell cycle pathway with Bcl-2 and XIAP, anti-apoptotic proteins, were analysed using a western blotting ( a ). Caspase-3/-7 activity, apoptotic execution factors, assessed using a Caspase-Glo ® 3/7 reagent (mimic conc.; 50 nM and *; 100 nM) ( b ). FACS analysis was performed using flow cytometry in A549 cells fixed or permeabilized 48 h post transfection. Cell cycle distribution was determined according to DNA stained with PI ( c ). Apoptotic cell death was sorted using Annexin V-FITC and PI staining. Annexin V-FITC (Annexin V + /PI − ) indicates that the distribution of apoptosis in the inner leaflet of the phospholipid bilayer of the plasma membrane is translocated to the outer leaflet where the plasma membrane is intact. Annexin V + /PI + indicates the distribution of late apoptotic cell death ( d ). Influence of miR-12528 on cell viability in vitro. In short-term proliferative rates, A549 cells were determined to be time-dependent via a XTT assay after transfection with 100 nM miRNA mimics. Cell morphology was imaged at 48 h (×100 magnification) ( e ). After mimic transfection, A549 cell-derived colonies were formed in soft agar for 3 weeks at 37 °C with 5% CO 2 . Graphical presentation shows the representative single colonies at high-power views (×100 magnification) and the numbers of colony-forming units ( f ). The experiments were performed in triplicate for independent samples (bars, mean ± S.E.M.; * p
Figure Legend Snippet: The anticancer effect of hsa-miR-12528 in cell cycle and apoptosis pathway. The signalling alteration and effect for miR-12528 on cell cycle and apoptosis pathway. Forty-eight hours post transfection, Cdk-2, Cdk-4, Rb and pRb proteins in cell cycle pathway with Bcl-2 and XIAP, anti-apoptotic proteins, were analysed using a western blotting ( a ). Caspase-3/-7 activity, apoptotic execution factors, assessed using a Caspase-Glo ® 3/7 reagent (mimic conc.; 50 nM and *; 100 nM) ( b ). FACS analysis was performed using flow cytometry in A549 cells fixed or permeabilized 48 h post transfection. Cell cycle distribution was determined according to DNA stained with PI ( c ). Apoptotic cell death was sorted using Annexin V-FITC and PI staining. Annexin V-FITC (Annexin V + /PI − ) indicates that the distribution of apoptosis in the inner leaflet of the phospholipid bilayer of the plasma membrane is translocated to the outer leaflet where the plasma membrane is intact. Annexin V + /PI + indicates the distribution of late apoptotic cell death ( d ). Influence of miR-12528 on cell viability in vitro. In short-term proliferative rates, A549 cells were determined to be time-dependent via a XTT assay after transfection with 100 nM miRNA mimics. Cell morphology was imaged at 48 h (×100 magnification) ( e ). After mimic transfection, A549 cell-derived colonies were formed in soft agar for 3 weeks at 37 °C with 5% CO 2 . Graphical presentation shows the representative single colonies at high-power views (×100 magnification) and the numbers of colony-forming units ( f ). The experiments were performed in triplicate for independent samples (bars, mean ± S.E.M.; * p

Techniques Used: Transfection, Western Blot, Activity Assay, FACS, Flow Cytometry, Cytometry, Staining, In Vitro, XTT Assay, Derivative Assay

7) Product Images from "CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis"

Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis

Journal: Cancer Cell International

doi: 10.1186/s12935-020-1097-2

GRHL2 mediated the regulatory effects of miR-144 on trophoblast cell proliferation, migration and invasion. Cell proliferation by MTS assay ( a , b ), cell migration by wound healing assay ( c , d ), cell invasion by transwell assay ( e , f ) in HTR-8/SVneo cells transfected with miR-con mimic, miR-144 mimic, miR-144 mimic + negative control plasmid (vector), miR-144 mimic + GRHL2 overexpression plasmid, anti-miR-con, anti-miR-144, anti-miR-144 + si-con, or anti-miR-144 + si-GRHL2. * P
Figure Legend Snippet: GRHL2 mediated the regulatory effects of miR-144 on trophoblast cell proliferation, migration and invasion. Cell proliferation by MTS assay ( a , b ), cell migration by wound healing assay ( c , d ), cell invasion by transwell assay ( e , f ) in HTR-8/SVneo cells transfected with miR-con mimic, miR-144 mimic, miR-144 mimic + negative control plasmid (vector), miR-144 mimic + GRHL2 overexpression plasmid, anti-miR-con, anti-miR-144, anti-miR-144 + si-con, or anti-miR-144 + si-GRHL2. * P

Techniques Used: Migration, MTS Assay, Wound Healing Assay, Transwell Assay, Transfection, Negative Control, Plasmid Preparation, Over Expression

CircZDHHC20 regulated GRHL2 expression through sponging miR-144. Correlations between GRHL2 mRNA expression and miR-144 level ( a ) or circZDHHC20 level ( b ) in 26 placental tissues from PE patients using the Spearman test. c , d GRHL2 protein level by western blot in HTR-8/SVneo cells transfected with negative control plasmid (vector), circZDHHC20 overexpression plasmid (circZDHHC20), circZDHHC20 + miR-con mimic, circZDHHC20 + miR-144 mimic, si-con, si-circZDHHC20, si-circZDHHC20 + anti-miR-con or si-circZDHHC20 + anti-miR-144. * P
Figure Legend Snippet: CircZDHHC20 regulated GRHL2 expression through sponging miR-144. Correlations between GRHL2 mRNA expression and miR-144 level ( a ) or circZDHHC20 level ( b ) in 26 placental tissues from PE patients using the Spearman test. c , d GRHL2 protein level by western blot in HTR-8/SVneo cells transfected with negative control plasmid (vector), circZDHHC20 overexpression plasmid (circZDHHC20), circZDHHC20 + miR-con mimic, circZDHHC20 + miR-144 mimic, si-con, si-circZDHHC20, si-circZDHHC20 + anti-miR-con or si-circZDHHC20 + anti-miR-144. * P

Techniques Used: Expressing, Western Blot, Transfection, Negative Control, Plasmid Preparation, Over Expression

CircZDHHC20 was up-regulated and miR-144 was down-regulated in PE placental tissues. a qRT-PCR for circZDHHC20 expression in placental tissues from 26 PE patients and 15 healthy volunteers. b qRT-PCR for the levels of circZDHHC20 and linear ZDHHC20 mRNA after RNase R digestion. c The expression of circZDHHC20 and linear ZDHHC20 mRNA by qRT-PCR in reverse transcription using Random and Oligo(dT) 18 primers. d CircZDHHC20 level by qRT-PCR in the nuclear and cytoplasm fractions of HTR-8/SVneo cells. 18S rRNA and U6 were used as internal controls. e The level of miR-144 in placental tissues from 26 PE patients and 15 healthy volunteers. f The correlation between circZDHHC20 level and miR-144 expression in placental tissues from 26 PE patients using the Spearman test. * P
Figure Legend Snippet: CircZDHHC20 was up-regulated and miR-144 was down-regulated in PE placental tissues. a qRT-PCR for circZDHHC20 expression in placental tissues from 26 PE patients and 15 healthy volunteers. b qRT-PCR for the levels of circZDHHC20 and linear ZDHHC20 mRNA after RNase R digestion. c The expression of circZDHHC20 and linear ZDHHC20 mRNA by qRT-PCR in reverse transcription using Random and Oligo(dT) 18 primers. d CircZDHHC20 level by qRT-PCR in the nuclear and cytoplasm fractions of HTR-8/SVneo cells. 18S rRNA and U6 were used as internal controls. e The level of miR-144 in placental tissues from 26 PE patients and 15 healthy volunteers. f The correlation between circZDHHC20 level and miR-144 expression in placental tissues from 26 PE patients using the Spearman test. * P

Techniques Used: Quantitative RT-PCR, Expressing

CircZDHHC20 acted as a molecular sponge of miR-144. a Schematic of circZDHHC20 illustrating position of the miR-144-binding site and mutated the miR-144-binding site. b , c Relative luciferase activity in HTR-8/SVneo cells cotransfected with circZDHHC20-WT or circZDHHC20-MUT and miR-con mimic, miR-144 mimic, anti-miR-con or anti-miR-144. d The enrichment of circZDHHC20 and miR-144 in the RISC of HTR-8/SVneo cells using anti-Ago2 or IgG antibody, with Input content as positive control. e The expression of miR-144 by qRT-PCR in HTR-8/SVneo cells transfected with negative control plasmid (vector), circZDHHC20 overexpression plasmid (circZDHHC20), si-con or si-circZDHHC20. * P
Figure Legend Snippet: CircZDHHC20 acted as a molecular sponge of miR-144. a Schematic of circZDHHC20 illustrating position of the miR-144-binding site and mutated the miR-144-binding site. b , c Relative luciferase activity in HTR-8/SVneo cells cotransfected with circZDHHC20-WT or circZDHHC20-MUT and miR-con mimic, miR-144 mimic, anti-miR-con or anti-miR-144. d The enrichment of circZDHHC20 and miR-144 in the RISC of HTR-8/SVneo cells using anti-Ago2 or IgG antibody, with Input content as positive control. e The expression of miR-144 by qRT-PCR in HTR-8/SVneo cells transfected with negative control plasmid (vector), circZDHHC20 overexpression plasmid (circZDHHC20), si-con or si-circZDHHC20. * P

Techniques Used: Binding Assay, Luciferase, Activity Assay, Positive Control, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Plasmid Preparation, Over Expression

GRHL2 was a direct target of miR-144. a Schematic of the complementary site for miR-144 in the 3′-UTR of GRHL2 and the mutation of the seed region. b , c Relative luciferase activity in HTR-8/SVneo cells introduced with GRHL2-WT or GRHL2-MUT together with miR-con mimic, miR-144 mimic, anti-miR-con or anti-miR-144. d The enrichment of GRHL2 and miR-144 in the RISC of HTR-8/SVneo cells using anti-Ago2 or IgG antibody, with Input content as positive control. e qRT-PCR for GRHL2 mRNA in placental tissues from 26 PE patients and 15 healthy volunteers. f Western blot for GRHL2 protein level in HTR-8/SVneo cells transfected with miR-con mimic, miR-144 mimic, anti-miR-con, or anti-miR-144. * P
Figure Legend Snippet: GRHL2 was a direct target of miR-144. a Schematic of the complementary site for miR-144 in the 3′-UTR of GRHL2 and the mutation of the seed region. b , c Relative luciferase activity in HTR-8/SVneo cells introduced with GRHL2-WT or GRHL2-MUT together with miR-con mimic, miR-144 mimic, anti-miR-con or anti-miR-144. d The enrichment of GRHL2 and miR-144 in the RISC of HTR-8/SVneo cells using anti-Ago2 or IgG antibody, with Input content as positive control. e qRT-PCR for GRHL2 mRNA in placental tissues from 26 PE patients and 15 healthy volunteers. f Western blot for GRHL2 protein level in HTR-8/SVneo cells transfected with miR-con mimic, miR-144 mimic, anti-miR-con, or anti-miR-144. * P

Techniques Used: Mutagenesis, Luciferase, Activity Assay, Positive Control, Quantitative RT-PCR, Western Blot, Transfection

MiR-144 was involved in the regulation of circZDHHC20 on trophoblast cell proliferation, migration, and invasion. MTS assay for cell proliferation ( a , b ), wound healing assay for cell migration ( c , d ), transwell assay for cell invasion ( e , f ) in HTR-8/SVneo cells transfected with si-con, si-circZDHHC20, si-circZDHHC20 + anti-miR-con, si-circZDHHC20 + anti-miR-144, negative control plasmid (vector), circZDHHC20 overexpression plasmid (circZDHHC20), circZDHHC20 + miR-con mimic, or circZDHHC20 + miR-144 mimic. * P
Figure Legend Snippet: MiR-144 was involved in the regulation of circZDHHC20 on trophoblast cell proliferation, migration, and invasion. MTS assay for cell proliferation ( a , b ), wound healing assay for cell migration ( c , d ), transwell assay for cell invasion ( e , f ) in HTR-8/SVneo cells transfected with si-con, si-circZDHHC20, si-circZDHHC20 + anti-miR-con, si-circZDHHC20 + anti-miR-144, negative control plasmid (vector), circZDHHC20 overexpression plasmid (circZDHHC20), circZDHHC20 + miR-con mimic, or circZDHHC20 + miR-144 mimic. * P

Techniques Used: Migration, MTS Assay, Wound Healing Assay, Transwell Assay, Transfection, Negative Control, Plasmid Preparation, Over Expression

8) Product Images from "Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil"

Article Title: Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0005048

Activation of the IFN-β promoter by poly I:C in cells over expressing ZIKV sfRNA. A549 cells were co-transfected with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP (sfRNA over-expression plasmids and MBP-HDVr control, respectively) and p125Luc IFN-β promoter reporter (expressing Firefly luciferase) along with pRL-CMV (internal control, expressing Renilla luciferase). The IFN-β promoter was stimulated by transfecting poly I:C 24 h after the primary transfection. The relative luciferase activity (Firefly/ Renilla ) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in triplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any poly I:C treatment. Asterisk (*) indicates significance (2-way ANOVA, p
Figure Legend Snippet: Activation of the IFN-β promoter by poly I:C in cells over expressing ZIKV sfRNA. A549 cells were co-transfected with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP (sfRNA over-expression plasmids and MBP-HDVr control, respectively) and p125Luc IFN-β promoter reporter (expressing Firefly luciferase) along with pRL-CMV (internal control, expressing Renilla luciferase). The IFN-β promoter was stimulated by transfecting poly I:C 24 h after the primary transfection. The relative luciferase activity (Firefly/ Renilla ) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in triplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any poly I:C treatment. Asterisk (*) indicates significance (2-way ANOVA, p

Techniques Used: Activation Assay, Expressing, Transfection, Over Expression, Luciferase, Activity Assay

Activation of the IFN-β promoter by RIG-I or MDA-5 agonists in cells over-expressing ZIKV sfRNA. A549 cells were co-transfected as described with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP and p125Luc IFN-β promoter reporter along with pRL-CMV. The IFN-β promoter was stimulated by transfecting either RIG-I agonist (Neo 1-99 IVT-RNA) (A) or MDA-5 agonist (Vero cell produced EMCV RNA) (B) 24 h after the primary transfection. The relative luciferase activity (Firefly/ Renilla ) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in duplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any agonist treatment. Asterisk (*) indicates significance (2-way ANOVA, p
Figure Legend Snippet: Activation of the IFN-β promoter by RIG-I or MDA-5 agonists in cells over-expressing ZIKV sfRNA. A549 cells were co-transfected as described with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP and p125Luc IFN-β promoter reporter along with pRL-CMV. The IFN-β promoter was stimulated by transfecting either RIG-I agonist (Neo 1-99 IVT-RNA) (A) or MDA-5 agonist (Vero cell produced EMCV RNA) (B) 24 h after the primary transfection. The relative luciferase activity (Firefly/ Renilla ) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in duplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any agonist treatment. Asterisk (*) indicates significance (2-way ANOVA, p

Techniques Used: Activation Assay, Multiple Displacement Amplification, Expressing, Transfection, Produced, Luciferase, Activity Assay

9) Product Images from "lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis"

Article Title: lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis

Journal: Cells

doi: 10.3390/cells7120243

miR-1611 interacts with lncRNA-Six1 to regulate Six1 expression. ( A ) The potential binding site of miR-1611 in the lncRNA-Six1 transcript and the Six1 3′ untranslated region (UTR). The mutant sequence in the miR-1611 binding site is highlighted in red. ( B – D ) A dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: ( B ) lncRNA-Six1 position 1, ( C ) lncRNA-Six1 position 2, or ( D ) Six1 3′ UTR with a miR-1611 mimic or mimic-NC in DF-1 cells. ( E ) Relative lncRNA-Six1 expression after overexpression and inhibition of miR-1611. ( F ) The mRNA and protein expression levels of Six1 from the miR-1611 mimic and inhibitor-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There is only one Western blot performed per treatment, and therefore, n = 1. ( G ) Cell cycle analysis of CPMs after co-transfection with the listed nucleic acids. ( H ) Relative mRNA levels of several myoblast differentiation marker genes from co-transfected CPMs. ( I , J ) The mRNA expression levels of several fast and slow muscle genes induced by the listed nucleic acids in CPMs. In all panels, data are presented as means ± S.E.M. of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: miR-1611 interacts with lncRNA-Six1 to regulate Six1 expression. ( A ) The potential binding site of miR-1611 in the lncRNA-Six1 transcript and the Six1 3′ untranslated region (UTR). The mutant sequence in the miR-1611 binding site is highlighted in red. ( B – D ) A dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: ( B ) lncRNA-Six1 position 1, ( C ) lncRNA-Six1 position 2, or ( D ) Six1 3′ UTR with a miR-1611 mimic or mimic-NC in DF-1 cells. ( E ) Relative lncRNA-Six1 expression after overexpression and inhibition of miR-1611. ( F ) The mRNA and protein expression levels of Six1 from the miR-1611 mimic and inhibitor-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There is only one Western blot performed per treatment, and therefore, n = 1. ( G ) Cell cycle analysis of CPMs after co-transfection with the listed nucleic acids. ( H ) Relative mRNA levels of several myoblast differentiation marker genes from co-transfected CPMs. ( I , J ) The mRNA expression levels of several fast and slow muscle genes induced by the listed nucleic acids in CPMs. In all panels, data are presented as means ± S.E.M. of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Over Expression, Inhibition, Transfection, Western Blot, Cell Cycle Assay, Cotransfection, Marker

lncRNA-Six1 functions to facilitate myoblast proliferation and differentiation. ( A ) The relative expression levels of lncRNA-Six1 with lncRNA-Six1 overexpression and knockdown. ( B ) The mRNA and protein expression levels of Six1 from pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( C ) Cell cycle analysis of CPMs after lncRNA-Six1 overexpression and knockdown. ( D ) EdU proliferation assays for CPMs with the overexpression and inhibition of lncRNA-Six1, ( E ) the numbers of proliferative cells were also counted. ( F , G ) CPM growth curves following the transfection of pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1. ( H ) Relative lncRNA-Six1 expression during CPM differentiation. ( I ) The mRNA and protein expression levels of myoblast differentiation marker genes with lncRNA-Six1 overexpression and knockdown in the CPMs. The numbers shown below the bands were fold-changes of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( J ) Immunofluorescence analysis of MyHC-staining cells with the overexpression and knockdown of lncRNA-Six1 in CPMs. ( K ) Myotube area (%) of CPMs with lncRNA-Six1 overexpression and knockdown. In all panels, results are expressed as the mean ± SEM of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: lncRNA-Six1 functions to facilitate myoblast proliferation and differentiation. ( A ) The relative expression levels of lncRNA-Six1 with lncRNA-Six1 overexpression and knockdown. ( B ) The mRNA and protein expression levels of Six1 from pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( C ) Cell cycle analysis of CPMs after lncRNA-Six1 overexpression and knockdown. ( D ) EdU proliferation assays for CPMs with the overexpression and inhibition of lncRNA-Six1, ( E ) the numbers of proliferative cells were also counted. ( F , G ) CPM growth curves following the transfection of pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1. ( H ) Relative lncRNA-Six1 expression during CPM differentiation. ( I ) The mRNA and protein expression levels of myoblast differentiation marker genes with lncRNA-Six1 overexpression and knockdown in the CPMs. The numbers shown below the bands were fold-changes of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( J ) Immunofluorescence analysis of MyHC-staining cells with the overexpression and knockdown of lncRNA-Six1 in CPMs. ( K ) Myotube area (%) of CPMs with lncRNA-Six1 overexpression and knockdown. In all panels, results are expressed as the mean ± SEM of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Over Expression, Allele-specific Oligonucleotide, Transfection, Western Blot, Cell Cycle Assay, Inhibition, Marker, Immunofluorescence, Staining

lncRNA-Six1 induces the fast-twitch muscle phenotype. ( A , B ) The relative mRNA expression of several fast and slow muscle genes induced by lncRNA-Six1 overexpression and inhibition in CPMs. In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: lncRNA-Six1 induces the fast-twitch muscle phenotype. ( A , B ) The relative mRNA expression of several fast and slow muscle genes induced by lncRNA-Six1 overexpression and inhibition in CPMs. In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Over Expression, Inhibition

10) Product Images from "The Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1 Transcription Factors in Human Colon Cancer"

Article Title: The Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1 Transcription Factors in Human Colon Cancer

Journal: Gene expression

doi: 10.3727/105221615X14399878166230

GCN5 is a target of the transcription factor E2F1 to suppress E2F1-induced cell death. (A, B) HCT116 cells were transfected with E2F1 expression plasmids in the presence of GCN5 knockdown siRNA. The protein expression levels of E2F1 (top), GCN5 (middle),
Figure Legend Snippet: GCN5 is a target of the transcription factor E2F1 to suppress E2F1-induced cell death. (A, B) HCT116 cells were transfected with E2F1 expression plasmids in the presence of GCN5 knockdown siRNA. The protein expression levels of E2F1 (top), GCN5 (middle),

Techniques Used: Transfection, Expressing

c-Myc promotes human colon cancer cell growth through GCN5. (A) HCT116 cells with c-Myc knockdown (KD) were transfected with or without GCN5. Their proliferation was measured by MTT assay. Error bars represent data from three independent experiments (mean
Figure Legend Snippet: c-Myc promotes human colon cancer cell growth through GCN5. (A) HCT116 cells with c-Myc knockdown (KD) were transfected with or without GCN5. Their proliferation was measured by MTT assay. Error bars represent data from three independent experiments (mean

Techniques Used: Transfection, MTT Assay

GCN5 suppression inhibits human colon cancer cell growth. (A, B) HCT116 or HT9 colon cancer cells were treated with GCN5 inhibitor at each indicated concentration. The histone H3 acetylation at the lysine residue 9 (H3K9) was determined by Western blotting
Figure Legend Snippet: GCN5 suppression inhibits human colon cancer cell growth. (A, B) HCT116 or HT9 colon cancer cells were treated with GCN5 inhibitor at each indicated concentration. The histone H3 acetylation at the lysine residue 9 (H3K9) was determined by Western blotting

Techniques Used: Concentration Assay, Western Blot

11) Product Images from "CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway"

Article Title: CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.10.015

The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P
Figure Legend Snippet: The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P

Techniques Used: Expressing

The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P
Figure Legend Snippet: The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P

Techniques Used: Stable Transfection, Expressing, Transfection, Cotransfection, Western Blot, Apoptosis Assay, Two Tailed Test

CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.
Figure Legend Snippet: CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.

Techniques Used: Expressing, Immunoprecipitation, Immunofluorescence, Activity Assay, Inhibition, Luciferase, Stable Transfection, Cotransfection, Transfection

MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.
Figure Legend Snippet: MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.

Techniques Used: Western Blot, Expressing, Inhibition, Activity Assay, Over Expression

12) Product Images from "CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway"

Article Title: CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.10.015

The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P
Figure Legend Snippet: The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P

Techniques Used: Stable Transfection, Expressing, Transfection, Cotransfection, Western Blot, Apoptosis Assay, Two Tailed Test

CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.
Figure Legend Snippet: CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.

Techniques Used: Expressing, Immunoprecipitation, Immunofluorescence, Activity Assay, Inhibition, Luciferase, Stable Transfection, Cotransfection, Transfection

13) Product Images from "Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression"

Article Title: Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression

Journal: Scientific Reports

doi: 10.1038/srep34034

MiR-95-3p inhibits p21 expression by directly targeting the 3′-UTR of the CDKN1A gene. ( a ) Bioinformatic analysis of the repressor element for the stability of CDKN1A mRNA at the region from +1,500 to +1,515 of the 3′-UTR identified a binding site for miR-95-3p . ( b ) Schematic diagram showing the wild type (wt) pMIR-p21-wt or mutant pMIR-p21-mut reporter with the miR-95-3p binding site mutated. ( c ) Luciferase activity of pMIR-p21-wt or mutant pMIR-p21-mut reporters in the presence of miR-95-3p mimics, a negative control miRNA mimics (Ncontrol), and a miR-95-3p specific inhibitor in HepG2 cells. ( d ) Similar luciferase assays as in ( c ) but in SMMC7721 cells.
Figure Legend Snippet: MiR-95-3p inhibits p21 expression by directly targeting the 3′-UTR of the CDKN1A gene. ( a ) Bioinformatic analysis of the repressor element for the stability of CDKN1A mRNA at the region from +1,500 to +1,515 of the 3′-UTR identified a binding site for miR-95-3p . ( b ) Schematic diagram showing the wild type (wt) pMIR-p21-wt or mutant pMIR-p21-mut reporter with the miR-95-3p binding site mutated. ( c ) Luciferase activity of pMIR-p21-wt or mutant pMIR-p21-mut reporters in the presence of miR-95-3p mimics, a negative control miRNA mimics (Ncontrol), and a miR-95-3p specific inhibitor in HepG2 cells. ( d ) Similar luciferase assays as in ( c ) but in SMMC7721 cells.

Techniques Used: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Negative Control

MiR-95-3p inhibits expression of p21 at the translational level and promotes SMMC7721 tumor cell proliferation and migration. ( a ) Western blot analysis for the expression level of the p21 protein in SMMC7721 cells transfected with miR-95-3p mimics compared with negative control miRNA mimics (Ncontrol). The Western blot images are shown at the left, quantified and graphed at the right. β-tubulin was used as a loading control. ( b ) Similar Western blot analysis as in ( a ) but with a miR-95-3p inhibitor vs. a negative control miRNA inhibitor (NC inhibitor). ( c ) Proliferation of SMMC7721 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( d ) Percentage of cells at the S phase during cell cycle in SMMC7721 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( e ) Migration of SMMC7721 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid, Ncontrol mimics, and miR-95-3p inhibitor using a wound assay.
Figure Legend Snippet: MiR-95-3p inhibits expression of p21 at the translational level and promotes SMMC7721 tumor cell proliferation and migration. ( a ) Western blot analysis for the expression level of the p21 protein in SMMC7721 cells transfected with miR-95-3p mimics compared with negative control miRNA mimics (Ncontrol). The Western blot images are shown at the left, quantified and graphed at the right. β-tubulin was used as a loading control. ( b ) Similar Western blot analysis as in ( a ) but with a miR-95-3p inhibitor vs. a negative control miRNA inhibitor (NC inhibitor). ( c ) Proliferation of SMMC7721 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( d ) Percentage of cells at the S phase during cell cycle in SMMC7721 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( e ) Migration of SMMC7721 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid, Ncontrol mimics, and miR-95-3p inhibitor using a wound assay.

Techniques Used: Expressing, Migration, Western Blot, Transfection, Negative Control, Plasmid Preparation

MiR-95-3p inhibits expression of p21 at the translational level and promotes HepG2 tumor cell proliferation and migration. ( a ) Western blot analysis for the expression level of the p21 protein in HepG2 cells transfected with miR-95-3p mimics compared with negative control miRNA mimics (Ncontrol). The Western blot images are shown at the left, quantified and graphed at the right. β-tubulin was used as a loading control. ( b ) Similar Western blot analysis as in ( a ) but with a miR-95-3p inhibitor vs. a negative control miRNA inhibitor (NC inhibitor). ( c ) Proliferation of HepG2 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( d ) Percentage of cells at the S phase during cell cycle in HepG2 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( e ) Migration of HepG2 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid, Ncontrol mimics, and miR-95-3p inhibitor using a wound assay.
Figure Legend Snippet: MiR-95-3p inhibits expression of p21 at the translational level and promotes HepG2 tumor cell proliferation and migration. ( a ) Western blot analysis for the expression level of the p21 protein in HepG2 cells transfected with miR-95-3p mimics compared with negative control miRNA mimics (Ncontrol). The Western blot images are shown at the left, quantified and graphed at the right. β-tubulin was used as a loading control. ( b ) Similar Western blot analysis as in ( a ) but with a miR-95-3p inhibitor vs. a negative control miRNA inhibitor (NC inhibitor). ( c ) Proliferation of HepG2 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( d ) Percentage of cells at the S phase during cell cycle in HepG2 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid vs. Ncontrol mimics, and miR-95-3p inhibitor vs. NC inhibitor. ( e ) Migration of HepG2 cells transfected with miR-95-3p mimics alone or with a p21 expression plasmid, Ncontrol mimics, and miR-95-3p inhibitor using a wound assay.

Techniques Used: Expressing, Migration, Western Blot, Transfection, Negative Control, Plasmid Preparation

14) Product Images from "lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis"

Article Title: lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis

Journal: Cells

doi: 10.3390/cells7120243

miR-1611 interacts with lncRNA-Six1 to regulate Six1 expression. ( A ) The potential binding site of miR-1611 in the lncRNA-Six1 transcript and the Six1 3′ untranslated region (UTR). The mutant sequence in the miR-1611 binding site is highlighted in red. ( B – D ) A dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: ( B ) lncRNA-Six1 position 1, ( C ) lncRNA-Six1 position 2, or ( D ) Six1 3′ UTR with a miR-1611 mimic or mimic-NC in DF-1 cells. ( E ) Relative lncRNA-Six1 expression after overexpression and inhibition of miR-1611. ( F ) The mRNA and protein expression levels of Six1 from the miR-1611 mimic and inhibitor-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There is only one Western blot performed per treatment, and therefore, n = 1. ( G ) Cell cycle analysis of CPMs after co-transfection with the listed nucleic acids. ( H ) Relative mRNA levels of several myoblast differentiation marker genes from co-transfected CPMs. ( I , J ) The mRNA expression levels of several fast and slow muscle genes induced by the listed nucleic acids in CPMs. In all panels, data are presented as means ± S.E.M. of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: miR-1611 interacts with lncRNA-Six1 to regulate Six1 expression. ( A ) The potential binding site of miR-1611 in the lncRNA-Six1 transcript and the Six1 3′ untranslated region (UTR). The mutant sequence in the miR-1611 binding site is highlighted in red. ( B – D ) A dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: ( B ) lncRNA-Six1 position 1, ( C ) lncRNA-Six1 position 2, or ( D ) Six1 3′ UTR with a miR-1611 mimic or mimic-NC in DF-1 cells. ( E ) Relative lncRNA-Six1 expression after overexpression and inhibition of miR-1611. ( F ) The mRNA and protein expression levels of Six1 from the miR-1611 mimic and inhibitor-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There is only one Western blot performed per treatment, and therefore, n = 1. ( G ) Cell cycle analysis of CPMs after co-transfection with the listed nucleic acids. ( H ) Relative mRNA levels of several myoblast differentiation marker genes from co-transfected CPMs. ( I , J ) The mRNA expression levels of several fast and slow muscle genes induced by the listed nucleic acids in CPMs. In all panels, data are presented as means ± S.E.M. of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Over Expression, Inhibition, Transfection, Western Blot, Cell Cycle Assay, Cotransfection, Marker

lncRNA-Six1 functions to facilitate myoblast proliferation and differentiation. ( A ) The relative expression levels of lncRNA-Six1 with lncRNA-Six1 overexpression and knockdown. ( B ) The mRNA and protein expression levels of Six1 from pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( C ) Cell cycle analysis of CPMs after lncRNA-Six1 overexpression and knockdown. ( D ) EdU proliferation assays for CPMs with the overexpression and inhibition of lncRNA-Six1, ( E ) the numbers of proliferative cells were also counted. ( F , G ) CPM growth curves following the transfection of pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1. ( H ) Relative lncRNA-Six1 expression during CPM differentiation. ( I ) The mRNA and protein expression levels of myoblast differentiation marker genes with lncRNA-Six1 overexpression and knockdown in the CPMs. The numbers shown below the bands were fold-changes of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( J ) Immunofluorescence analysis of MyHC-staining cells with the overexpression and knockdown of lncRNA-Six1 in CPMs. ( K ) Myotube area (%) of CPMs with lncRNA-Six1 overexpression and knockdown. In all panels, results are expressed as the mean ± SEM of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: lncRNA-Six1 functions to facilitate myoblast proliferation and differentiation. ( A ) The relative expression levels of lncRNA-Six1 with lncRNA-Six1 overexpression and knockdown. ( B ) The mRNA and protein expression levels of Six1 from pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( C ) Cell cycle analysis of CPMs after lncRNA-Six1 overexpression and knockdown. ( D ) EdU proliferation assays for CPMs with the overexpression and inhibition of lncRNA-Six1, ( E ) the numbers of proliferative cells were also counted. ( F , G ) CPM growth curves following the transfection of pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1. ( H ) Relative lncRNA-Six1 expression during CPM differentiation. ( I ) The mRNA and protein expression levels of myoblast differentiation marker genes with lncRNA-Six1 overexpression and knockdown in the CPMs. The numbers shown below the bands were fold-changes of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( J ) Immunofluorescence analysis of MyHC-staining cells with the overexpression and knockdown of lncRNA-Six1 in CPMs. ( K ) Myotube area (%) of CPMs with lncRNA-Six1 overexpression and knockdown. In all panels, results are expressed as the mean ± SEM of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Over Expression, Allele-specific Oligonucleotide, Transfection, Western Blot, Cell Cycle Assay, Inhibition, Marker, Immunofluorescence, Staining

lncRNA-Six1 induces the fast-twitch muscle phenotype. ( A , B ) The relative mRNA expression of several fast and slow muscle genes induced by lncRNA-Six1 overexpression and inhibition in CPMs. In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: lncRNA-Six1 induces the fast-twitch muscle phenotype. ( A , B ) The relative mRNA expression of several fast and slow muscle genes induced by lncRNA-Six1 overexpression and inhibition in CPMs. In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Over Expression, Inhibition

15) Product Images from "The Transcriptional Factor PPARαb Positively Regulates Elovl5 Elongase in Golden Pompano Trachinotus ovatus (Linnaeus 1758)"

Article Title: The Transcriptional Factor PPARαb Positively Regulates Elovl5 Elongase in Golden Pompano Trachinotus ovatus (Linnaeus 1758)

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.01340

Promoter activity analysis of the ToElovl5 gene. (A) The structure and transcriptional activity of ToElovl5 promoters. Five recombinant plasmids, denoted Elovl5-1 (–382 to +89), Elovl5-2 (–793 to +89), Elovl5-3 (–1262 to +89), Elovl5-4 (–146 to +265) and Elovl5-5 (–146 to +459) were constructed and transfected with transcription factor PPARαb into HEK 293T cells. (B) Dual-luciferase activity driven by the ToElovl5-5 core promoter upon the transfection of pcDNA3.1-PPAR-α and pcDNA3.1 in HEK 293T cells. All values are presented as the means ± SD ( n = 3). Asterisks indicate that the values are significantly different from the individual controls ( ∗ p
Figure Legend Snippet: Promoter activity analysis of the ToElovl5 gene. (A) The structure and transcriptional activity of ToElovl5 promoters. Five recombinant plasmids, denoted Elovl5-1 (–382 to +89), Elovl5-2 (–793 to +89), Elovl5-3 (–1262 to +89), Elovl5-4 (–146 to +265) and Elovl5-5 (–146 to +459) were constructed and transfected with transcription factor PPARαb into HEK 293T cells. (B) Dual-luciferase activity driven by the ToElovl5-5 core promoter upon the transfection of pcDNA3.1-PPAR-α and pcDNA3.1 in HEK 293T cells. All values are presented as the means ± SD ( n = 3). Asterisks indicate that the values are significantly different from the individual controls ( ∗ p

Techniques Used: Activity Assay, Recombinant, Construct, Transfection, Luciferase

ToPPARαb (A) and ToElovl5 (B) mRNA expression levels by qRT-PCR after the transfection of either control RNA (control) or siRNA (RNAi). TOCF cells were stimulated with 0.1, 1, and 4 mM of PPARαb agonist (WY-14643) (C) and inhibitor (GW6471) (D) for 24 h, and the expression levels of ToPPARαb and ToElovl5 were significantly increased and decreased, respectively, in a concentration-dependent manner. All values are expressed as the means ± SD ( n = 3). Bars on the same group with different letters are statistically significant from one another ( p
Figure Legend Snippet: ToPPARαb (A) and ToElovl5 (B) mRNA expression levels by qRT-PCR after the transfection of either control RNA (control) or siRNA (RNAi). TOCF cells were stimulated with 0.1, 1, and 4 mM of PPARαb agonist (WY-14643) (C) and inhibitor (GW6471) (D) for 24 h, and the expression levels of ToPPARαb and ToElovl5 were significantly increased and decreased, respectively, in a concentration-dependent manner. All values are expressed as the means ± SD ( n = 3). Bars on the same group with different letters are statistically significant from one another ( p

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Concentration Assay

16) Product Images from "Critical Roles of Glucocorticoid-Induced Leucine Zipper in Infectious Bursal Disease Virus (IBDV)-Induced Suppression of Type I Interferon Expression and Enhancement of IBDV Growth in Host Cells via Interaction with VP4"

Article Title: Critical Roles of Glucocorticoid-Induced Leucine Zipper in Infectious Bursal Disease Virus (IBDV)-Induced Suppression of Type I Interferon Expression and Enhancement of IBDV Growth in Host Cells via Interaction with VP4

Journal: Journal of Virology

doi: 10.1128/JVI.02421-12

Expression of VP4 inhibits TNF-α- or SeV-induced activation of IFN-α, IFN-β, and NF-κB promoters. (A to C) Effects of VP4 on TNF-induced activation of IFN-α, IFN-β, and NF-κB promoters. HEK293T cells
Figure Legend Snippet: Expression of VP4 inhibits TNF-α- or SeV-induced activation of IFN-α, IFN-β, and NF-κB promoters. (A to C) Effects of VP4 on TNF-induced activation of IFN-α, IFN-β, and NF-κB promoters. HEK293T cells

Techniques Used: Expressing, Activation Assay

GILZ mediates the inhibitory effect of VP4 on TNF-α or SeV-induced activation of IFN-α, IFN-β, and NF-κB promoters. (A and B) Effects of GILZ RNAi on the expression of endogenous GILZ. HEK293T cells (2.0 × 10 5 )
Figure Legend Snippet: GILZ mediates the inhibitory effect of VP4 on TNF-α or SeV-induced activation of IFN-α, IFN-β, and NF-κB promoters. (A and B) Effects of GILZ RNAi on the expression of endogenous GILZ. HEK293T cells (2.0 × 10 5 )

Techniques Used: Activation Assay, Expressing

17) Product Images from "Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells"

Article Title: Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-10-117

Functional microRNA is shuttled from macrophages to breast cancer cells . (A) Schematic illustration of a miRNA transfer model. Macrophages were preloaded with Cy3 fluorescently-labeled miR-223 or the non-mammalian miRNA lin-4. A transwell system with a 0.4- μm pore size film, which allows small size materials (e.g., miRNAs), but not cells, to pass through, was used to separate SKBR3 cells from macrophages. (B) and (C) SKBR3 cells were cultured alone (blank) or co-cultured with macrophages that were pre-transfected with reagent (mock), unlabeled miR-223 or Cy3-miR-223. The Cy3 fluorescence signal in SKBR3 cells was determined by fluorescence microscopy (B), and the percentage of Cy3-positive cells in the images was calculated in (C). Arrowheads indicate Cy3-positive SKBR3 cells, and images are shown at 200×. (D) SKBR3 cells were cultured as shown in (B), and Cy3-positive cells (%) were quantified by flow cytometry. (E) A lin-4-targeting luciferase reporter gene carrying a lin-4 complementary sequence in the 3'-UTR was transfected into SKBR3 cells. SKBR3 cells were then co-cultured in a transwell with macrophages pre-transfected with miR-NC or lin-4. SKBR3 cells that were directly transfected with lin-4 were used as a control. Luciferase activities in SKBR3 cells were measured, and relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p
Figure Legend Snippet: Functional microRNA is shuttled from macrophages to breast cancer cells . (A) Schematic illustration of a miRNA transfer model. Macrophages were preloaded with Cy3 fluorescently-labeled miR-223 or the non-mammalian miRNA lin-4. A transwell system with a 0.4- μm pore size film, which allows small size materials (e.g., miRNAs), but not cells, to pass through, was used to separate SKBR3 cells from macrophages. (B) and (C) SKBR3 cells were cultured alone (blank) or co-cultured with macrophages that were pre-transfected with reagent (mock), unlabeled miR-223 or Cy3-miR-223. The Cy3 fluorescence signal in SKBR3 cells was determined by fluorescence microscopy (B), and the percentage of Cy3-positive cells in the images was calculated in (C). Arrowheads indicate Cy3-positive SKBR3 cells, and images are shown at 200×. (D) SKBR3 cells were cultured as shown in (B), and Cy3-positive cells (%) were quantified by flow cytometry. (E) A lin-4-targeting luciferase reporter gene carrying a lin-4 complementary sequence in the 3'-UTR was transfected into SKBR3 cells. SKBR3 cells were then co-cultured in a transwell with macrophages pre-transfected with miR-NC or lin-4. SKBR3 cells that were directly transfected with lin-4 were used as a control. Luciferase activities in SKBR3 cells were measured, and relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p

Techniques Used: Functional Assay, Labeling, Cell Culture, Transfection, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Luciferase, Sequencing, Activity Assay

miR-223 was up-regulated in breast cancer cell lines co-cultured with IL-4-activated macrophages . (A) miRNA profiling in macrophages and the breast cancer cell line SKBR3 using the DiscovArray miRNA array. Three of the miRNAs that were present at high levels in macrophages but absent in SKBR3 cells are shown. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages). (B) Quantitative real-time PCR (qRT-PCR) confirmed a high expression level of hsa-miR-223 in macrophages but not in SKBR3 or MDA-MB-231 cells. * p
Figure Legend Snippet: miR-223 was up-regulated in breast cancer cell lines co-cultured with IL-4-activated macrophages . (A) miRNA profiling in macrophages and the breast cancer cell line SKBR3 using the DiscovArray miRNA array. Three of the miRNAs that were present at high levels in macrophages but absent in SKBR3 cells are shown. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages). (B) Quantitative real-time PCR (qRT-PCR) confirmed a high expression level of hsa-miR-223 in macrophages but not in SKBR3 or MDA-MB-231 cells. * p

Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Multiple Displacement Amplification

Exosomal shuttling of miR-223 from macrophages to breast cancer cells promotes breast cancer cell invasion . (A) IL-4-activated macrophages promote breast cancer cell invasion. The breast cancer cell lines SKBR3 and MDA-MB-231 were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages. Approximately 24 to 48 h after co-culture, SKBR3 and MDA-MB-231 cells were subjected to an invasion assay. Data are averages of triplicates from more than three independent experiments and are presented as the number of invading cells per field. (B) miR-223 mimics enhanced the invasion of SKBR3 and MDA-MB-231 breast cancer cells. miR-223 was transfected into SKBR3 and MDA-MB-231 cells. Cell invasion was then determined by a transwell invasion assay. Relative invasion activities are presented as fold increases in the miR-223 group. The miR-NC group was normalized to 1.0. (C) Exposure to exosomes derived from macrophages resulted in similar effects on breast cancer invasion as the actual macrophages. SKBR3 cells were incubated with exosomes derived from unactivated or IL-4-activated macrophages and then treated with miR-NC ASO or miR-223 ASO. Cell invasion assays were performed as shown in (A). Data are averages of triplicates from three independent experiments and are presented as the number of invading cells per field. * p
Figure Legend Snippet: Exosomal shuttling of miR-223 from macrophages to breast cancer cells promotes breast cancer cell invasion . (A) IL-4-activated macrophages promote breast cancer cell invasion. The breast cancer cell lines SKBR3 and MDA-MB-231 were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages. Approximately 24 to 48 h after co-culture, SKBR3 and MDA-MB-231 cells were subjected to an invasion assay. Data are averages of triplicates from more than three independent experiments and are presented as the number of invading cells per field. (B) miR-223 mimics enhanced the invasion of SKBR3 and MDA-MB-231 breast cancer cells. miR-223 was transfected into SKBR3 and MDA-MB-231 cells. Cell invasion was then determined by a transwell invasion assay. Relative invasion activities are presented as fold increases in the miR-223 group. The miR-NC group was normalized to 1.0. (C) Exposure to exosomes derived from macrophages resulted in similar effects on breast cancer invasion as the actual macrophages. SKBR3 cells were incubated with exosomes derived from unactivated or IL-4-activated macrophages and then treated with miR-NC ASO or miR-223 ASO. Cell invasion assays were performed as shown in (A). Data are averages of triplicates from three independent experiments and are presented as the number of invading cells per field. * p

Techniques Used: Multiple Displacement Amplification, Cell Culture, Co-Culture Assay, Invasion Assay, Transfection, Transwell Invasion Assay, Derivative Assay, Incubation, Allele-specific Oligonucleotide

18) Product Images from "CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway"

Article Title: CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.10.015

The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P
Figure Legend Snippet: The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P

Techniques Used: Stable Transfection, Expressing, Transfection, Cotransfection, Western Blot, Apoptosis Assay, Two Tailed Test

CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.
Figure Legend Snippet: CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.

Techniques Used: Expressing, Immunoprecipitation, Immunofluorescence, Activity Assay, Inhibition, Luciferase, Stable Transfection, Cotransfection, Transfection

MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.
Figure Legend Snippet: MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.

Techniques Used: Western Blot, Expressing, Inhibition, Activity Assay, Over Expression

19) Product Images from "Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway"

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.00588-19

DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  
Figure Legend Snippet: DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  

Techniques Used: Inhibition, Transfection, Infection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.
Figure Legend Snippet: FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

Techniques Used: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Western Blot, Translocation Assay, Clear Native PAGE, Staining, Confocal Microscopy

20) Product Images from "lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis"

Article Title: lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis

Journal: Cells

doi: 10.3390/cells7120243

miR-1611 interacts with lncRNA-Six1 to regulate Six1 expression. ( A ) The potential binding site of miR-1611 in the lncRNA-Six1 transcript and the Six1 3′ untranslated region (UTR). The mutant sequence in the miR-1611 binding site is highlighted in red. ( B – D ) A dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: ( B ) lncRNA-Six1 position 1, ( C ) lncRNA-Six1 position 2, or ( D ) Six1 3′ UTR with a miR-1611 mimic or mimic-NC in DF-1 cells. ( E ) Relative lncRNA-Six1 expression after overexpression and inhibition of miR-1611. ( F ) The mRNA and protein expression levels of Six1 from the miR-1611 mimic and inhibitor-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There is only one Western blot performed per treatment, and therefore, n = 1. ( G ) Cell cycle analysis of CPMs after co-transfection with the listed nucleic acids. ( H ) Relative mRNA levels of several myoblast differentiation marker genes from co-transfected CPMs. ( I , J ) The mRNA expression levels of several fast and slow muscle genes induced by the listed nucleic acids in CPMs. In all panels, data are presented as means ± S.E.M. of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: miR-1611 interacts with lncRNA-Six1 to regulate Six1 expression. ( A ) The potential binding site of miR-1611 in the lncRNA-Six1 transcript and the Six1 3′ untranslated region (UTR). The mutant sequence in the miR-1611 binding site is highlighted in red. ( B – D ) A dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: ( B ) lncRNA-Six1 position 1, ( C ) lncRNA-Six1 position 2, or ( D ) Six1 3′ UTR with a miR-1611 mimic or mimic-NC in DF-1 cells. ( E ) Relative lncRNA-Six1 expression after overexpression and inhibition of miR-1611. ( F ) The mRNA and protein expression levels of Six1 from the miR-1611 mimic and inhibitor-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There is only one Western blot performed per treatment, and therefore, n = 1. ( G ) Cell cycle analysis of CPMs after co-transfection with the listed nucleic acids. ( H ) Relative mRNA levels of several myoblast differentiation marker genes from co-transfected CPMs. ( I , J ) The mRNA expression levels of several fast and slow muscle genes induced by the listed nucleic acids in CPMs. In all panels, data are presented as means ± S.E.M. of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Over Expression, Inhibition, Transfection, Western Blot, Cell Cycle Assay, Cotransfection, Marker

lncRNA-Six1 functions to facilitate myoblast proliferation and differentiation. ( A ) The relative expression levels of lncRNA-Six1 with lncRNA-Six1 overexpression and knockdown. ( B ) The mRNA and protein expression levels of Six1 from pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( C ) Cell cycle analysis of CPMs after lncRNA-Six1 overexpression and knockdown. ( D ) EdU proliferation assays for CPMs with the overexpression and inhibition of lncRNA-Six1, ( E ) the numbers of proliferative cells were also counted. ( F , G ) CPM growth curves following the transfection of pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1. ( H ) Relative lncRNA-Six1 expression during CPM differentiation. ( I ) The mRNA and protein expression levels of myoblast differentiation marker genes with lncRNA-Six1 overexpression and knockdown in the CPMs. The numbers shown below the bands were fold-changes of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( J ) Immunofluorescence analysis of MyHC-staining cells with the overexpression and knockdown of lncRNA-Six1 in CPMs. ( K ) Myotube area (%) of CPMs with lncRNA-Six1 overexpression and knockdown. In all panels, results are expressed as the mean ± SEM of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: lncRNA-Six1 functions to facilitate myoblast proliferation and differentiation. ( A ) The relative expression levels of lncRNA-Six1 with lncRNA-Six1 overexpression and knockdown. ( B ) The mRNA and protein expression levels of Six1 from pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to the control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( C ) Cell cycle analysis of CPMs after lncRNA-Six1 overexpression and knockdown. ( D ) EdU proliferation assays for CPMs with the overexpression and inhibition of lncRNA-Six1, ( E ) the numbers of proliferative cells were also counted. ( F , G ) CPM growth curves following the transfection of pSDS-lncRNA-Six1 and si-ASO-lncRNA-Six1. ( H ) Relative lncRNA-Six1 expression during CPM differentiation. ( I ) The mRNA and protein expression levels of myoblast differentiation marker genes with lncRNA-Six1 overexpression and knockdown in the CPMs. The numbers shown below the bands were fold-changes of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. There was only one Western blot performed per treatment, and therefore n = 1. ( J ) Immunofluorescence analysis of MyHC-staining cells with the overexpression and knockdown of lncRNA-Six1 in CPMs. ( K ) Myotube area (%) of CPMs with lncRNA-Six1 overexpression and knockdown. In all panels, results are expressed as the mean ± SEM of three independent experiments, and the statistical significance of differences between means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Over Expression, Allele-specific Oligonucleotide, Transfection, Western Blot, Cell Cycle Assay, Inhibition, Marker, Immunofluorescence, Staining

lncRNA-Six1 induces the fast-twitch muscle phenotype. ( A , B ) The relative mRNA expression of several fast and slow muscle genes induced by lncRNA-Six1 overexpression and inhibition in CPMs. In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student’s t -test. (* p
Figure Legend Snippet: lncRNA-Six1 induces the fast-twitch muscle phenotype. ( A , B ) The relative mRNA expression of several fast and slow muscle genes induced by lncRNA-Six1 overexpression and inhibition in CPMs. In all panels, data are presented as means ± S.E.M. of three independent assays. Statistical significance of differences between the means was assessed using an unpaired Student’s t -test. (* p

Techniques Used: Expressing, Over Expression, Inhibition

21) Product Images from "Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression"

Article Title: Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression

Journal: Scientific Reports

doi: 10.1038/srep34034

MiR-95-3p inhibits p21 expression by directly targeting the 3′-UTR of the CDKN1A gene. ( a ) Bioinformatic analysis of the repressor element for the stability of CDKN1A mRNA at the region from +1,500 to +1,515 of the 3′-UTR identified a binding site for miR-95-3p . ( b ) Schematic diagram showing the wild type (wt) pMIR-p21-wt or mutant pMIR-p21-mut reporter with the miR-95-3p binding site mutated. ( c ) Luciferase activity of pMIR-p21-wt or mutant pMIR-p21-mut reporters in the presence of miR-95-3p mimics, a negative control miRNA mimics (Ncontrol), and a miR-95-3p specific inhibitor in HepG2 cells. ( d ) Similar luciferase assays as in ( c ) but in SMMC7721 cells.
Figure Legend Snippet: MiR-95-3p inhibits p21 expression by directly targeting the 3′-UTR of the CDKN1A gene. ( a ) Bioinformatic analysis of the repressor element for the stability of CDKN1A mRNA at the region from +1,500 to +1,515 of the 3′-UTR identified a binding site for miR-95-3p . ( b ) Schematic diagram showing the wild type (wt) pMIR-p21-wt or mutant pMIR-p21-mut reporter with the miR-95-3p binding site mutated. ( c ) Luciferase activity of pMIR-p21-wt or mutant pMIR-p21-mut reporters in the presence of miR-95-3p mimics, a negative control miRNA mimics (Ncontrol), and a miR-95-3p specific inhibitor in HepG2 cells. ( d ) Similar luciferase assays as in ( c ) but in SMMC7721 cells.

Techniques Used: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Negative Control

Identification of a critical regulatory element at the 3′-UTR of the CDKN1A gene (encoding p21) for regulation of p21 expression. ( a ) Schematic diagram showing a luciferase reporter with the 3′-UTR of the CDKN1A gene (a 1,539 bp fragment from the stop codon) sub-cloned after the luciferase gene (pMIR-p21-wt or pMIR-1). ( b ) Serial deletions of the 3′-UTR were created in pMIR-1, resulting in pMIR-2 to pMIR-9. ( c ) Luciferase activity for different luciferase reporters. Note that a repressor element was present at the region from +1,500 to +1,515 of the 3′-UTR of the CDKN1A gene.
Figure Legend Snippet: Identification of a critical regulatory element at the 3′-UTR of the CDKN1A gene (encoding p21) for regulation of p21 expression. ( a ) Schematic diagram showing a luciferase reporter with the 3′-UTR of the CDKN1A gene (a 1,539 bp fragment from the stop codon) sub-cloned after the luciferase gene (pMIR-p21-wt or pMIR-1). ( b ) Serial deletions of the 3′-UTR were created in pMIR-1, resulting in pMIR-2 to pMIR-9. ( c ) Luciferase activity for different luciferase reporters. Note that a repressor element was present at the region from +1,500 to +1,515 of the 3′-UTR of the CDKN1A gene.

Techniques Used: Expressing, Luciferase, Clone Assay, Activity Assay

22) Product Images from "CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway"

Article Title: CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.10.015

The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P
Figure Legend Snippet: The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P

Techniques Used: Stable Transfection, Expressing, Transfection, Cotransfection, Western Blot, Apoptosis Assay, Two Tailed Test

23) Product Images from "miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein"

Article Title: miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Journal: Scientific Reports

doi: 10.1038/s41598-018-22767-y

miR-3140 directly targeted BRD4 and BRD3 by binding their coding regions. ( a ) Luciferase reporter assay. MIAPaCa2 cells were transfected with the pmirGLO Dual Luciferase vectors containing Wt or Mt BRD4 , or empty vector (EV), and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding sequence of miR-3140 within the CDS of BRD4 and mutant sequences are indicated. Bottom, the results of luciferase assay; * P
Figure Legend Snippet: miR-3140 directly targeted BRD4 and BRD3 by binding their coding regions. ( a ) Luciferase reporter assay. MIAPaCa2 cells were transfected with the pmirGLO Dual Luciferase vectors containing Wt or Mt BRD4 , or empty vector (EV), and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding sequence of miR-3140 within the CDS of BRD4 and mutant sequences are indicated. Bottom, the results of luciferase assay; * P

Techniques Used: Binding Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Sequencing, Mutagenesis

miR-3140 targeted CDK2 and EGFR by binding their 3′UTR regions. ( a ) Left, identification of downregulated genes after miR-3140 transfection by a gene expression array. The Venn diagram shows that 228 genes were commonly downregulated (fold change > 2) by transfection of miR-3140 in Panc1, MIAPaCa2, and MDA-MB-231 cells. Right, prediction of candidate target genes regulated by miR-3140 via their 3′UTR. The Venn diagram shows that 99 genes were predicted as candidate 3′UTR-targets of miR-3140 by the TargetScan program. ( b ) Western blot analysis of CDK2, CDK6, and EGFR in Panc1 and MIAPaCa2 cells 72 hours after transfection with 10 nmol/L of miR-NC or miR-3140 . ( c ) Luciferase reporter assays. Panc1 cells were transfected with the pmirGLO Dual Luciferase vectors containing wild type (Wt) CDK2 and EGFR or mutant (Mt) 3’UTR target sites of these genes, and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding site of miR-3140 within the 3′UTR of each gene and mutant sequences. Bottom, results of the luciferase assay; * P
Figure Legend Snippet: miR-3140 targeted CDK2 and EGFR by binding their 3′UTR regions. ( a ) Left, identification of downregulated genes after miR-3140 transfection by a gene expression array. The Venn diagram shows that 228 genes were commonly downregulated (fold change > 2) by transfection of miR-3140 in Panc1, MIAPaCa2, and MDA-MB-231 cells. Right, prediction of candidate target genes regulated by miR-3140 via their 3′UTR. The Venn diagram shows that 99 genes were predicted as candidate 3′UTR-targets of miR-3140 by the TargetScan program. ( b ) Western blot analysis of CDK2, CDK6, and EGFR in Panc1 and MIAPaCa2 cells 72 hours after transfection with 10 nmol/L of miR-NC or miR-3140 . ( c ) Luciferase reporter assays. Panc1 cells were transfected with the pmirGLO Dual Luciferase vectors containing wild type (Wt) CDK2 and EGFR or mutant (Mt) 3’UTR target sites of these genes, and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding site of miR-3140 within the 3′UTR of each gene and mutant sequences. Bottom, results of the luciferase assay; * P

Techniques Used: Binding Assay, Transfection, Expressing, Multiple Displacement Amplification, Western Blot, Luciferase, Mutagenesis

24) Product Images from "MicroRNA-1271 inhibits cellular proliferation of hepatocellular carcinoma"

Article Title: MicroRNA-1271 inhibits cellular proliferation of hepatocellular carcinoma

Journal: Oncology Letters

doi: 10.3892/ol.2017.7052

) demonstrated that FOXQ1 was a direct target of miR-1271. (B) HepG-2 cells were co-transfected with miR-1271 mimics, the control and reporter plasmid or the Mut 3′UTR, together with the controls. (C) HepG-2 cells were transfected with miR-1271 mimics. Subsequent to 48 h, western blot analysis was performed to determine the expression level of FOXQ1 protein. (D) FOXQ1 mRNA expression levels in 11 HCC tissues were examined by reverse transcription-quantitative polymerase chain reaction. The correlation between FOXQ1 mRNA and miR-1271 expression levels were determined by two-tailed Pearson's correlation coefficient analysis. All data are presented as the mean ± standard deviation of three separate experiments. *P
Figure Legend Snippet: ) demonstrated that FOXQ1 was a direct target of miR-1271. (B) HepG-2 cells were co-transfected with miR-1271 mimics, the control and reporter plasmid or the Mut 3′UTR, together with the controls. (C) HepG-2 cells were transfected with miR-1271 mimics. Subsequent to 48 h, western blot analysis was performed to determine the expression level of FOXQ1 protein. (D) FOXQ1 mRNA expression levels in 11 HCC tissues were examined by reverse transcription-quantitative polymerase chain reaction. The correlation between FOXQ1 mRNA and miR-1271 expression levels were determined by two-tailed Pearson's correlation coefficient analysis. All data are presented as the mean ± standard deviation of three separate experiments. *P

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Standard Deviation

Lower expression level of miR-1271 in HCC tissues. A total of 22 pairs of HCC tissues and corresponding tumor-adjacent normal tissues were collected from The Fourth Hospital of Huaian City (Jiangsu, China). (A) The miR-1271 expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction. The difference in expression level of miR-1271 between tumor tissues and matched normal tissues was determined. (B) The mean miR-1271 expression level in the HCC tissues and the corresponding tumor-adjacent normal tissues. Data are expressed as the mean ± standard deviation of three separate experiments. *P
Figure Legend Snippet: Lower expression level of miR-1271 in HCC tissues. A total of 22 pairs of HCC tissues and corresponding tumor-adjacent normal tissues were collected from The Fourth Hospital of Huaian City (Jiangsu, China). (A) The miR-1271 expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction. The difference in expression level of miR-1271 between tumor tissues and matched normal tissues was determined. (B) The mean miR-1271 expression level in the HCC tissues and the corresponding tumor-adjacent normal tissues. Data are expressed as the mean ± standard deviation of three separate experiments. *P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Transfection with miR-1271 mimics inhibited HepG-2 and Huh-7 cell growth and induced apoptosis. The total RNA of normal liver tissues, HepG2 and Huh-7 were extracted. The miR-1271 expression levels were analyzed by RT-qPCR analysis. (A) The miR-1271 expression levels of normal liver tissues were treated as 100%. Following a 48 h miR-1271 mimic transfection, the expression levels of miR-1271 in HepG-2 and Huh-7 cells were examined by RT-qPCR. (B) The expression level of miR-1271 in the miR-NC transfected group was deliberately treated as 100%. (C) The rate of cell growth was evaluated by MTT analysis at the indicated time point. (D) Following miR-1271 mimic transfection, cell apoptosis was examined by apoptosis FACS analysis. All data are presented as the mean ± standard deviation of three separate experiments. *P
Figure Legend Snippet: Transfection with miR-1271 mimics inhibited HepG-2 and Huh-7 cell growth and induced apoptosis. The total RNA of normal liver tissues, HepG2 and Huh-7 were extracted. The miR-1271 expression levels were analyzed by RT-qPCR analysis. (A) The miR-1271 expression levels of normal liver tissues were treated as 100%. Following a 48 h miR-1271 mimic transfection, the expression levels of miR-1271 in HepG-2 and Huh-7 cells were examined by RT-qPCR. (B) The expression level of miR-1271 in the miR-NC transfected group was deliberately treated as 100%. (C) The rate of cell growth was evaluated by MTT analysis at the indicated time point. (D) Following miR-1271 mimic transfection, cell apoptosis was examined by apoptosis FACS analysis. All data are presented as the mean ± standard deviation of three separate experiments. *P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, MTT Assay, FACS, Standard Deviation

Transfection with miR-1271 ASO promoted HepG-2 and Huh-7 growth and inhibited apoptosis. Following miR-1271 ASO transfection, the expression levels of miR-1271 in HepG-2 and Huh-7 cells were evaluated by reverse transcription-quantitative polymerase chain reaction. (A) The expression level of miR-1271 in the miR-NC ASO treated group was deliberately treated as 100%. (B) The rate of cell growth was determined by MTT analysis at the indicated time point. (C) Following miR-1271 mimic transfection, cell apoptosis was examined by apoptosis FACS analysis. All data are presented as the mean ± standard deviation of three separate experiments. *P
Figure Legend Snippet: Transfection with miR-1271 ASO promoted HepG-2 and Huh-7 growth and inhibited apoptosis. Following miR-1271 ASO transfection, the expression levels of miR-1271 in HepG-2 and Huh-7 cells were evaluated by reverse transcription-quantitative polymerase chain reaction. (A) The expression level of miR-1271 in the miR-NC ASO treated group was deliberately treated as 100%. (B) The rate of cell growth was determined by MTT analysis at the indicated time point. (C) Following miR-1271 mimic transfection, cell apoptosis was examined by apoptosis FACS analysis. All data are presented as the mean ± standard deviation of three separate experiments. *P

Techniques Used: Transfection, Allele-specific Oligonucleotide, Expressing, Real-time Polymerase Chain Reaction, MTT Assay, FACS, Standard Deviation

25) Product Images from "Bufalin down-regulates Axl expression to inhibit cell proliferation and induce apoptosis in non-small-cell lung cancer cells"

Article Title: Bufalin down-regulates Axl expression to inhibit cell proliferation and induce apoptosis in non-small-cell lung cancer cells

Journal: Bioscience Reports

doi: 10.1042/BSR20193959

Bufalin reduces Axl protein level and suppresses promoter activity ( A ) Cells (3 × 10 5 cells/ 60 mm dish) were treated with 20, 40, or 80 nM bufalin for 24 h. Axl protein levels were determined by Western blot analysis. ( B ) For RT-PCR, total RNAs extracted from the cells treated with the indicated concentrations of bufalin for 8 h were used to measure Axl mRNA levels. ( C ) To examine the effect of bufalin on Axl promoter activity, H460/pGL3-Axl cells (3 × 10 4 cells) were incubated with 20, 40, or 80 nM bufalin for 4 or 8 h, and the luciferase activities were then measured (** P
Figure Legend Snippet: Bufalin reduces Axl protein level and suppresses promoter activity ( A ) Cells (3 × 10 5 cells/ 60 mm dish) were treated with 20, 40, or 80 nM bufalin for 24 h. Axl protein levels were determined by Western blot analysis. ( B ) For RT-PCR, total RNAs extracted from the cells treated with the indicated concentrations of bufalin for 8 h were used to measure Axl mRNA levels. ( C ) To examine the effect of bufalin on Axl promoter activity, H460/pGL3-Axl cells (3 × 10 4 cells) were incubated with 20, 40, or 80 nM bufalin for 4 or 8 h, and the luciferase activities were then measured (** P

Techniques Used: Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Luciferase

26) Product Images from "miR-342 overexpression results in a synthetic lethal phenotype in BRCA1-mutant HCC1937 breast cancer cells"

Article Title: miR-342 overexpression results in a synthetic lethal phenotype in BRCA1-mutant HCC1937 breast cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.7617

miR-342 targets BIRC6 and down-modulates Apollon/BRUCE protein in HCC1937 cells A. Schematic representation of the interaction of miR-342 at nucleotides 204-210 of the wild-type (wt) and mutated (mut) BIRC6 3′ UTR cloned into pGL3 promoter vectors (pLuc–BIRC6 and pLuc-MUT-BIRC6). B. Quantification of relative luciferase activity (RLU) in 293T cells upon transfection with pre-miR-342 or scramble. Data are given as the ratio between luciferase activity detected in pre-miR-342 vs scramble-transfected cells and represent mean ± SD from at least three independent determinations. *P
Figure Legend Snippet: miR-342 targets BIRC6 and down-modulates Apollon/BRUCE protein in HCC1937 cells A. Schematic representation of the interaction of miR-342 at nucleotides 204-210 of the wild-type (wt) and mutated (mut) BIRC6 3′ UTR cloned into pGL3 promoter vectors (pLuc–BIRC6 and pLuc-MUT-BIRC6). B. Quantification of relative luciferase activity (RLU) in 293T cells upon transfection with pre-miR-342 or scramble. Data are given as the ratio between luciferase activity detected in pre-miR-342 vs scramble-transfected cells and represent mean ± SD from at least three independent determinations. *P

Techniques Used: Clone Assay, Luciferase, Activity Assay, Transfection

27) Product Images from "Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression"

Article Title: Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression

Journal: Scientific Reports

doi: 10.1038/srep34034

MiR-95-3p inhibits p21 expression by directly targeting the 3′-UTR of the CDKN1A gene. ( a ) Bioinformatic analysis of the repressor element for the stability of CDKN1A mRNA at the region from +1,500 to +1,515 of the 3′-UTR identified a binding site for miR-95-3p . ( b ) Schematic diagram showing the wild type (wt) pMIR-p21-wt or mutant pMIR-p21-mut reporter with the miR-95-3p binding site mutated. ( c ) Luciferase activity of pMIR-p21-wt or mutant pMIR-p21-mut reporters in the presence of miR-95-3p mimics, a negative control miRNA mimics (Ncontrol), and a miR-95-3p specific inhibitor in HepG2 cells. ( d ) Similar luciferase assays as in ( c ) but in SMMC7721 cells.
Figure Legend Snippet: MiR-95-3p inhibits p21 expression by directly targeting the 3′-UTR of the CDKN1A gene. ( a ) Bioinformatic analysis of the repressor element for the stability of CDKN1A mRNA at the region from +1,500 to +1,515 of the 3′-UTR identified a binding site for miR-95-3p . ( b ) Schematic diagram showing the wild type (wt) pMIR-p21-wt or mutant pMIR-p21-mut reporter with the miR-95-3p binding site mutated. ( c ) Luciferase activity of pMIR-p21-wt or mutant pMIR-p21-mut reporters in the presence of miR-95-3p mimics, a negative control miRNA mimics (Ncontrol), and a miR-95-3p specific inhibitor in HepG2 cells. ( d ) Similar luciferase assays as in ( c ) but in SMMC7721 cells.

Techniques Used: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Negative Control

Identification of a critical regulatory element at the 3′-UTR of the CDKN1A gene (encoding p21) for regulation of p21 expression. ( a ) Schematic diagram showing a luciferase reporter with the 3′-UTR of the CDKN1A gene (a 1,539 bp fragment from the stop codon) sub-cloned after the luciferase gene (pMIR-p21-wt or pMIR-1). ( b ) Serial deletions of the 3′-UTR were created in pMIR-1, resulting in pMIR-2 to pMIR-9. ( c ) Luciferase activity for different luciferase reporters. Note that a repressor element was present at the region from +1,500 to +1,515 of the 3′-UTR of the CDKN1A gene.
Figure Legend Snippet: Identification of a critical regulatory element at the 3′-UTR of the CDKN1A gene (encoding p21) for regulation of p21 expression. ( a ) Schematic diagram showing a luciferase reporter with the 3′-UTR of the CDKN1A gene (a 1,539 bp fragment from the stop codon) sub-cloned after the luciferase gene (pMIR-p21-wt or pMIR-1). ( b ) Serial deletions of the 3′-UTR were created in pMIR-1, resulting in pMIR-2 to pMIR-9. ( c ) Luciferase activity for different luciferase reporters. Note that a repressor element was present at the region from +1,500 to +1,515 of the 3′-UTR of the CDKN1A gene.

Techniques Used: Expressing, Luciferase, Clone Assay, Activity Assay

28) Product Images from "Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer"

Article Title: Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer

Journal: Oncology Letters

doi: 10.3892/ol.2017.7038

(A) Matrigel invasion assay of Sawano cells transfected with three sets of siRNA, A, B and C, LSR. Scale bar, 100 µm. The results are presented below as a histogram. Control vs. siRNA, **P
Figure Legend Snippet: (A) Matrigel invasion assay of Sawano cells transfected with three sets of siRNA, A, B and C, LSR. Scale bar, 100 µm. The results are presented below as a histogram. Control vs. siRNA, **P

Techniques Used: Invasion Assay, Transfection

(A) Matrigel invasion assay of Sawano cells transfected with or without siRNAs of lipolysis-stimulated lipoprotein receptor (LSR) and claudin-1 (CLDN-1). Scale bar, 200 µm. The results are presented below as a histogram. Control vs. siRNA-LSR, **P
Figure Legend Snippet: (A) Matrigel invasion assay of Sawano cells transfected with or without siRNAs of lipolysis-stimulated lipoprotein receptor (LSR) and claudin-1 (CLDN-1). Scale bar, 200 µm. The results are presented below as a histogram. Control vs. siRNA-LSR, **P

Techniques Used: Invasion Assay, Transfection

29) Product Images from "Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway"

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.00588-19

The K208 of VP1 is critical for its interaction with DNAJA3. (A, C, E, and G) Schematic representations of individual DNAJA3 mutants (A) and VP1 mutants (C, E, and G). (B) The D-1-168 (J domain) of DNAJA3 is required for its association with VP1. HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with Flag antibodies, followed by immunoblotting with Myc and Flag antibodies. Asterisks represent target proteins. (D) V-188-211 (C-terminal domain) of VP1 interacts with DNAJA3. The method is the same as for panel B. (F) The VP1-207-211 alanine mutant lost the ability to interact with DNAJA3. The method is the same as for panel B. (H) The VP1-K208A alanine mutant lost the ability to interact with DNAJA3. The method is the same as for panel B. (I) Co-IP of FMDV VP1 with endogenous DNAJA3. FMDV-infected (+) or mock-infected (–) BHK-21 cells were used for immunoprecipitation with mouse anti-VP1 antibody and immunoblotted with rabbit anti-DNAJA3 antibody. (J) The K208A mutant FMDV lost the ability to interact with endogenous DNAJA3. rO-VP1K208A-infected, rO-WT-infected, or mock-infected BHK-21 cells were used for immunoprecipitation with rabbit anti-DNAJA3 and were immunoblotted with mouse anti-VP1 antibody.
Figure Legend Snippet: The K208 of VP1 is critical for its interaction with DNAJA3. (A, C, E, and G) Schematic representations of individual DNAJA3 mutants (A) and VP1 mutants (C, E, and G). (B) The D-1-168 (J domain) of DNAJA3 is required for its association with VP1. HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with Flag antibodies, followed by immunoblotting with Myc and Flag antibodies. Asterisks represent target proteins. (D) V-188-211 (C-terminal domain) of VP1 interacts with DNAJA3. The method is the same as for panel B. (F) The VP1-207-211 alanine mutant lost the ability to interact with DNAJA3. The method is the same as for panel B. (H) The VP1-K208A alanine mutant lost the ability to interact with DNAJA3. The method is the same as for panel B. (I) Co-IP of FMDV VP1 with endogenous DNAJA3. FMDV-infected (+) or mock-infected (–) BHK-21 cells were used for immunoprecipitation with mouse anti-VP1 antibody and immunoblotted with rabbit anti-DNAJA3 antibody. (J) The K208A mutant FMDV lost the ability to interact with endogenous DNAJA3. rO-VP1K208A-infected, rO-WT-infected, or mock-infected BHK-21 cells were used for immunoprecipitation with rabbit anti-DNAJA3 and were immunoblotted with mouse anti-VP1 antibody.

Techniques Used: Transfection, Immunoprecipitation, Mutagenesis, Co-Immunoprecipitation Assay, Infection

DNAJA3 increases the levels of LC3-II through interaction with LC3. (A) Myc-DNAJA3 plasmids were transfected into HEK293T cells in a dose-dependent manner, and total protein lysates were collected and analyzed with appropriate antibodies. β-Actin was used as protein loading control. (B) The HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with a Flag antibody, followed by immunoblotting with Myc and Flag antibodies. (C) HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with a Flag antibody, followed by immunoblotting with Myc and Flag antibodies. (D) Truncated Myc-DNAJA3 plasmids were transfected into HEK293T cells, and total protein lysates were collected and analyzed with the appropriate antibodies. β-Actin was used as protein loading control.
Figure Legend Snippet: DNAJA3 increases the levels of LC3-II through interaction with LC3. (A) Myc-DNAJA3 plasmids were transfected into HEK293T cells in a dose-dependent manner, and total protein lysates were collected and analyzed with appropriate antibodies. β-Actin was used as protein loading control. (B) The HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with a Flag antibody, followed by immunoblotting with Myc and Flag antibodies. (C) HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with a Flag antibody, followed by immunoblotting with Myc and Flag antibodies. (D) Truncated Myc-DNAJA3 plasmids were transfected into HEK293T cells, and total protein lysates were collected and analyzed with the appropriate antibodies. β-Actin was used as protein loading control.

Techniques Used: Transfection, Immunoprecipitation

DNAJA3 induced FMDV VP1 protein degradation in a lysosome-dependent manner. (A) DNAJA3 induces the reduction of FMDV VP1 proteins in a dose-dependent manner. HEK293T cells were transfected with Flag-VP1- or HA-VP1-expressing plasmid, along with increasing quantities of Myc-DNAJA3-expressing plasmid. The expression of Myc-DNAJA3 and Flag-VP1 or HA-VP1 was detected by Western blotting. (B) The expression of VP1 mRNA was determined by RT-PCR analysis. (C) HEK293T cells were transfected with the indicated plasmids. The expression of Myc-DNAJA3- and Flag-tagged viral proteins was detected by Western blotting. (D) DNAJA3 regulates the half-life of the VP1 protein. HEK293T cells were cotransfected with Myc-DNAJA3 and Flag-VP1 plasmids. After 24 h, the cells were treated with CHX (100 μg/ml) or actinomycin D (10 μg/ml) before immunoblot analysis was performed. The relative fold change in the abundance of VP1 was determined by densitometric analysis. (E) HEK293T cells were cotransfected with Flag-VP1 plasmids and empty vector or Myc-DNAJA3 plasmids and maintained in the presence or absence of MG132 (2 or 20 μM), NH 4 Cl (10 or 20 mM), Z-VAD-FMK (20 or 50 μM), CQ (50 or 100 μM), or 3-MA (5 or 10 mM) for 36 h. The expression of Myc-DNAJA3 and Flag-VP1 proteins was detected by Western blotting. The relative fold change in abundance of VP1 was determined by densitometric analysis. (F) HEK293T cells were cotransfected with Flag-VP1 plasmids and empty vector or Myc-DNAJA3 or truncated Myc-DNAJA3 plasmids. The expression of these proteins was detected by Western blotting.
Figure Legend Snippet: DNAJA3 induced FMDV VP1 protein degradation in a lysosome-dependent manner. (A) DNAJA3 induces the reduction of FMDV VP1 proteins in a dose-dependent manner. HEK293T cells were transfected with Flag-VP1- or HA-VP1-expressing plasmid, along with increasing quantities of Myc-DNAJA3-expressing plasmid. The expression of Myc-DNAJA3 and Flag-VP1 or HA-VP1 was detected by Western blotting. (B) The expression of VP1 mRNA was determined by RT-PCR analysis. (C) HEK293T cells were transfected with the indicated plasmids. The expression of Myc-DNAJA3- and Flag-tagged viral proteins was detected by Western blotting. (D) DNAJA3 regulates the half-life of the VP1 protein. HEK293T cells were cotransfected with Myc-DNAJA3 and Flag-VP1 plasmids. After 24 h, the cells were treated with CHX (100 μg/ml) or actinomycin D (10 μg/ml) before immunoblot analysis was performed. The relative fold change in the abundance of VP1 was determined by densitometric analysis. (E) HEK293T cells were cotransfected with Flag-VP1 plasmids and empty vector or Myc-DNAJA3 plasmids and maintained in the presence or absence of MG132 (2 or 20 μM), NH 4 Cl (10 or 20 mM), Z-VAD-FMK (20 or 50 μM), CQ (50 or 100 μM), or 3-MA (5 or 10 mM) for 36 h. The expression of Myc-DNAJA3 and Flag-VP1 proteins was detected by Western blotting. The relative fold change in abundance of VP1 was determined by densitometric analysis. (F) HEK293T cells were cotransfected with Flag-VP1 plasmids and empty vector or Myc-DNAJA3 or truncated Myc-DNAJA3 plasmids. The expression of these proteins was detected by Western blotting.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction

VP1 interacts with DNAJA3. (A) Yeast two-hybrid assay. The indicated plasmids were cotransformed into Y2HGold yeast strain. (B) Co-IP analysis of DNAJA3 and VP1. HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with an Flag or Myc antibodies, followed by immunoblotting with Myc and Flag antibodies. HC, heavy chain; LC, light chain. (C) Colocalization of VP1 with endogenous DNAJA3. PK-15 cells were infected with or without FMDV. Cells were then analyzed using immunofluorescence staining with anti-VP1 (green), anti-DNAJA3 (red) and DAPI (blue) and microscopy.
Figure Legend Snippet: VP1 interacts with DNAJA3. (A) Yeast two-hybrid assay. The indicated plasmids were cotransformed into Y2HGold yeast strain. (B) Co-IP analysis of DNAJA3 and VP1. HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with an Flag or Myc antibodies, followed by immunoblotting with Myc and Flag antibodies. HC, heavy chain; LC, light chain. (C) Colocalization of VP1 with endogenous DNAJA3. PK-15 cells were infected with or without FMDV. Cells were then analyzed using immunofluorescence staining with anti-VP1 (green), anti-DNAJA3 (red) and DAPI (blue) and microscopy.

Techniques Used: Y2H Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Infection, Immunofluorescence, Staining, Microscopy

DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋅C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P
Figure Legend Snippet: DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋅C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P

Techniques Used: Inhibition, Transfection, Infection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.
Figure Legend Snippet: FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

Techniques Used: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Western Blot, Translocation Assay, Clear Native PAGE, Staining, Confocal Microscopy

30) Product Images from "Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83"

Article Title: Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83

Journal: Journal of Virology

doi: 10.1128/JVI.00366-18

Recombinant sCD83 inhibits the expression of TAP1 and ERp57 in MoDCs. (A) Protein electrophoresis showing GST-sCD83 expression and purification. M, protein markers. Lane 1, GST-sCD83 after purification by GST affinity columns; lane 2, soluble component of induced BL21(DE3) with recombinant plasmid pGEX-6P-1/sCD83. (B) Optical scan of thin-layer gel (SDS-PAGE) before (lane 1) and after (lane 2) GST-sCD83 purification. (C) GST-Cap, which was purified in the same way as GST-sCD83, was used as a control. GST-Cap fusion protein and GST-sCD83 fusion protein were analyzed by SDS-PAGE. M, protein markers; lane 1, GST-Cap fusion protein; lane 2, GST-sCD83 fusion protein. (D) Sandwich ELISA analysis demonstrating that GST-sCD83 protein reacts with anti-CD83 antibody. GST-sCD83 protein was used at 0, 0.1, 0.5, 1, 2, 5, and 10 μg/ml. GST-Cap (5 μg/ml) was used as a negative control. (E) Effect of sCD83 protein on TAP1 and ERp57 expression in MoDCs. MoDCs (1.0 × 10 6 ) were incubated with 10 μg/ml LPS and 0.1, 1, 5, and 10 μg/ml of sCD83 protein for 24 h. Cell lysates were examined by Western blotting with anti-TAP1 and anti-ERp57 antibodies. PBS treatment and GST-Cap (5 μg/ml) were used as negative controls, and endogenous β-actin expression was used as an internal control. TAP1 (F) and ERp57 (G) mRNA expression was analyzed by qRT-PCR. mRNA levels were calculated relative to known amounts of template and normalized to β-actin expression. Results are representative of three independent experiments. Data are represented as means ± SEM.
Figure Legend Snippet: Recombinant sCD83 inhibits the expression of TAP1 and ERp57 in MoDCs. (A) Protein electrophoresis showing GST-sCD83 expression and purification. M, protein markers. Lane 1, GST-sCD83 after purification by GST affinity columns; lane 2, soluble component of induced BL21(DE3) with recombinant plasmid pGEX-6P-1/sCD83. (B) Optical scan of thin-layer gel (SDS-PAGE) before (lane 1) and after (lane 2) GST-sCD83 purification. (C) GST-Cap, which was purified in the same way as GST-sCD83, was used as a control. GST-Cap fusion protein and GST-sCD83 fusion protein were analyzed by SDS-PAGE. M, protein markers; lane 1, GST-Cap fusion protein; lane 2, GST-sCD83 fusion protein. (D) Sandwich ELISA analysis demonstrating that GST-sCD83 protein reacts with anti-CD83 antibody. GST-sCD83 protein was used at 0, 0.1, 0.5, 1, 2, 5, and 10 μg/ml. GST-Cap (5 μg/ml) was used as a negative control. (E) Effect of sCD83 protein on TAP1 and ERp57 expression in MoDCs. MoDCs (1.0 × 10 6 ) were incubated with 10 μg/ml LPS and 0.1, 1, 5, and 10 μg/ml of sCD83 protein for 24 h. Cell lysates were examined by Western blotting with anti-TAP1 and anti-ERp57 antibodies. PBS treatment and GST-Cap (5 μg/ml) were used as negative controls, and endogenous β-actin expression was used as an internal control. TAP1 (F) and ERp57 (G) mRNA expression was analyzed by qRT-PCR. mRNA levels were calculated relative to known amounts of template and normalized to β-actin expression. Results are representative of three independent experiments. Data are represented as means ± SEM.

Techniques Used: Recombinant, Expressing, Protein Electrophoresis, Purification, Plasmid Preparation, SDS Page, Sandwich ELISA, Negative Control, Incubation, Western Blot, Quantitative RT-PCR

Anti-CD83 antibody blocks the ability of PRRSV to depress immunoregulatory activity of MoDCs. (A and B) MoDCs were pretreated with rabbit anti-CD83 antibody (A) to remove sCD83, or with isotype (rabbit IgG) antibody (B) as a negative control, at 20, 2, 1, and 0. 4 μg/ml in the cell culture medium. MoDCs were then infected with PRRSV at an MOI of 1 in the presence of LPS (10 μg/ml) for 24 h. Cell lysates were analyzed for TAP1, ERp57, and N proteins using Western blotting. β-Actin was used as a loading control. Treatment with LPS (10 μg/ml) alone served as a positive control. (C) T cells are characterized by high CD3 expression. Approximately 50% of freshly isolated PBMCs are T cells. The panel shows a representative dot plot of the flow cytometry gating strategy for T cell selection, using side scatter (SSC) and CD3. (D) Sorting by FACS yields a highly enriched T cell population (98%). Shown is a representative dot plot for enriched CD3 + T lymphocytes. (E) MoDCs were either left untreated or pretreated with anti-CD83 antibody (20 μg/ml) or isotype (20 μg/ml) and infected with PRRSV at an MOI of 0.1, 1, and 2 in the presence or absence of LPS (10 μg/ml). After 24 h, supernatants from these cultures were added to allogeneic T cells. Treatment with LPS alone at 10 μg/ml served as a positive control. T cell proliferation was restored when supernatants were added from PRRSV-infected MoDCs pretreated with rabbit anti-CD83 antibody compared with those pretreated with isotype (20 μg/ml). (F) MoDCs were stimulated with recombinant GST-sCD83 at 0, 0.1, 1, and 5 μg/ml for 24 h. Cell-free supernatants were then transferred to cultures containing purified T cells. MoDCs treated with PBS (MOCK) and GST-Cap fusion protein at 0.1, 1, and 5 μg/ml were used as negative controls. Meanwhile, LPS at 0, 1, 5, and 10 μg/ml was used as a positive control. Cell proliferation was measured using the CCK-8 (absorbance at 450 nm). Data are representative of at least three independent experiments. *** , P
Figure Legend Snippet: Anti-CD83 antibody blocks the ability of PRRSV to depress immunoregulatory activity of MoDCs. (A and B) MoDCs were pretreated with rabbit anti-CD83 antibody (A) to remove sCD83, or with isotype (rabbit IgG) antibody (B) as a negative control, at 20, 2, 1, and 0. 4 μg/ml in the cell culture medium. MoDCs were then infected with PRRSV at an MOI of 1 in the presence of LPS (10 μg/ml) for 24 h. Cell lysates were analyzed for TAP1, ERp57, and N proteins using Western blotting. β-Actin was used as a loading control. Treatment with LPS (10 μg/ml) alone served as a positive control. (C) T cells are characterized by high CD3 expression. Approximately 50% of freshly isolated PBMCs are T cells. The panel shows a representative dot plot of the flow cytometry gating strategy for T cell selection, using side scatter (SSC) and CD3. (D) Sorting by FACS yields a highly enriched T cell population (98%). Shown is a representative dot plot for enriched CD3 + T lymphocytes. (E) MoDCs were either left untreated or pretreated with anti-CD83 antibody (20 μg/ml) or isotype (20 μg/ml) and infected with PRRSV at an MOI of 0.1, 1, and 2 in the presence or absence of LPS (10 μg/ml). After 24 h, supernatants from these cultures were added to allogeneic T cells. Treatment with LPS alone at 10 μg/ml served as a positive control. T cell proliferation was restored when supernatants were added from PRRSV-infected MoDCs pretreated with rabbit anti-CD83 antibody compared with those pretreated with isotype (20 μg/ml). (F) MoDCs were stimulated with recombinant GST-sCD83 at 0, 0.1, 1, and 5 μg/ml for 24 h. Cell-free supernatants were then transferred to cultures containing purified T cells. MoDCs treated with PBS (MOCK) and GST-Cap fusion protein at 0.1, 1, and 5 μg/ml were used as negative controls. Meanwhile, LPS at 0, 1, 5, and 10 μg/ml was used as a positive control. Cell proliferation was measured using the CCK-8 (absorbance at 450 nm). Data are representative of at least three independent experiments. *** , P

Techniques Used: Activity Assay, Negative Control, Cell Culture, Infection, Western Blot, Positive Control, Expressing, Isolation, Flow Cytometry, Cytometry, Selection, FACS, Recombinant, Purification, CCK-8 Assay

Effect of nsp1α mutations on CD83 expression. (A) MoDCs were mock infected or infected with PRRSV mutants [rL5-2A, rG45A/G48A, rL61-6A, rNsp1α-2m, rNsp1α-3m, rL5-2A(R), rL61-6A(R), rG45A/G48A(R), rNsp1α-2m(R), and rNsp1α-3m(R)] at an MOI of 1 in the presence of LPS (10 μg/ml). After 24 h, cells were analyzed for surface CD83 (mCD83) expression by flow cytometry. Cells were stained with an isotype-matched control antibody. (B) Mean fluorescence intensity (MFI; y axis) values are shown for each virus. (C) Culture supernatants were collected and sCD83 was analyzed by ELISA. (D) CD83 mRNA levels were determined by qRT-PCR. All assays were repeated at least three times, with each experiment performed in triplicate. Bars represent means ± SEM from three independent experiments. *** , P
Figure Legend Snippet: Effect of nsp1α mutations on CD83 expression. (A) MoDCs were mock infected or infected with PRRSV mutants [rL5-2A, rG45A/G48A, rL61-6A, rNsp1α-2m, rNsp1α-3m, rL5-2A(R), rL61-6A(R), rG45A/G48A(R), rNsp1α-2m(R), and rNsp1α-3m(R)] at an MOI of 1 in the presence of LPS (10 μg/ml). After 24 h, cells were analyzed for surface CD83 (mCD83) expression by flow cytometry. Cells were stained with an isotype-matched control antibody. (B) Mean fluorescence intensity (MFI; y axis) values are shown for each virus. (C) Culture supernatants were collected and sCD83 was analyzed by ELISA. (D) CD83 mRNA levels were determined by qRT-PCR. All assays were repeated at least three times, with each experiment performed in triplicate. Bars represent means ± SEM from three independent experiments. *** , P

Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Inhibitory effects of PRRSV infection on MoDC activity are mediated by soluble CD83. Infection of MoDCs by PRRSV increases CD83 production and especially the release of soluble CD83. sCD83 strongly decreases the expression of the MHC-peptide complex proteins TAP1 and ERp57 and then inhibits the ability of MoDCs to stimulate T cell proliferation. Viruses containing mutations in L5A, D6A, G45A, G48A, L61A, P62A, R63A, F65A, and P66A do not affect CD83 expression or depress the ability of MoDCs to stimulate T cell proliferation.
Figure Legend Snippet: Inhibitory effects of PRRSV infection on MoDC activity are mediated by soluble CD83. Infection of MoDCs by PRRSV increases CD83 production and especially the release of soluble CD83. sCD83 strongly decreases the expression of the MHC-peptide complex proteins TAP1 and ERp57 and then inhibits the ability of MoDCs to stimulate T cell proliferation. Viruses containing mutations in L5A, D6A, G45A, G48A, L61A, P62A, R63A, F65A, and P66A do not affect CD83 expression or depress the ability of MoDCs to stimulate T cell proliferation.

Techniques Used: Infection, Activity Assay, Expressing

CD83 promoter activity is increased by PRRSV Nsp1α. (A and B) Effects of Nsp1 and its autocleaved products on the activity of the pCD83 promoter. Human embryonic kidney (HEK) 293T cells and MARC-145 cells were cotransfected with plasmids pCI (negative control), pCI-Nsp1, pCI-Nsp1α, or pCI-Nsp1β, along with pCD83-luc and pRL-TK. After incubation for 36 h, CD83 promoter activity was analyzed using a Dual-Luciferase reporter assay. Extracts from transfected cells were also subjected to Western blotting to detect Nsp1, Nsp1α, and Nsp1β expression. pCI-transfected cells served as a negative control, and pCI-transfected cells stimulated with LPS 20 h prior to harvest served as a positive control. (C) pCD83 promoter activity is induced by Nsp1α in a dose-dependent manner. (D) pCD83 promoter activity increases with time after transfection. All assays were repeated at least three times, with each experiment performed in triplicate. Bars represent means ± SEMs from three independent experiments. Three asterisks indicate significant difference between groups ( P
Figure Legend Snippet: CD83 promoter activity is increased by PRRSV Nsp1α. (A and B) Effects of Nsp1 and its autocleaved products on the activity of the pCD83 promoter. Human embryonic kidney (HEK) 293T cells and MARC-145 cells were cotransfected with plasmids pCI (negative control), pCI-Nsp1, pCI-Nsp1α, or pCI-Nsp1β, along with pCD83-luc and pRL-TK. After incubation for 36 h, CD83 promoter activity was analyzed using a Dual-Luciferase reporter assay. Extracts from transfected cells were also subjected to Western blotting to detect Nsp1, Nsp1α, and Nsp1β expression. pCI-transfected cells served as a negative control, and pCI-transfected cells stimulated with LPS 20 h prior to harvest served as a positive control. (C) pCD83 promoter activity is induced by Nsp1α in a dose-dependent manner. (D) pCD83 promoter activity increases with time after transfection. All assays were repeated at least three times, with each experiment performed in triplicate. Bars represent means ± SEMs from three independent experiments. Three asterisks indicate significant difference between groups ( P

Techniques Used: Activity Assay, Negative Control, Incubation, Luciferase, Reporter Assay, Transfection, Western Blot, Expressing, Positive Control

PRRSV downregulates TAP1 and ERp57 and upregulates CD83 in MoDCs. (A) Porcine monocytes (PMBCs) were cultured for 0 days (left) and 7 days (right) in the presence of GM-CSF and IL-4. After staining with an isotype-matched control antibody, the cells were analyzed for CD86 expression at the cell surface using FACS. (B) MoDCs were infected with PRRSV at an MOI of 0.1, 1, and 2 in the presence or absence of LPS (10 μg/ml) for 24 h. Cell lysates were analyzed for TAP1, ERp57, and N proteins using Western blotting. β-Actin served as a loading control. TAP1 (C) and ERp57 (D) mRNA levels were analyzed by qRT-PCR. mRNA levels were calculated relative to known amounts of template and normalized to β-actin expression. (E) The cells were also analyzed for surface CD83 (mCD83) expression by flow cytometric analysis. (F) Mean fluorescence intensity (MFI) was quantified as a measure of mCD83 production for each analyzed sample. Culture supernatants were also collected, and sCD83 expression was analyzed by ELISA (G) and qRT-PCR (H). MoDCs were inoculated with PRRSV (MOI of 1) at 6, 12, 18, 24, 30, 36, and 48 hpi. All assays were repeated at least three times, with each experiment performed in triplicate. Bars represent means ± SEM from three independent experiments. *** , P
Figure Legend Snippet: PRRSV downregulates TAP1 and ERp57 and upregulates CD83 in MoDCs. (A) Porcine monocytes (PMBCs) were cultured for 0 days (left) and 7 days (right) in the presence of GM-CSF and IL-4. After staining with an isotype-matched control antibody, the cells were analyzed for CD86 expression at the cell surface using FACS. (B) MoDCs were infected with PRRSV at an MOI of 0.1, 1, and 2 in the presence or absence of LPS (10 μg/ml) for 24 h. Cell lysates were analyzed for TAP1, ERp57, and N proteins using Western blotting. β-Actin served as a loading control. TAP1 (C) and ERp57 (D) mRNA levels were analyzed by qRT-PCR. mRNA levels were calculated relative to known amounts of template and normalized to β-actin expression. (E) The cells were also analyzed for surface CD83 (mCD83) expression by flow cytometric analysis. (F) Mean fluorescence intensity (MFI) was quantified as a measure of mCD83 production for each analyzed sample. Culture supernatants were also collected, and sCD83 expression was analyzed by ELISA (G) and qRT-PCR (H). MoDCs were inoculated with PRRSV (MOI of 1) at 6, 12, 18, 24, 30, 36, and 48 hpi. All assays were repeated at least three times, with each experiment performed in triplicate. Bars represent means ± SEM from three independent experiments. *** , P

Techniques Used: Cell Culture, Staining, Expressing, FACS, Infection, Western Blot, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Effect of Nsp1α mutant viruses on CD83 promoter activity. MARC-145 cells were cotransfected with pCD83-luc (1 μg) and pRL-TK (0.1 μg). Twenty-four h later, they were inoculated with PRRSV rBB/wt, rL5-2A, rG45A/G48A, rL61-6A, rNsp1α-2m, rNsp1α-3m, rL5-2A(R), rL61-6A(R), rG45A/G48A(R), rNsp1α-2m(R), and rNsp1α-3m(R) at an MOI of 1. Lysates from LPS-treated and mock-infected cells were used as positive and negative controls, respectively. CD83 promoter activation was analyzed using a Dual-Luciferase reporter assay. The panel below the bar graph shows immunoblots of proteins from infected cells probed with anti-N and anti-β-actin. Data represent the average relative luciferase units from three independent experiments (means ± SEM).
Figure Legend Snippet: Effect of Nsp1α mutant viruses on CD83 promoter activity. MARC-145 cells were cotransfected with pCD83-luc (1 μg) and pRL-TK (0.1 μg). Twenty-four h later, they were inoculated with PRRSV rBB/wt, rL5-2A, rG45A/G48A, rL61-6A, rNsp1α-2m, rNsp1α-3m, rL5-2A(R), rL61-6A(R), rG45A/G48A(R), rNsp1α-2m(R), and rNsp1α-3m(R) at an MOI of 1. Lysates from LPS-treated and mock-infected cells were used as positive and negative controls, respectively. CD83 promoter activation was analyzed using a Dual-Luciferase reporter assay. The panel below the bar graph shows immunoblots of proteins from infected cells probed with anti-N and anti-β-actin. Data represent the average relative luciferase units from three independent experiments (means ± SEM).

Techniques Used: Mutagenesis, Activity Assay, Infection, Activation Assay, Luciferase, Reporter Assay, Western Blot

Identification of the Nsp1α domain responsible for CD83 modulation. (A) Mutations affecting one or two cysteine residues were constructed in the Nsp1α ZF and PCP domains. (B to Q) HEK 293T or MARC-145 cells were seeded in 12-well plates and cotransfected with 1 μg pCD83 and 1 μg pCI-Nsp1α or mutant plasmid together with 100 ng pRL-TK. Forty-eight h after transfection, cells were lysed and analyzed using a Dual-Luciferase reporter assay for CD83 promoter activation. Cells transfected with pCI served as a negative control, and cells transfected with pCI and stimulated with LPS 20 h prior to harvest served as a positive control. Nsp1α levels in cells lysates were analyzed by Western blotting. β-Actin was used as a loading control. (B and C) Mutants affecting the ZF domain; (D and G) mutants affecting the PCP domain; (E and H) Nsp1α truncation mutants; (F and I) constructs containing adjacent alanine substitutions; (J and N) individual alanine substitutions affecting residues 2 to 7; (K and O) individual alanine substitutions affecting residues 41 to 45; (L and P) individual alanine substitutions affecting residues 46 to 50; (M and Q) individual alanine substitutions affecting residues 61 to 66.
Figure Legend Snippet: Identification of the Nsp1α domain responsible for CD83 modulation. (A) Mutations affecting one or two cysteine residues were constructed in the Nsp1α ZF and PCP domains. (B to Q) HEK 293T or MARC-145 cells were seeded in 12-well plates and cotransfected with 1 μg pCD83 and 1 μg pCI-Nsp1α or mutant plasmid together with 100 ng pRL-TK. Forty-eight h after transfection, cells were lysed and analyzed using a Dual-Luciferase reporter assay for CD83 promoter activation. Cells transfected with pCI served as a negative control, and cells transfected with pCI and stimulated with LPS 20 h prior to harvest served as a positive control. Nsp1α levels in cells lysates were analyzed by Western blotting. β-Actin was used as a loading control. (B and C) Mutants affecting the ZF domain; (D and G) mutants affecting the PCP domain; (E and H) Nsp1α truncation mutants; (F and I) constructs containing adjacent alanine substitutions; (J and N) individual alanine substitutions affecting residues 2 to 7; (K and O) individual alanine substitutions affecting residues 41 to 45; (L and P) individual alanine substitutions affecting residues 46 to 50; (M and Q) individual alanine substitutions affecting residues 61 to 66.

Techniques Used: Construct, Mutagenesis, Plasmid Preparation, Transfection, Luciferase, Reporter Assay, Activation Assay, Negative Control, Positive Control, Western Blot

Nsp1α mutations impair the ability of PRRSV to depress immunoregulatory activity of MoDCs. (A to C) MoDCs were mock infected or infected with recombinant PRRSV [rL5-2A, rG45A/G48A, rL61-6A, rNsp1α-2m, rNsp1α-3m, rL5-2A(R), rL61-6A(R), rG45A/G48A(R), rNsp1α-2m(R), and rNsp1α-3m(R)] at an MOI of 1 in the presence of LPS (10 μg/ml) with (A) or without (B) culture medium preprocessed with anti-CD83 or rabbit-IgG (C). After incubation for 24 h, cell lysates were examined by Western blotting with anti-TAP1 or anti-ERp57 antibodies. Replication of PRRSV was analyzed by Western blotting using anti-N protein. Endogenous β-actin expression was used as an internal control. Data are representative of three experiments. (D and E) TAP1 and ERp57 mRNA levels were determined by qPCR. (F to H) MoDCs were treated as described above, and supernatants were added to T cells at 10%, vol/vol. T cell proliferation stimulated by MoDCs increased significantly when the supernatants were generated using PRRSV Nsp1 mutant viruses (F) or treated with anti-CD83 antibody (G) or rabbit-IgG (H). Proliferation was measured as absorbance at 450 nm using a microplate reader. Results are representative of three independent experiments. Data are represented as means ± SEM. ***, P
Figure Legend Snippet: Nsp1α mutations impair the ability of PRRSV to depress immunoregulatory activity of MoDCs. (A to C) MoDCs were mock infected or infected with recombinant PRRSV [rL5-2A, rG45A/G48A, rL61-6A, rNsp1α-2m, rNsp1α-3m, rL5-2A(R), rL61-6A(R), rG45A/G48A(R), rNsp1α-2m(R), and rNsp1α-3m(R)] at an MOI of 1 in the presence of LPS (10 μg/ml) with (A) or without (B) culture medium preprocessed with anti-CD83 or rabbit-IgG (C). After incubation for 24 h, cell lysates were examined by Western blotting with anti-TAP1 or anti-ERp57 antibodies. Replication of PRRSV was analyzed by Western blotting using anti-N protein. Endogenous β-actin expression was used as an internal control. Data are representative of three experiments. (D and E) TAP1 and ERp57 mRNA levels were determined by qPCR. (F to H) MoDCs were treated as described above, and supernatants were added to T cells at 10%, vol/vol. T cell proliferation stimulated by MoDCs increased significantly when the supernatants were generated using PRRSV Nsp1 mutant viruses (F) or treated with anti-CD83 antibody (G) or rabbit-IgG (H). Proliferation was measured as absorbance at 450 nm using a microplate reader. Results are representative of three independent experiments. Data are represented as means ± SEM. ***, P

Techniques Used: Activity Assay, Infection, Recombinant, Incubation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Generated, Mutagenesis

31) Product Images from "Identification and characterization of regulatory elements in the promoter of ACVR1, the gene mutated in Fibrodysplasia Ossificans Progressiva"

Article Title: Identification and characterization of regulatory elements in the promoter of ACVR1, the gene mutated in Fibrodysplasia Ossificans Progressiva

Journal: Orphanet Journal of Rare Diseases

doi: 10.1186/1750-1172-8-145

Functional characterization of the 2. 9 kb promoter region of human ACVR1 gene. A) Schematic representation of the deletion constructs derived from the ACVR1 2.9 kb promoter region. These reporter constructs were transiently transfected in U2OS (B) , HeLa (C) , C2C12 (D) and ATDC5 cells (E) , together with the pRL-TK-Renilla plasmid as a control for transfection efficiency. The corresponding observed activities are shown as relative to the activity of the Pr-2.9 ACVR1 promoter construct (100%). The data represent the mean ± SD (error bars) of five independent experiments conducted in triplicate, with p
Figure Legend Snippet: Functional characterization of the 2. 9 kb promoter region of human ACVR1 gene. A) Schematic representation of the deletion constructs derived from the ACVR1 2.9 kb promoter region. These reporter constructs were transiently transfected in U2OS (B) , HeLa (C) , C2C12 (D) and ATDC5 cells (E) , together with the pRL-TK-Renilla plasmid as a control for transfection efficiency. The corresponding observed activities are shown as relative to the activity of the Pr-2.9 ACVR1 promoter construct (100%). The data represent the mean ± SD (error bars) of five independent experiments conducted in triplicate, with p

Techniques Used: Functional Assay, Construct, Derivative Assay, Transfection, Plasmid Preparation, Activity Assay

Effects of Egr - 1, Hey - 1 and ZBTB7A / LRF / pokemon on ACVR 1 promoter activity. Expression vectors carrying the cDNA of Egr-1, Hey-1 or ZBTB7A/LRF/pokemon transcription factors were transiently transfected in the HeLa (A) and ATDC5 (B) cell lines together with the three reporter ACVR1 promoter deletion constructs (Pr-2.9, Pr-0.7 and Pr-0.3) and the pRL-TK-Renilla plasmid as control for transfection efficiency. Detected activities are expressed relative to those of the same promoter construct co-transfected with empty expression vector considered as 100% (RLU, Relative Light Unit). The data represent the mean ± SD (error bars) of independent experiments (n=2 in HeLa cells, n=3 in ATDC5 cells) carried out in triplicate with p
Figure Legend Snippet: Effects of Egr - 1, Hey - 1 and ZBTB7A / LRF / pokemon on ACVR 1 promoter activity. Expression vectors carrying the cDNA of Egr-1, Hey-1 or ZBTB7A/LRF/pokemon transcription factors were transiently transfected in the HeLa (A) and ATDC5 (B) cell lines together with the three reporter ACVR1 promoter deletion constructs (Pr-2.9, Pr-0.7 and Pr-0.3) and the pRL-TK-Renilla plasmid as control for transfection efficiency. Detected activities are expressed relative to those of the same promoter construct co-transfected with empty expression vector considered as 100% (RLU, Relative Light Unit). The data represent the mean ± SD (error bars) of independent experiments (n=2 in HeLa cells, n=3 in ATDC5 cells) carried out in triplicate with p

Techniques Used: Activity Assay, Expressing, Transfection, Construct, Plasmid Preparation

32) Product Images from "Cadherin 6 is activated by Epstein–Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma"

Article Title: Cadherin 6 is activated by Epstein–Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma

Journal: Oncogenesis

doi: 10.1038/s41389-017-0005-7

NF-κB inhibits miR-203 by binding to its promoter region a Schematic diagram of the predicted binding sites for NF-κB on the miR-203 promoter region. P3 and P4 were combined as one site (P3 + P4) for the detection because they are close to each other. Different miR-203 promoter region fragments as indicated were inserted into the vector pGL3-enhancer for the luciferase activity assay. b Luciferase assay for the promoter binding activity of NF-κB. Luciferase activity was measured at 24 h post-transfection of the pGL3E- plasmids. The pGL3-enhancer vector served as a negative control. Ctrl, the empty vector (pCMV3); pCMV3-p65, the overexpression vector of NF-κB (pCMV3-p65). All the fragments containing the P3 + P4 site showed NF-κB (p65) binding activity, and the one (939bp) not containing P3 + P4 did not show binding activity. c , d ChIP assay was performed using the antibody against NF-κB p65 with IgG as a control in 293 or 293 with p65-overexpressing cells. Ctrl was the empty vector (pCMV3), and the plasmid pCMV3-p65 was constructed for NF-κB overexpression. The PCR amplification positions containing the p65-binding sites are indicated. Based on the result from c , the P1 site with negative activity was neglected in d . e Different protein expression levels of CDH6 and RUNX2 corresponding to the different levels of activated NF-κB (p-p65) in the cells containing EBV genomes (p2089 and its derivative). 293-BAC was a negative control. f Different mRNA expression levels of miR-203 and CDH6 in the cells containing EBV genomes (p2089 and its derivative). 293-BAC is a negative control. g - i CTAR1 and CTAR2 in LMP1 are known to be responsible for miR-203 downregulation and CDH6 activation via NF-κB. The 293 cells were transfected with LMP1 and the mutant plasmids, the expression of the indicated molecules were detected by western blotting or qPCR. g The detection of LMP1, p-p65, RUNX2 and CDH6 expression by western blotting. h The detection of miR-203 by qPCR. i The detection of CDH6 using qPCR. Results are means ± SD, n = 3. * p
Figure Legend Snippet: NF-κB inhibits miR-203 by binding to its promoter region a Schematic diagram of the predicted binding sites for NF-κB on the miR-203 promoter region. P3 and P4 were combined as one site (P3 + P4) for the detection because they are close to each other. Different miR-203 promoter region fragments as indicated were inserted into the vector pGL3-enhancer for the luciferase activity assay. b Luciferase assay for the promoter binding activity of NF-κB. Luciferase activity was measured at 24 h post-transfection of the pGL3E- plasmids. The pGL3-enhancer vector served as a negative control. Ctrl, the empty vector (pCMV3); pCMV3-p65, the overexpression vector of NF-κB (pCMV3-p65). All the fragments containing the P3 + P4 site showed NF-κB (p65) binding activity, and the one (939bp) not containing P3 + P4 did not show binding activity. c , d ChIP assay was performed using the antibody against NF-κB p65 with IgG as a control in 293 or 293 with p65-overexpressing cells. Ctrl was the empty vector (pCMV3), and the plasmid pCMV3-p65 was constructed for NF-κB overexpression. The PCR amplification positions containing the p65-binding sites are indicated. Based on the result from c , the P1 site with negative activity was neglected in d . e Different protein expression levels of CDH6 and RUNX2 corresponding to the different levels of activated NF-κB (p-p65) in the cells containing EBV genomes (p2089 and its derivative). 293-BAC was a negative control. f Different mRNA expression levels of miR-203 and CDH6 in the cells containing EBV genomes (p2089 and its derivative). 293-BAC is a negative control. g - i CTAR1 and CTAR2 in LMP1 are known to be responsible for miR-203 downregulation and CDH6 activation via NF-κB. The 293 cells were transfected with LMP1 and the mutant plasmids, the expression of the indicated molecules were detected by western blotting or qPCR. g The detection of LMP1, p-p65, RUNX2 and CDH6 expression by western blotting. h The detection of miR-203 by qPCR. i The detection of CDH6 using qPCR. Results are means ± SD, n = 3. * p

Techniques Used: Binding Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Negative Control, Over Expression, Chromatin Immunoprecipitation, Construct, Polymerase Chain Reaction, Amplification, Expressing, BAC Assay, Activation Assay, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction

The miR-203 inhibits CDH6-induced EMT and LMP1 promotes EMT in NPC cells a , b The effect of miR-203 overexpression on the expression of CDH6, RUNX2 and EMT markers in NPC cells. The 5-8F and HK-1 were transfected with pMIR-GFP-miR-203 or pMIR-GFP-NC, respectively, at 36 h post-transfection, proteins and RNA were collected and used for the detections by western blotting or qPCR, respectively. Ctrl, the empty vector (pMIR-GFP-NC); miR-203, the overexpression of miR-203 (pMIR-GFP-miR-203). Results are means ± SD; n = 3. * p
Figure Legend Snippet: The miR-203 inhibits CDH6-induced EMT and LMP1 promotes EMT in NPC cells a , b The effect of miR-203 overexpression on the expression of CDH6, RUNX2 and EMT markers in NPC cells. The 5-8F and HK-1 were transfected with pMIR-GFP-miR-203 or pMIR-GFP-NC, respectively, at 36 h post-transfection, proteins and RNA were collected and used for the detections by western blotting or qPCR, respectively. Ctrl, the empty vector (pMIR-GFP-NC); miR-203, the overexpression of miR-203 (pMIR-GFP-miR-203). Results are means ± SD; n = 3. * p

Techniques Used: Over Expression, Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation

33) Product Images from "CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway"

Article Title: CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.10.015

The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P
Figure Legend Snippet: The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P

Techniques Used: Expressing

The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P
Figure Legend Snippet: The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P

Techniques Used: Stable Transfection, Expressing, Transfection, Cotransfection, Western Blot, Apoptosis Assay, Two Tailed Test

CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.
Figure Legend Snippet: CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.

Techniques Used: Expressing, Immunoprecipitation, Immunofluorescence, Activity Assay, Inhibition, Luciferase, Stable Transfection, Cotransfection, Transfection

MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.
Figure Legend Snippet: MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.

Techniques Used: Western Blot, Expressing, Inhibition, Activity Assay, Over Expression

34) Product Images from "CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway"

Article Title: CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway), CREB‐binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.10.015

The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P
Figure Legend Snippet: The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P

Techniques Used: Expressing

The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P
Figure Legend Snippet: The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P

Techniques Used: Stable Transfection, Expressing, Transfection, Cotransfection, Western Blot, Apoptosis Assay, Two Tailed Test

CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.
Figure Legend Snippet: CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.

Techniques Used: Expressing, Immunoprecipitation, Immunofluorescence, Activity Assay, Inhibition, Luciferase, Stable Transfection, Cotransfection, Transfection

MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.
Figure Legend Snippet: MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.

Techniques Used: Western Blot, Expressing, Inhibition, Activity Assay, Over Expression

35) Product Images from "MicroRNA 628 suppresses migration and invasion of breast cancer stem cells through targeting SOS1"

Article Title: MicroRNA 628 suppresses migration and invasion of breast cancer stem cells through targeting SOS1

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S164575

miR-628 directly targets SOS1. Notes: ( A ) Schematic diagram of human SOS1 3′-UTR luciferase constructs with wild type and mutant (MT) (SOS1-3′-UTR) miR-628 target sequences. The breast CSCs of MDA-MB-231 ( B ) and MCF-7 ( C ) cells were transfected with luciferase constructs and the miR-628 mimic. Firefly luciferase activity was normalized to Renilla luciferase activity. * P
Figure Legend Snippet: miR-628 directly targets SOS1. Notes: ( A ) Schematic diagram of human SOS1 3′-UTR luciferase constructs with wild type and mutant (MT) (SOS1-3′-UTR) miR-628 target sequences. The breast CSCs of MDA-MB-231 ( B ) and MCF-7 ( C ) cells were transfected with luciferase constructs and the miR-628 mimic. Firefly luciferase activity was normalized to Renilla luciferase activity. * P

Techniques Used: Luciferase, Construct, Mutagenesis, Multiple Displacement Amplification, Transfection, Activity Assay

36) Product Images from "Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells"

Article Title: Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/1472-6882-14-231

Effect of MME on the translocation and activation of NF-κB in LPS-stimulated BV-2 cells. (A) Cells were treated with and without MME for 2 h followed by LPS stimulation for 30 min. NF-κB/p65 subunits were probed by anti-NF-κB antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were stained by DAPI and the images were captured by confocal microscopy (×40). (B) Cells pretreated with different concentrations of MME for 2 h were stimulated with LPS for 30 min. Cytosolic and nuclear extracts were prepared and analyzed using Western blot by using corresponding antibodies. (C) Cells were co-transfected with 2 μg of NF-κB promoter-containing luciferase DNA along with 40 ng of control pRL-TK DNA for 40 h. Transfected cells were pretreated with various concentrations of MME for 2 h and then stimulated with LPS for 6 h. Cell lysates were prepared and used for reporter gene assay. Data are means ± SDs of three independent experiments. a p
Figure Legend Snippet: Effect of MME on the translocation and activation of NF-κB in LPS-stimulated BV-2 cells. (A) Cells were treated with and without MME for 2 h followed by LPS stimulation for 30 min. NF-κB/p65 subunits were probed by anti-NF-κB antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were stained by DAPI and the images were captured by confocal microscopy (×40). (B) Cells pretreated with different concentrations of MME for 2 h were stimulated with LPS for 30 min. Cytosolic and nuclear extracts were prepared and analyzed using Western blot by using corresponding antibodies. (C) Cells were co-transfected with 2 μg of NF-κB promoter-containing luciferase DNA along with 40 ng of control pRL-TK DNA for 40 h. Transfected cells were pretreated with various concentrations of MME for 2 h and then stimulated with LPS for 6 h. Cell lysates were prepared and used for reporter gene assay. Data are means ± SDs of three independent experiments. a p

Techniques Used: Translocation Assay, Activation Assay, Staining, Confocal Microscopy, Western Blot, Transfection, Luciferase, Reporter Gene Assay

37) Product Images from "Herpes Simplex Virus 1-Encoded Tegument Protein VP16 Abrogates the Production of Beta Interferon (IFN) by Inhibiting NF-?B Activation and Blocking IFN Regulatory Factor 3 To Recruit Its Coactivator CBP"

Article Title: Herpes Simplex Virus 1-Encoded Tegument Protein VP16 Abrogates the Production of Beta Interferon (IFN) by Inhibiting NF-?B Activation and Blocking IFN Regulatory Factor 3 To Recruit Its Coactivator CBP

Journal: Journal of Virology

doi: 10.1128/JVI.01440-13

VP16 inhibits SeV-induced IFN-β production and the activation of the NF-κB promoter induced by SeV or TNF-α treatment. (A and C) HEK 293T cells were transfected with 500 ng of the IFN-β promoter reporter plasmid pIFN-β-luc (A) or p(PRDIII-I)4-Luc (C), together with Renilla luciferase plasmid pRL-TK (50 ng) and pCMV-Flag empty vector or plasmids encoding the indicated viral proteins (1,000 ng). At 24 h after transfection, cells were mock infected or infected with 100 HAU ml −1 SeV as indicated and luciferase activity was measured 16 h postinfection. (B) Results of an experiment similar to that shown in panel A, except an increased amount of VP16-Flag expression plasmid was used, as indicated. The expression of VP16 was analyzed by Western blotting using anti-Flag and anti-β-actin (as a control) MAbs. Data are the relative luciferase activity, with standard deviations of three independent experiments performed in duplicate. (D) HEK 293T cells were transfected with pCMV-Flag empty vector or VP16-Flag expression plasmid. At 24 h posttransfection, cells were mock infected or infected with 100 HAU ml −1 SeV for 16 h and total RNA was analyzed by qRT-PCR. GAPDH levels were quantified for normalization of the data. The fold change in gene expression was determined by comparison to the mock-treated samples. The data represent means + standard deviations of three replicates. (E) Medium from infected cells in shown in panel D was isolated and analyzed by ELISA for IFN-β secretion as described in Materials and Methods. The data represent means + standard deviations of three replicates. (F) HEK 293T cells were transfected with the NF-κB promoter reporter plasmid NF-κB-luc, together with pRL-TK and pCMV-Flag empty vector or plasmid pVP16-Flag. At 24 h posttransfection, cells were treated or not with 10 ng/ml recombinant human TNF-α and incubated for an additional 6 h or infected with SeV for 16 h. NF-κB-driven luciferase activity was determined as described for panel A.
Figure Legend Snippet: VP16 inhibits SeV-induced IFN-β production and the activation of the NF-κB promoter induced by SeV or TNF-α treatment. (A and C) HEK 293T cells were transfected with 500 ng of the IFN-β promoter reporter plasmid pIFN-β-luc (A) or p(PRDIII-I)4-Luc (C), together with Renilla luciferase plasmid pRL-TK (50 ng) and pCMV-Flag empty vector or plasmids encoding the indicated viral proteins (1,000 ng). At 24 h after transfection, cells were mock infected or infected with 100 HAU ml −1 SeV as indicated and luciferase activity was measured 16 h postinfection. (B) Results of an experiment similar to that shown in panel A, except an increased amount of VP16-Flag expression plasmid was used, as indicated. The expression of VP16 was analyzed by Western blotting using anti-Flag and anti-β-actin (as a control) MAbs. Data are the relative luciferase activity, with standard deviations of three independent experiments performed in duplicate. (D) HEK 293T cells were transfected with pCMV-Flag empty vector or VP16-Flag expression plasmid. At 24 h posttransfection, cells were mock infected or infected with 100 HAU ml −1 SeV for 16 h and total RNA was analyzed by qRT-PCR. GAPDH levels were quantified for normalization of the data. The fold change in gene expression was determined by comparison to the mock-treated samples. The data represent means + standard deviations of three replicates. (E) Medium from infected cells in shown in panel D was isolated and analyzed by ELISA for IFN-β secretion as described in Materials and Methods. The data represent means + standard deviations of three replicates. (F) HEK 293T cells were transfected with the NF-κB promoter reporter plasmid NF-κB-luc, together with pRL-TK and pCMV-Flag empty vector or plasmid pVP16-Flag. At 24 h posttransfection, cells were treated or not with 10 ng/ml recombinant human TNF-α and incubated for an additional 6 h or infected with SeV for 16 h. NF-κB-driven luciferase activity was determined as described for panel A.

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Activity Assay, Expressing, Western Blot, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation

VP16 interacts with IRF-3 and blocks the expression of NF-κB-dependent genes through interaction with p65. (A and B) VP16 associates with IRF-3 but not IRF-7. HEK 293T cells (∼5 × 10 6 ) were cotransfected with expression plasmids VP16-Flag and HA-IRF-3 or plasmids VP16-HA and Flag-IRF-7. At 36 h posttransfection, cells were lysed and the samples were then subjected to immunoprecipitation assays (IP) using anti-HA MAb or nonspecific mouse monoclonal antibody (IgG2b). Cell lysates and immunoprecipitated proteins were separated in denaturing 10% polyacrylamide gels and transferred to nitrocellulose membranes. The transferred proteins were probed with anti-HA and anti-Flag MAbs. (C) VP16 interacts with endogenous IRF-3 in HSV-1-infected cells. HEK 293T cells were infected with WT HSV-1 at a multiplicity of infection of 1 for 16 h. The cells were then lysed, and the extracts were subjected to immunoprecipitation using anti-IRF-3 PAb or control IgG. Precipitates were analyzed by Western blotting using anti-VP16 MAb. (D) VP16 interacts with NF-κB subunit p65. HEK 293T cells were cotransfected with expression plasmids VP16-HA and p65-Flag. At 36 h posttransfection, immunoprecipitation assays was performed using anti-HA MAb as described for panel A. (E) HEK 293T cells were transfected with pCMV-Flag empty vector or VP16-Flag expression plasmid. At 24 h posttransfection, the cells were treated with TNF-α (15 ng/ml) for 4 h, and total RNA was analyzed by qRT-PCR. GAPDH levels were quantified for normalization of the data. The fold change in gene expression of IL-6 or IL-8 was determined by comparison to the mock-treated samples. The data represent means + standard deviations of three replicates.
Figure Legend Snippet: VP16 interacts with IRF-3 and blocks the expression of NF-κB-dependent genes through interaction with p65. (A and B) VP16 associates with IRF-3 but not IRF-7. HEK 293T cells (∼5 × 10 6 ) were cotransfected with expression plasmids VP16-Flag and HA-IRF-3 or plasmids VP16-HA and Flag-IRF-7. At 36 h posttransfection, cells were lysed and the samples were then subjected to immunoprecipitation assays (IP) using anti-HA MAb or nonspecific mouse monoclonal antibody (IgG2b). Cell lysates and immunoprecipitated proteins were separated in denaturing 10% polyacrylamide gels and transferred to nitrocellulose membranes. The transferred proteins were probed with anti-HA and anti-Flag MAbs. (C) VP16 interacts with endogenous IRF-3 in HSV-1-infected cells. HEK 293T cells were infected with WT HSV-1 at a multiplicity of infection of 1 for 16 h. The cells were then lysed, and the extracts were subjected to immunoprecipitation using anti-IRF-3 PAb or control IgG. Precipitates were analyzed by Western blotting using anti-VP16 MAb. (D) VP16 interacts with NF-κB subunit p65. HEK 293T cells were cotransfected with expression plasmids VP16-HA and p65-Flag. At 36 h posttransfection, immunoprecipitation assays was performed using anti-HA MAb as described for panel A. (E) HEK 293T cells were transfected with pCMV-Flag empty vector or VP16-Flag expression plasmid. At 24 h posttransfection, the cells were treated with TNF-α (15 ng/ml) for 4 h, and total RNA was analyzed by qRT-PCR. GAPDH levels were quantified for normalization of the data. The fold change in gene expression of IL-6 or IL-8 was determined by comparison to the mock-treated samples. The data represent means + standard deviations of three replicates.

Techniques Used: Expressing, Immunoprecipitation, Infection, Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR

38) Product Images from "The Transcriptional Factor PPARαb Positively Regulates Elovl5 Elongase in Golden Pompano Trachinotus ovatus (Linnaeus 1758)"

Article Title: The Transcriptional Factor PPARαb Positively Regulates Elovl5 Elongase in Golden Pompano Trachinotus ovatus (Linnaeus 1758)

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.01340

Promoter activity analysis of the ToElovl5 gene. (A) The structure and transcriptional activity of ToElovl5 promoters. Five recombinant plasmids, denoted Elovl5-1 (–382 to +89), Elovl5-2 (–793 to +89), Elovl5-3 (–1262 to +89), Elovl5-4 (–146 to +265) and Elovl5-5 (–146 to +459) were constructed and transfected with transcription factor PPARαb into HEK 293T cells. (B) Dual-luciferase activity driven by the ToElovl5-5 core promoter upon the transfection of pcDNA3.1-PPAR-α and pcDNA3.1 in HEK 293T cells. All values are presented as the means ± SD ( n = 3). Asterisks indicate that the values are significantly different from the individual controls ( ∗ p
Figure Legend Snippet: Promoter activity analysis of the ToElovl5 gene. (A) The structure and transcriptional activity of ToElovl5 promoters. Five recombinant plasmids, denoted Elovl5-1 (–382 to +89), Elovl5-2 (–793 to +89), Elovl5-3 (–1262 to +89), Elovl5-4 (–146 to +265) and Elovl5-5 (–146 to +459) were constructed and transfected with transcription factor PPARαb into HEK 293T cells. (B) Dual-luciferase activity driven by the ToElovl5-5 core promoter upon the transfection of pcDNA3.1-PPAR-α and pcDNA3.1 in HEK 293T cells. All values are presented as the means ± SD ( n = 3). Asterisks indicate that the values are significantly different from the individual controls ( ∗ p

Techniques Used: Activity Assay, Recombinant, Construct, Transfection, Luciferase

ToPPARαb (A) and ToElovl5 (B) mRNA expression levels by qRT-PCR after the transfection of either control RNA (control) or siRNA (RNAi). TOCF cells were stimulated with 0.1, 1, and 4 mM of PPARαb agonist (WY-14643) (C) and inhibitor (GW6471) (D) for 24 h, and the expression levels of ToPPARαb and ToElovl5 were significantly increased and decreased, respectively, in a concentration-dependent manner. All values are expressed as the means ± SD ( n = 3). Bars on the same group with different letters are statistically significant from one another ( p
Figure Legend Snippet: ToPPARαb (A) and ToElovl5 (B) mRNA expression levels by qRT-PCR after the transfection of either control RNA (control) or siRNA (RNAi). TOCF cells were stimulated with 0.1, 1, and 4 mM of PPARαb agonist (WY-14643) (C) and inhibitor (GW6471) (D) for 24 h, and the expression levels of ToPPARαb and ToElovl5 were significantly increased and decreased, respectively, in a concentration-dependent manner. All values are expressed as the means ± SD ( n = 3). Bars on the same group with different letters are statistically significant from one another ( p

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Concentration Assay

Amino acid sequences of Elovl5 (A) and PPARαb (B) homologs in vertebrate. (A) Indicated are the highly conserved domains (CD1-3), five putative membrane-spanning domains (MS1-5) and the ER retrieval signal. (B) The four domains indicated by arrows are the N-terminal hypervariable region (A/B), DNA-binding domain (C), flexible hinge domain (D), and ligand-binding domain (E/F). Yellow and blue outlines indicate the eight zinc-binding sites in the DBD and the nine ligand-binding sites in the LBD, respectively. Moreover, the 12 α-helices (H) and four parts of the β-sheet (S) are indicated by a red oval and box, respectively. The accession numbers of the Elovl5 and PPARαb sequences used and species abbreviation are listed in Supplementary Table S3 .
Figure Legend Snippet: Amino acid sequences of Elovl5 (A) and PPARαb (B) homologs in vertebrate. (A) Indicated are the highly conserved domains (CD1-3), five putative membrane-spanning domains (MS1-5) and the ER retrieval signal. (B) The four domains indicated by arrows are the N-terminal hypervariable region (A/B), DNA-binding domain (C), flexible hinge domain (D), and ligand-binding domain (E/F). Yellow and blue outlines indicate the eight zinc-binding sites in the DBD and the nine ligand-binding sites in the LBD, respectively. Moreover, the 12 α-helices (H) and four parts of the β-sheet (S) are indicated by a red oval and box, respectively. The accession numbers of the Elovl5 and PPARαb sequences used and species abbreviation are listed in Supplementary Table S3 .

Techniques Used: Binding Assay, Ligand Binding Assay

39) Product Images from "MicroRNA 628 suppresses migration and invasion of breast cancer stem cells through targeting SOS1"

Article Title: MicroRNA 628 suppresses migration and invasion of breast cancer stem cells through targeting SOS1

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S164575

miR-628 directly targets SOS1. Notes: ( A ) Schematic diagram of human SOS1 3′-UTR luciferase constructs with wild type and mutant (MT) (SOS1-3′-UTR) miR-628 target sequences. The breast CSCs of MDA-MB-231 ( B ) and MCF-7 ( C ) cells were transfected with luciferase constructs and the miR-628 mimic. Firefly luciferase activity was normalized to Renilla luciferase activity. * P
Figure Legend Snippet: miR-628 directly targets SOS1. Notes: ( A ) Schematic diagram of human SOS1 3′-UTR luciferase constructs with wild type and mutant (MT) (SOS1-3′-UTR) miR-628 target sequences. The breast CSCs of MDA-MB-231 ( B ) and MCF-7 ( C ) cells were transfected with luciferase constructs and the miR-628 mimic. Firefly luciferase activity was normalized to Renilla luciferase activity. * P

Techniques Used: Luciferase, Construct, Mutagenesis, Multiple Displacement Amplification, Transfection, Activity Assay

40) Product Images from "Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production"

Article Title: Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00940

Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B) . (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C) , or NF-κB-Luc (D) . At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F) , IFIT2 (G) , IFIT3 (H) , IL-6 (I) , and IL-8 (J) . The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. * P
Figure Legend Snippet: Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B) . (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C) , or NF-κB-Luc (D) . At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F) , IFIT2 (G) , IFIT3 (H) , IL-6 (I) , and IL-8 (J) . The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. * P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Software

Related Articles

Transfection:

Article Title: Long Noncoding RNA Plasmacytoma Variant Translocation 1 (PVT1) Promotes Colon Cancer Progression via Endogenous Sponging miR-26b
Article Snippet: .. Then, HCT116 cells were cultured in 24 well plates and co-transfected constructed luciferase plasmids together with the internal control vector containing Renilla luciferase, pRL-TK (Promega), and miR-26b or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen). .. After 48-hour post-transfection, HCT116 cells were lysed and the Dual-Luciferase Reporter Assay System (Promega) were used for monitoring the luciferase activity.

Article Title: TRIM16 controls assembly and degradation of protein aggregates by modulating the p62‐NRF2 axis and autophagy
Article Snippet: .. For overexpression experiments, HEK293T cells were transfected using calcium phosphate method as per the manufacturer's instructions (Profection, Promega). .. Other cells are transfected using Effectene (Qiagen) or Viafect (Promega) or Interference (Polyplus) as per the manufacturer's instruction.

Luciferase:

Article Title: Suppression of GPR56 expression by pyrrole-imidazole polyamide represents a novel therapeutic drug for AML with high EVI1 expression
Article Snippet: .. Firefly and Renilla luciferase activities were measured in 10 μl of cell lysates using the Dual-Luciferase reporter assay system (Promega) and a Lumat 9507 Luminometer (Berthold, Germany). .. Relative luciferase activities were defined as the ratio of firefly luciferase activity to renilla luciferase activity.

Article Title: Structure and ubiquitination-dependent activation of Tank-Binding Kinase 1
Article Snippet: .. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in . .. Lysates were prepared 60 h following transient transfection of HEK293T cells with TBK1 constructs using standard RIPA buffer.

Article Title: Long Noncoding RNA Plasmacytoma Variant Translocation 1 (PVT1) Promotes Colon Cancer Progression via Endogenous Sponging miR-26b
Article Snippet: .. Then, HCT116 cells were cultured in 24 well plates and co-transfected constructed luciferase plasmids together with the internal control vector containing Renilla luciferase, pRL-TK (Promega), and miR-26b or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen). .. After 48-hour post-transfection, HCT116 cells were lysed and the Dual-Luciferase Reporter Assay System (Promega) were used for monitoring the luciferase activity.

Article Title: Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA
Article Snippet: .. Two days post-transfection, luciferase expression was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. .. The ratio of Firefly to Renilla was calculated as the relative Luc activity.

Reporter Assay:

Article Title: Suppression of GPR56 expression by pyrrole-imidazole polyamide represents a novel therapeutic drug for AML with high EVI1 expression
Article Snippet: .. Firefly and Renilla luciferase activities were measured in 10 μl of cell lysates using the Dual-Luciferase reporter assay system (Promega) and a Lumat 9507 Luminometer (Berthold, Germany). .. Relative luciferase activities were defined as the ratio of firefly luciferase activity to renilla luciferase activity.

Article Title: Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA
Article Snippet: .. Two days post-transfection, luciferase expression was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. .. The ratio of Firefly to Renilla was calculated as the relative Luc activity.

Construct:

Article Title: Long Noncoding RNA Plasmacytoma Variant Translocation 1 (PVT1) Promotes Colon Cancer Progression via Endogenous Sponging miR-26b
Article Snippet: .. Then, HCT116 cells were cultured in 24 well plates and co-transfected constructed luciferase plasmids together with the internal control vector containing Renilla luciferase, pRL-TK (Promega), and miR-26b or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen). .. After 48-hour post-transfection, HCT116 cells were lysed and the Dual-Luciferase Reporter Assay System (Promega) were used for monitoring the luciferase activity.

other:

Article Title: HIV-1 Vpr and p21 restrict LINE-1 mobility
Article Snippet: In addition, we confirmed that p21 co-immunoprecipitated with FLAG-tagged ORF2p (pTMO2F3) ( ) in 293T cells (Figure ).

Activity Assay:

Article Title: Structure and ubiquitination-dependent activation of Tank-Binding Kinase 1
Article Snippet: .. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in . .. Lysates were prepared 60 h following transient transfection of HEK293T cells with TBK1 constructs using standard RIPA buffer.

Article Title: HIV-1 Vpr and p21 restrict LINE-1 mobility
Article Snippet: .. Since Vpr is known to enhance p21 promoter activity ( , ), we wished to confirm this regulation in 293T cells. .. Indeed, we observed that Vpr weakly enhanced p21 promoter activity (Figure ).

Cell Culture:

Article Title: Long Noncoding RNA Plasmacytoma Variant Translocation 1 (PVT1) Promotes Colon Cancer Progression via Endogenous Sponging miR-26b
Article Snippet: .. Then, HCT116 cells were cultured in 24 well plates and co-transfected constructed luciferase plasmids together with the internal control vector containing Renilla luciferase, pRL-TK (Promega), and miR-26b or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen). .. After 48-hour post-transfection, HCT116 cells were lysed and the Dual-Luciferase Reporter Assay System (Promega) were used for monitoring the luciferase activity.

Expressing:

Article Title: Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA
Article Snippet: .. Two days post-transfection, luciferase expression was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. .. The ratio of Firefly to Renilla was calculated as the relative Luc activity.

Article Title: HIV-1 Vpr and p21 restrict LINE-1 mobility
Article Snippet: .. To test this possibility, several plasmids expressing tumor suppressor-related proteins were independently co-transfected with pL1RP -EGFP plasmid into 293T cells. .. GFP fluorescence was then analyzed by flow cytometry to monitor the impact on L1 retrotransposition.

Quantitative RT-PCR:

Article Title: Structure and ubiquitination-dependent activation of Tank-Binding Kinase 1
Article Snippet: .. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in . .. Lysates were prepared 60 h following transient transfection of HEK293T cells with TBK1 constructs using standard RIPA buffer.

Over Expression:

Article Title: TRIM16 controls assembly and degradation of protein aggregates by modulating the p62‐NRF2 axis and autophagy
Article Snippet: .. For overexpression experiments, HEK293T cells were transfected using calcium phosphate method as per the manufacturer's instructions (Profection, Promega). .. Other cells are transfected using Effectene (Qiagen) or Viafect (Promega) or Interference (Polyplus) as per the manufacturer's instruction.

Plasmid Preparation:

Article Title: Long Noncoding RNA Plasmacytoma Variant Translocation 1 (PVT1) Promotes Colon Cancer Progression via Endogenous Sponging miR-26b
Article Snippet: .. Then, HCT116 cells were cultured in 24 well plates and co-transfected constructed luciferase plasmids together with the internal control vector containing Renilla luciferase, pRL-TK (Promega), and miR-26b or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen). .. After 48-hour post-transfection, HCT116 cells were lysed and the Dual-Luciferase Reporter Assay System (Promega) were used for monitoring the luciferase activity.

Article Title: HIV-1 Vpr and p21 restrict LINE-1 mobility
Article Snippet: .. To test this possibility, several plasmids expressing tumor suppressor-related proteins were independently co-transfected with pL1RP -EGFP plasmid into 293T cells. .. GFP fluorescence was then analyzed by flow cytometry to monitor the impact on L1 retrotransposition.

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    Promega dual specific luciferase assay kit
    DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before <t>luciferase</t> assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a <t>dual-specific</t> luciferase <t>assay</t> <t>kit.</t> (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  
    Dual Specific Luciferase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  

    Journal: Journal of Virology

    Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

    doi: 10.1128/JVI.00588-19

    Figure Lengend Snippet: DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  

    Article Snippet: At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega).

    Techniques: Inhibition, Transfection, Infection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

    Journal: Journal of Virology

    Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

    doi: 10.1128/JVI.00588-19

    Figure Lengend Snippet: FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

    Article Snippet: At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega).

    Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Western Blot, Translocation Assay, Clear Native PAGE, Staining, Confocal Microscopy

    Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B) . (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C) , or NF-κB-Luc (D) . At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F) , IFIT2 (G) , IFIT3 (H) , IL-6 (I) , and IL-8 (J) . The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. * P

    Journal: Frontiers in Immunology

    Article Title: Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production

    doi: 10.3389/fimmu.2017.00940

    Figure Lengend Snippet: Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B) . (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C) , or NF-κB-Luc (D) . At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F) , IFIT2 (G) , IFIT3 (H) , IL-6 (I) , and IL-8 (J) . The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. * P

    Article Snippet: Cell extracts were collected at the indicated time points and luciferase activity was measured with a dual-specific luciferase assay kit (Promega).

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Software

    2B protein induces the reduction of RIG-I and suppresses RIG-I-mediated signal transduction. (A) PK-15 cells (5 × 10 5 cells in each well) were grown in six-well plates, and the monolayer cells were transfected with different doses of Flag-2B-expressing plasmids (0, 0.5, 1, or 2 μg). The expression of endogenous RIG-I and Flag-2B proteins was detected by Western blotting at 48 hpt. RIG-I was detected by using rabbit anti-RIG-I polyclonal antibody. (B) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg). The cells were collected at 0, 4, 8, 12, 24, 36, and 48 hpt, and the cell lysates were analyzed by Western blotting to detect the expression levels of RIG-I and any possible cleaved bands. (C) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg) in the presence or absence of 5′ppp-dsRNA control or 5′ppp-dsRNA (1 μg/ml; InvivoGen). The expression of two ISGs (ISG15 and GBP1) was determined by qPCR assay at 24 hpt. The data represent results from one of the triplicate experiments. (D) HEK293T cells (5 × 10 5 cells in each well) were transfected with HA-RIG-I-, HA-VISA-, HA-TBK1-, HA-MITA-, or HA-IRF3-expressing plasmids (2 μg), along with Flag-2B-expressing plasmid or empty vector (2 μg). The expression of HA-RIG-I, HA-VISA, HA-TBK1, HA-MITA, HA-IRF3, and Flag-2B was detected by Western blotting at 48 hpt. Mouse anti-HA antibody was used to detect HA-tagged proteins. (E) HEK293T cells (10 5 cells in each well) were seeded in 24-well plates, and the monolayer cells were transfected with Flag-2B-expressing plasmid (0.1 μg) or empty vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.05 μg). pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. At 24 h after transfection, the cells were left infected or uninfected with SeV (100 HAU/ml) for 16 h. A dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla . Empty vector plasmid was used in the transfection process to ensure that the cells received the same amounts of total plasmids. The data represent the means and standard deviations from three independent experiments. (F) HEK293T cells (10 5 cells in each well) were cotransfected with HA-RIG-I-expressing plasmid (0.1 μg) or empty vector (0.1 μg) and Flag-2B-expressing plasmid (0.1 μg) or empty Flag vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.1 μg). The pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. The dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla at 24 hpt as described for panel E. (G) FMDV 2B enhances virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg). At 24 h after transfection, the cells were infected or uninfected with FMDV (MOI of 0.5) for 12 h. The expression of viral RNA was determined by qPCR assay. The viral VP1 proteins were detected by Western blotting. (H) FMDV 2B, L pro , and 3C pro enhance virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with empty vector, Flag-2B-, Flag-3C-, or Flag-L-expressing plasmids (2 μg). At 24 h after transfection, the cells were infected with FMDV (MOI of 0.5) for 12 h. The viral titers were determined by using a TCID 50 assay. All of the above-described experiments were repeated three times, with similar results. ** , P

    Journal: Journal of Virology

    Article Title: Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression

    doi: 10.1128/JVI.01310-16

    Figure Lengend Snippet: 2B protein induces the reduction of RIG-I and suppresses RIG-I-mediated signal transduction. (A) PK-15 cells (5 × 10 5 cells in each well) were grown in six-well plates, and the monolayer cells were transfected with different doses of Flag-2B-expressing plasmids (0, 0.5, 1, or 2 μg). The expression of endogenous RIG-I and Flag-2B proteins was detected by Western blotting at 48 hpt. RIG-I was detected by using rabbit anti-RIG-I polyclonal antibody. (B) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg). The cells were collected at 0, 4, 8, 12, 24, 36, and 48 hpt, and the cell lysates were analyzed by Western blotting to detect the expression levels of RIG-I and any possible cleaved bands. (C) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg) in the presence or absence of 5′ppp-dsRNA control or 5′ppp-dsRNA (1 μg/ml; InvivoGen). The expression of two ISGs (ISG15 and GBP1) was determined by qPCR assay at 24 hpt. The data represent results from one of the triplicate experiments. (D) HEK293T cells (5 × 10 5 cells in each well) were transfected with HA-RIG-I-, HA-VISA-, HA-TBK1-, HA-MITA-, or HA-IRF3-expressing plasmids (2 μg), along with Flag-2B-expressing plasmid or empty vector (2 μg). The expression of HA-RIG-I, HA-VISA, HA-TBK1, HA-MITA, HA-IRF3, and Flag-2B was detected by Western blotting at 48 hpt. Mouse anti-HA antibody was used to detect HA-tagged proteins. (E) HEK293T cells (10 5 cells in each well) were seeded in 24-well plates, and the monolayer cells were transfected with Flag-2B-expressing plasmid (0.1 μg) or empty vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.05 μg). pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. At 24 h after transfection, the cells were left infected or uninfected with SeV (100 HAU/ml) for 16 h. A dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla . Empty vector plasmid was used in the transfection process to ensure that the cells received the same amounts of total plasmids. The data represent the means and standard deviations from three independent experiments. (F) HEK293T cells (10 5 cells in each well) were cotransfected with HA-RIG-I-expressing plasmid (0.1 μg) or empty vector (0.1 μg) and Flag-2B-expressing plasmid (0.1 μg) or empty Flag vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.1 μg). The pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. The dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla at 24 hpt as described for panel E. (G) FMDV 2B enhances virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg). At 24 h after transfection, the cells were infected or uninfected with FMDV (MOI of 0.5) for 12 h. The expression of viral RNA was determined by qPCR assay. The viral VP1 proteins were detected by Western blotting. (H) FMDV 2B, L pro , and 3C pro enhance virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with empty vector, Flag-2B-, Flag-3C-, or Flag-L-expressing plasmids (2 μg). At 24 h after transfection, the cells were infected with FMDV (MOI of 0.5) for 12 h. The viral titers were determined by using a TCID 50 assay. All of the above-described experiments were repeated three times, with similar results. ** , P

    Article Snippet: The cells were lysed at 16 hpi, and the dual-specific luciferase assay kit (Promega) was used to analyze the firefly and Renilla luciferase activities according to the manufacturer's instructions.

    Techniques: Transduction, Transfection, Expressing, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Infection