Structured Review

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Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with <t>pCDNA-transfected</t> COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.*** P
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1) Product Images from "The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway"

Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl1128

Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with pCDNA-transfected COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.*** P
Figure Legend Snippet: Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with pCDNA-transfected COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.*** P

Techniques Used: Co-Culture Assay, Expressing, Cell Culture, Marker, Transfection, Immunostaining

2) Product Images from "Long Non Coding RNA SNHG16 Facilitates Proliferation, Migration, Invasion and Autophagy of Neuroblastoma Cells via Sponging miR-542-3p and Upregulating ATG5 Expression"

Article Title: Long Non Coding RNA SNHG16 Facilitates Proliferation, Migration, Invasion and Autophagy of Neuroblastoma Cells via Sponging miR-542-3p and Upregulating ATG5 Expression

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S226915

Effects of ATG5 on SNHG16 or miR-542-3p knockdown-mediated proliferation, migration, invasion and autophagy of NB cells. SK-N-SH and IMR-32 cells were transfected with si-SNHG16#2+pcDNA, si-SNHG16#2+pcDNA-ATG5, anti-miR-542-3p+anti-NC or anti-miR-542-3p+anti-ATG5, respectively. ( A and B ) ATG5 protein expression level in SK-N-SH and IMR-32 cells was assessed by Western blot analysis. ( C and D ) MTT assay was conducted for the determination of the proliferation of SK-N-SH and IMR-32 cells. ( E and F ) Transwell assay was performed for the assessment of the migration and invasion of SK-N-SH and IMR-32 cells. ( G and H ) Western blot analysis was used for the detection of the protein expression levels of LC3-I, LC3-II and p62 in SK-N-SH and IMR-32 cells. * P
Figure Legend Snippet: Effects of ATG5 on SNHG16 or miR-542-3p knockdown-mediated proliferation, migration, invasion and autophagy of NB cells. SK-N-SH and IMR-32 cells were transfected with si-SNHG16#2+pcDNA, si-SNHG16#2+pcDNA-ATG5, anti-miR-542-3p+anti-NC or anti-miR-542-3p+anti-ATG5, respectively. ( A and B ) ATG5 protein expression level in SK-N-SH and IMR-32 cells was assessed by Western blot analysis. ( C and D ) MTT assay was conducted for the determination of the proliferation of SK-N-SH and IMR-32 cells. ( E and F ) Transwell assay was performed for the assessment of the migration and invasion of SK-N-SH and IMR-32 cells. ( G and H ) Western blot analysis was used for the detection of the protein expression levels of LC3-I, LC3-II and p62 in SK-N-SH and IMR-32 cells. * P

Techniques Used: Migration, Transfection, Expressing, Western Blot, MTT Assay, Transwell Assay

3) Product Images from "Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition"

Article Title: Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

Journal: Mobile DNA

doi: 10.1186/s13100-016-0079-3

p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc ( left ) and Rluc ( right ) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in ( c ) by first averaging technical replicates for each biological sample ( n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate averages in ( b ); technical replicates are side-by-side; biological replicates are indicated with subscripts
Figure Legend Snippet: p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc ( left ) and Rluc ( right ) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in ( c ) by first averaging technical replicates for each biological sample ( n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate averages in ( b ); technical replicates are side-by-side; biological replicates are indicated with subscripts

Techniques Used: Transfection, Expressing, Plasmid Preparation, Derivative Assay

Effects of p38δ on two different L1 reporter assays. a Top rows show duplicate wells of Giemsa-stained G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT), p38δ-F324S (FS) or p38δ-D176A (DA). Bottom row shows the effect of each pcDNA expression vector on cell growth. The right panel indicates fluorescence intensities obtained from cotransfection of EGFP with each indicated p38δ construct or empty vector; results from duplicate wells are shown. b Relative Fluc/Rluc luminescence ratios obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX015 or pYX017 in the presence of indicated pcDNA mammalian expression vectors. Three biological replicates are shown for each experimental condition; error bars represent the SEM from two technical replicates (defined as two distinct samples taken from each biological sample). The graph at right shows the average of three biological replicates shown separately in the left panel; error bars indicate the SEM, n = 3 biological replicates. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate the Fluc/Rluc ratios from pYX017 in ( b ); technical replicates are side-by-side; biological replicates are indicated in subscript. d Mean Fluc and Rluc luminescence values were derived by first averaging the technical replicates for each biological sample ( n = 2), and then averaging the resulting values of each biological replicate; error bars represent the SEM of biological replicates, n = 3
Figure Legend Snippet: Effects of p38δ on two different L1 reporter assays. a Top rows show duplicate wells of Giemsa-stained G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT), p38δ-F324S (FS) or p38δ-D176A (DA). Bottom row shows the effect of each pcDNA expression vector on cell growth. The right panel indicates fluorescence intensities obtained from cotransfection of EGFP with each indicated p38δ construct or empty vector; results from duplicate wells are shown. b Relative Fluc/Rluc luminescence ratios obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX015 or pYX017 in the presence of indicated pcDNA mammalian expression vectors. Three biological replicates are shown for each experimental condition; error bars represent the SEM from two technical replicates (defined as two distinct samples taken from each biological sample). The graph at right shows the average of three biological replicates shown separately in the left panel; error bars indicate the SEM, n = 3 biological replicates. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate the Fluc/Rluc ratios from pYX017 in ( b ); technical replicates are side-by-side; biological replicates are indicated in subscript. d Mean Fluc and Rluc luminescence values were derived by first averaging the technical replicates for each biological sample ( n = 2), and then averaging the resulting values of each biological replicate; error bars represent the SEM of biological replicates, n = 3

Techniques Used: Staining, Transfection, Expressing, Plasmid Preparation, Fluorescence, Cotransfection, Construct, Derivative Assay

4) Product Images from "Nuclear Translocation of Acinetobacter baumannii Transposase Induces DNA Methylation of CpG Regions in the Promoters of E-cadherin Gene"

Article Title: Nuclear Translocation of Acinetobacter baumannii Transposase Induces DNA Methylation of CpG Regions in the Promoters of E-cadherin Gene

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038974

A. baumannii transposase targets in the nucleus of host cells via NLSs. COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp 1–362 and Tnp 1–230 , were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp 1–37 and Tnp 1–224 , were located in the cytoplasm.
Figure Legend Snippet: A. baumannii transposase targets in the nucleus of host cells via NLSs. COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp 1–362 and Tnp 1–230 , were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp 1–37 and Tnp 1–224 , were located in the cytoplasm.

Techniques Used: Transfection, Plasmid Preparation, Construct, Clone Assay, Incubation, Microscopy

Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene. (A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods . Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp 1–37 ; 7, A549 cells transfected with plasmid constructs of Tnp 1–224 ; 8, A549 cells transfected with plasmid constructs of Tnp 1–230 ; 9, A549 cells transfected with plasmid constructs of Tnp 1–362 . (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods . Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p
Figure Legend Snippet: Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene. (A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods . Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp 1–37 ; 7, A549 cells transfected with plasmid constructs of Tnp 1–224 ; 8, A549 cells transfected with plasmid constructs of Tnp 1–230 ; 9, A549 cells transfected with plasmid constructs of Tnp 1–362 . (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods . Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p

Techniques Used: DNA Methylation Assay, Expressing, Transfection, Clone Assay, Incubation, Purification, Methylation, Polymerase Chain Reaction, Marker, Plasmid Preparation, Construct, Quantitative RT-PCR

5) Product Images from "Serine 28 Phosphorylation of NRIF3 Confers its Coactivator Function for Estrogen Receptor-α Transactivation"

Article Title: Serine 28 Phosphorylation of NRIF3 Confers its Coactivator Function for Estrogen Receptor-α Transactivation

Journal: Oncogene

doi: 10.1038/onc.2008.151

NRIF3 enhances expression and promoter activity of other ER target genes and associates with their gene promoters ( a ) Expression of PS2 and cyclinD1 in control pcDNA3.1 and other NRIF3 stable clones were analyzed by RT-PCR. Actin was used as an internal control. ( b ) Expression of cyclinD1 was measured by Western blot analysis; vinculin was used as internal control. ( c ) CyclinD1 luciferase activity was measured in the overexpressing NRIF3 clones; in some cases, estrogen was added for the last 8 hours. All data are the mean value of three independent experiments. ( d–e ) The association of NRIF3 with E2-responsive genes as analyzed by ChIP assay. Stable transfectants of pcDNA-, NRIF3-, NRIF3-S28A, or NRIF3-S28E-MCF-7 cells were treated with estrogen (10 nM for 45 min), and chromatin lysates were immunoprecipitated with anti-T7 antibody or control IgG; samples were processed as described in Methods. Upper panels show PCR analysis of pS2 (d) and cycD1(e) promoter fragments associated with T7–NRIF3.
Figure Legend Snippet: NRIF3 enhances expression and promoter activity of other ER target genes and associates with their gene promoters ( a ) Expression of PS2 and cyclinD1 in control pcDNA3.1 and other NRIF3 stable clones were analyzed by RT-PCR. Actin was used as an internal control. ( b ) Expression of cyclinD1 was measured by Western blot analysis; vinculin was used as internal control. ( c ) CyclinD1 luciferase activity was measured in the overexpressing NRIF3 clones; in some cases, estrogen was added for the last 8 hours. All data are the mean value of three independent experiments. ( d–e ) The association of NRIF3 with E2-responsive genes as analyzed by ChIP assay. Stable transfectants of pcDNA-, NRIF3-, NRIF3-S28A, or NRIF3-S28E-MCF-7 cells were treated with estrogen (10 nM for 45 min), and chromatin lysates were immunoprecipitated with anti-T7 antibody or control IgG; samples were processed as described in Methods. Upper panels show PCR analysis of pS2 (d) and cycD1(e) promoter fragments associated with T7–NRIF3.

Techniques Used: Expressing, Activity Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction

6) Product Images from "ORF9p Phosphorylation by ORF47p Is Crucial for the Formation and Egress of Varicella-Zoster Virus Viral Particles"

Article Title: ORF9p Phosphorylation by ORF47p Is Crucial for the Formation and Egress of Varicella-Zoster Virus Viral Particles

Journal: Journal of Virology

doi: 10.1128/JVI.02757-12

ORF9p is highly phosphorylated during infection. (A) Cellular extracts of MeWo cells transfected with the pCDNA-ORF9-V5 vector for 48 h or infected with BAC-VZV-ORF9-V5 for 24 h were resolved by SDS-PAGE and immunoblotted using specific antibodies against
Figure Legend Snippet: ORF9p is highly phosphorylated during infection. (A) Cellular extracts of MeWo cells transfected with the pCDNA-ORF9-V5 vector for 48 h or infected with BAC-VZV-ORF9-V5 for 24 h were resolved by SDS-PAGE and immunoblotted using specific antibodies against

Techniques Used: Infection, Transfection, Plasmid Preparation, BAC Assay, SDS Page

pCDNA-ORF9-V5 vector.
Figure Legend Snippet: pCDNA-ORF9-V5 vector.

Techniques Used: Plasmid Preparation

7) Product Images from "Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation"

Article Title: Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

Journal: The FASEB Journal

doi: 10.1096/fj.201600330RR

sihBVR inhibits Akt1 activation and stimulates glucose uptake. A ) sihBVR treatment inhibits Akt1 activation by insulin and IGF-1. Cells were transfected with siRNA against hBVR or with pcDNA-HA-hBVR, and treated with IGF-1 or insulin. Akt1 was immunoprecipitated
Figure Legend Snippet: sihBVR inhibits Akt1 activation and stimulates glucose uptake. A ) sihBVR treatment inhibits Akt1 activation by insulin and IGF-1. Cells were transfected with siRNA against hBVR or with pcDNA-HA-hBVR, and treated with IGF-1 or insulin. Akt1 was immunoprecipitated

Techniques Used: Activation Assay, Transfection, Immunoprecipitation

A ) hBVR binds and stimulates Akt1 kinase activity in cell. Cells were transfected with pcDNA-Akt1 with or without pcDNA-HA-hBVR or pcDNA3 vector. Some cells, as indicated, were treated with 40 ng/ml IGF-1. Immunoprecipitates were prepared using anti-Akt1
Figure Legend Snippet: A ) hBVR binds and stimulates Akt1 kinase activity in cell. Cells were transfected with pcDNA-Akt1 with or without pcDNA-HA-hBVR or pcDNA3 vector. Some cells, as indicated, were treated with 40 ng/ml IGF-1. Immunoprecipitates were prepared using anti-Akt1

Techniques Used: Activity Assay, Transfection, Plasmid Preparation

8) Product Images from "Glucocorticoid Receptor (GR)-Associated SMRT Binding to C/EBP? TAD and Nrf2 Neh4/5: Role of SMRT Recruited to GR in GSTA2 Gene Repression"

Article Title: Glucocorticoid Receptor (GR)-Associated SMRT Binding to C/EBP? TAD and Nrf2 Neh4/5: Role of SMRT Recruited to GR in GSTA2 Gene Repression

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.10.4150-4165.2005

Direct interaction of C/EBPβ or Nrf2 with the RID or SD of SMRT. (A) In vitro translation of 35 S-C/EBPβ or 35 S-Nrf2. 35 S-C/EBPβ or 35 S-Nrf2 was expressed by in vitro translation using the pCDNA, pCDNA-C/EBPβ, or pCDNA-Nrf2
Figure Legend Snippet: Direct interaction of C/EBPβ or Nrf2 with the RID or SD of SMRT. (A) In vitro translation of 35 S-C/EBPβ or 35 S-Nrf2. 35 S-C/EBPβ or 35 S-Nrf2 was expressed by in vitro translation using the pCDNA, pCDNA-C/EBPβ, or pCDNA-Nrf2

Techniques Used: In Vitro

9) Product Images from "Metastasis-Associated Protein 1 Interacts with NRIF3, an Estrogen-Inducible Nuclear Receptor Coregulator"

Article Title: Metastasis-Associated Protein 1 Interacts with NRIF3, an Estrogen-Inducible Nuclear Receptor Coregulator

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.15.6581-6591.2004

Modulation of transactivation function of ER by NRIF3 and MTA1. (A) MCF-7 cells were cotransfected with ERE-luciferase on six-well plates (200 ng/well) and pcDNA vector or T7-MTA1 (500 ng/well) in the absence or presence of NRIF3 (150 ng/well). The luciferase assay was done 48 h posttransfection ( n = 3). (B) MTA1 represses f E2-mediated induction of ERE-luciferase. Stable clones of pcDNA or NRIF3 were transfected with ERE-luciferase in the absence or presence of T7-MTA1 (500 ng/well) and treated with E2 (10 −9 M) or ICI (10 −8 M) for 15 h, and luciferase activity was measured 48 h after transfection ( n = 3). (C) NRIF3 promotes ERE-luciferase activity in stable clones of pcDNA and T7-MTA1. Cells were cotransfected with ERE-luciferase in the absence or in presence of NRIF3 (500 ng/well) plus E2, and luciferase activity was measured 48 h posttransfection ( n = 3). (D) MTA1 modifies NRIF3's association with the target chromatin. MCF7 cells were transfected with T7-NRIF3 or Myc-MTA1 or both. Subsequently, cells were treated with or without E2 (10 −9 M) for 3 h; chromatin immunoprecipitation analysis of the pS2 (304 bp) promoter was performed.
Figure Legend Snippet: Modulation of transactivation function of ER by NRIF3 and MTA1. (A) MCF-7 cells were cotransfected with ERE-luciferase on six-well plates (200 ng/well) and pcDNA vector or T7-MTA1 (500 ng/well) in the absence or presence of NRIF3 (150 ng/well). The luciferase assay was done 48 h posttransfection ( n = 3). (B) MTA1 represses f E2-mediated induction of ERE-luciferase. Stable clones of pcDNA or NRIF3 were transfected with ERE-luciferase in the absence or presence of T7-MTA1 (500 ng/well) and treated with E2 (10 −9 M) or ICI (10 −8 M) for 15 h, and luciferase activity was measured 48 h after transfection ( n = 3). (C) NRIF3 promotes ERE-luciferase activity in stable clones of pcDNA and T7-MTA1. Cells were cotransfected with ERE-luciferase in the absence or in presence of NRIF3 (500 ng/well) plus E2, and luciferase activity was measured 48 h posttransfection ( n = 3). (D) MTA1 modifies NRIF3's association with the target chromatin. MCF7 cells were transfected with T7-NRIF3 or Myc-MTA1 or both. Subsequently, cells were treated with or without E2 (10 −9 M) for 3 h; chromatin immunoprecipitation analysis of the pS2 (304 bp) promoter was performed.

Techniques Used: Luciferase, Plasmid Preparation, Clone Assay, Transfection, Activity Assay, Chromatin Immunoprecipitation

NRIF3 modulates the growth of breast cancer cells. (A) NRIF3 promotes cell proliferation. MCF-7 pooled clones expressing NRIF3 or pcDNA were plated onto six-well plates (10,000 cells/well) in triplicate. After 24 h, the medium was replaced by 3% phenol red-free medium supplemented with charcoal-stripped serum for another 24 h and treated with E2 (10 −9 M) for 24 h. Cell growth was measured by the MTT assay. (B) NRIF3 enhances anchorage-independent growth. Colony growth assays were performed as described in the text. ZR-75 stable pooled cells were plated in triplicate and maintained for 28 days in the absence or presence of E2 (10 −9 M), and the total number of colonies was recorded.
Figure Legend Snippet: NRIF3 modulates the growth of breast cancer cells. (A) NRIF3 promotes cell proliferation. MCF-7 pooled clones expressing NRIF3 or pcDNA were plated onto six-well plates (10,000 cells/well) in triplicate. After 24 h, the medium was replaced by 3% phenol red-free medium supplemented with charcoal-stripped serum for another 24 h and treated with E2 (10 −9 M) for 24 h. Cell growth was measured by the MTT assay. (B) NRIF3 enhances anchorage-independent growth. Colony growth assays were performed as described in the text. ZR-75 stable pooled cells were plated in triplicate and maintained for 28 days in the absence or presence of E2 (10 −9 M), and the total number of colonies was recorded.

Techniques Used: Clone Assay, Expressing, MTT Assay

Identification of NRIF3 as an MTA1 binding protein. (A) Yeast cells were cotransfected with the GAD-NRIF3 and GBD control vector or GBD-MTA1 (amino acids 1 to 715) or GBD-C-terminal MTA1 (ctMTA1, amino acids 255 to 715), GBD-N-terminal MAT1 (amino acids 1 to 173), or GBD-MTA1s. Cotransformants were plated on selection plates lacking leucine and tryptophan (-LT) (bottom panel) or lacking adenine, histidine, leucine, and tryptophan (-AHLT) (middle panel). Growth was recorded after 72 h. For the β-galactosidase assay, filter lift assays were performed. Blue indicates specific interaction of two proteins (upper panel, second lane). (B and C) MTA1 interact with NRIF3 in vitro in GST pulldown assays. In vitro-translated, 35 S-labeled MTA1 protein was incubated with either GST-NRIF3 or GST alone (B) or in vitro-translated, 35 S-labeled NRIF3 protein was incubated with either GST-MTA1 or GST alone (C) and analyzed by SDS-PAGE and autoradiography. (D) MCF-7 cells were cotransfected with c-Myc-MTA1 and T7-NRIF3 or T7-MTA1 and the pcDNA vector. Cells were lysed after 36 h, immunoprecipitated with anti-c-Myc monoclonal antibody to pull down the c-Myc-MTA1 immune complex, resolved on SDS-12% PAGE, and immunoblotted with anti-T7 and anti-c-Myc antibodies.
Figure Legend Snippet: Identification of NRIF3 as an MTA1 binding protein. (A) Yeast cells were cotransfected with the GAD-NRIF3 and GBD control vector or GBD-MTA1 (amino acids 1 to 715) or GBD-C-terminal MTA1 (ctMTA1, amino acids 255 to 715), GBD-N-terminal MAT1 (amino acids 1 to 173), or GBD-MTA1s. Cotransformants were plated on selection plates lacking leucine and tryptophan (-LT) (bottom panel) or lacking adenine, histidine, leucine, and tryptophan (-AHLT) (middle panel). Growth was recorded after 72 h. For the β-galactosidase assay, filter lift assays were performed. Blue indicates specific interaction of two proteins (upper panel, second lane). (B and C) MTA1 interact with NRIF3 in vitro in GST pulldown assays. In vitro-translated, 35 S-labeled MTA1 protein was incubated with either GST-NRIF3 or GST alone (B) or in vitro-translated, 35 S-labeled NRIF3 protein was incubated with either GST-MTA1 or GST alone (C) and analyzed by SDS-PAGE and autoradiography. (D) MCF-7 cells were cotransfected with c-Myc-MTA1 and T7-NRIF3 or T7-MTA1 and the pcDNA vector. Cells were lysed after 36 h, immunoprecipitated with anti-c-Myc monoclonal antibody to pull down the c-Myc-MTA1 immune complex, resolved on SDS-12% PAGE, and immunoblotted with anti-T7 and anti-c-Myc antibodies.

Techniques Used: Binding Assay, Plasmid Preparation, Selection, In Vitro, Labeling, Incubation, SDS Page, Autoradiography, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

NRIF3 stimulates the expression of E2-responsive genes and associates with the ERE-responsive chromatin. (A) NRIF3 stable cell lines. Upper panels show expression of T7-NRIF3 protein by Western blotting with T7 monoclonal antibody. Vinculin expression was used as a loading control. Lower panels show Northern blot analysis of NRIF3 mRNA expression. Glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) and total RNA agarose gel picture were used as loading controls. (B) Equal amounts of cDNA probes from pcDNA and NRIF3 clone 19 were generated by reverse transcription-PCR, hybridized with two identical human estrogen signaling pathway GE array blots, and detected by phosphorimager scanning. The signals of the two blots were normalized to β-actin. Relative expression of E2-responsive genes in the NRIF3 clone was compared with that of the pcDNA clone. (C) Total RNA was made from MCF-7 stable pooled clones expressing pcDNA and NRIF3, hybridized with pS2 cDNA (upper panel), and reprobed with the HMG1 cDNA probe (middle panel). The same blot was hybridized with the glyceraldehhyde-3-phosphate dehydrogenase cDNA probe (bottom panel). (D) E2 induces while tamoxifen inhibits the expression of PS2 and HMG1 mRNA expression. (E) Recruitment of NRIF3 to estrogen target gene promoter. Stable clones expressing either T7-NRIF3 or control vector in MCF-7 cells were treated with estrogen (10 −9 M) or with ICI (10 −7 M) or both for the indicated times. Cells were cross-linked and processed for the chromatin immunoprecipitation assay by immunoprecipitation with anti-T7 antibody, and PCR was performed on the pS2 promoter.
Figure Legend Snippet: NRIF3 stimulates the expression of E2-responsive genes and associates with the ERE-responsive chromatin. (A) NRIF3 stable cell lines. Upper panels show expression of T7-NRIF3 protein by Western blotting with T7 monoclonal antibody. Vinculin expression was used as a loading control. Lower panels show Northern blot analysis of NRIF3 mRNA expression. Glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) and total RNA agarose gel picture were used as loading controls. (B) Equal amounts of cDNA probes from pcDNA and NRIF3 clone 19 were generated by reverse transcription-PCR, hybridized with two identical human estrogen signaling pathway GE array blots, and detected by phosphorimager scanning. The signals of the two blots were normalized to β-actin. Relative expression of E2-responsive genes in the NRIF3 clone was compared with that of the pcDNA clone. (C) Total RNA was made from MCF-7 stable pooled clones expressing pcDNA and NRIF3, hybridized with pS2 cDNA (upper panel), and reprobed with the HMG1 cDNA probe (middle panel). The same blot was hybridized with the glyceraldehhyde-3-phosphate dehydrogenase cDNA probe (bottom panel). (D) E2 induces while tamoxifen inhibits the expression of PS2 and HMG1 mRNA expression. (E) Recruitment of NRIF3 to estrogen target gene promoter. Stable clones expressing either T7-NRIF3 or control vector in MCF-7 cells were treated with estrogen (10 −9 M) or with ICI (10 −7 M) or both for the indicated times. Cells were cross-linked and processed for the chromatin immunoprecipitation assay by immunoprecipitation with anti-T7 antibody, and PCR was performed on the pS2 promoter.

Techniques Used: Expressing, Stable Transfection, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Generated, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Immunoprecipitation

10) Product Images from "SOCS2 overexpression alleviates diabetic nephropathy in rats by inhibiting the TLR4/NF-κB pathway"

Article Title: SOCS2 overexpression alleviates diabetic nephropathy in rats by inhibiting the TLR4/NF-κB pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.20434

Effect of SOCS2 overexpression on the TLR4/NF-κB pathway and the JAK/STAT pathway in HG-stimulated podocytes (A) Western blot was carried out to determine the expression of TLR4, p-p65, p65, p-IκBα and IκBα in control podocytes, HG-stimulated podocytes or HG-stimulated podocytes transfected with pcDNA-SOCS2 or pcDNA. (B) Western blot was conducted to analyze the protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in control podocytes, HG-stimulated podocytes or HG-stimulated podocytes transfected with pcDNA-SOCS2 or pcDNA. (C) The decrease fold of the protein levels of TLR4, p-p65, p-IκBα, p-JAK2, and p-STAT3 in HG-stimulated podocytes or HG-stimulated podocytes transfected with pcDNA-SOCS2 or pcDNA. * P
Figure Legend Snippet: Effect of SOCS2 overexpression on the TLR4/NF-κB pathway and the JAK/STAT pathway in HG-stimulated podocytes (A) Western blot was carried out to determine the expression of TLR4, p-p65, p65, p-IκBα and IκBα in control podocytes, HG-stimulated podocytes or HG-stimulated podocytes transfected with pcDNA-SOCS2 or pcDNA. (B) Western blot was conducted to analyze the protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in control podocytes, HG-stimulated podocytes or HG-stimulated podocytes transfected with pcDNA-SOCS2 or pcDNA. (C) The decrease fold of the protein levels of TLR4, p-p65, p-IκBα, p-JAK2, and p-STAT3 in HG-stimulated podocytes or HG-stimulated podocytes transfected with pcDNA-SOCS2 or pcDNA. * P

Techniques Used: Over Expression, Western Blot, Expressing, Transfection

Effect of SOCS2 overexpression on the expression of IL-6, IL-1β and MCP-1 in HG-stimulated podocytes qRT-PCR was used to detect the mRNA expression of IL-6 (A) , IL-1β (B) and MCP-1 (C) in control group, HG group, HG + pcDNA group and HG + pcDNA-SOCS2 group * P
Figure Legend Snippet: Effect of SOCS2 overexpression on the expression of IL-6, IL-1β and MCP-1 in HG-stimulated podocytes qRT-PCR was used to detect the mRNA expression of IL-6 (A) , IL-1β (B) and MCP-1 (C) in control group, HG group, HG + pcDNA group and HG + pcDNA-SOCS2 group * P

Techniques Used: Over Expression, Expressing, Quantitative RT-PCR

Effects of TAK-242 and AG490 on apoptosis and inflammatory cytokines expressions in HG-stimulated podocytes transfected with pcDNA-SOCS2 Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. (A) Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. Western blot was then performed to detect the levels of TLR4, p-p65, p65, p-IκBα and IκBα in treated podocytes. (B) Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. The levels of Bcl-2 and Cleaved caspase-3 in treated podocytes were examined by western blot. (C) Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. Apoptosis of treated podocytes was assessed by flow cytometry. The mRNA expressions of IL-6 (D) , IL-1β (E) and MCP-1 (F) in treated podocytes were determined by qRT-PCR. (G) Podocytes were pretreated with or without 20 μM AG490 for 30 min, and then stimulated with 30 mM glucose for 24 h. The effect of AG490 on the JAK/STAT pathway in treated podocytes was explored by western blot. (H) The decrease fold of the protein levels of TLR4, p-p65, p-IκBα in TAK-242-treated podocytes, and the protein levels of p-JAK2, and p-STAT3 in AG490-treated podocytes. (I) The decrease fold of apoptosis and mRNA expressions of inflammatory cytokines (IL-6, IL-1β and MCP-1) in TAK-242 or AG490-treated podocytes. * P
Figure Legend Snippet: Effects of TAK-242 and AG490 on apoptosis and inflammatory cytokines expressions in HG-stimulated podocytes transfected with pcDNA-SOCS2 Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. (A) Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. Western blot was then performed to detect the levels of TLR4, p-p65, p65, p-IκBα and IκBα in treated podocytes. (B) Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. The levels of Bcl-2 and Cleaved caspase-3 in treated podocytes were examined by western blot. (C) Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were pretreated with or without 1 μM TAK-242 for 2 h, and then stimulated with 30 mM glucose for 24 h. Apoptosis of treated podocytes was assessed by flow cytometry. The mRNA expressions of IL-6 (D) , IL-1β (E) and MCP-1 (F) in treated podocytes were determined by qRT-PCR. (G) Podocytes were pretreated with or without 20 μM AG490 for 30 min, and then stimulated with 30 mM glucose for 24 h. The effect of AG490 on the JAK/STAT pathway in treated podocytes was explored by western blot. (H) The decrease fold of the protein levels of TLR4, p-p65, p-IκBα in TAK-242-treated podocytes, and the protein levels of p-JAK2, and p-STAT3 in AG490-treated podocytes. (I) The decrease fold of apoptosis and mRNA expressions of inflammatory cytokines (IL-6, IL-1β and MCP-1) in TAK-242 or AG490-treated podocytes. * P

Techniques Used: Transfection, Western Blot, Flow Cytometry, Cytometry, Quantitative RT-PCR

Effect of SOCS2 overexpression on apoptosis in HG-stimulated podocytes Podocytes transfected with pcDNA-SOCS2 or pcDNA were treated with HG for 24 h. (A) Western blot analysis was conducted to measure the levels of SOCS2 in podocytes after transfection for 48 h. (B) Flow cytometry was performed to assess the apoptosis of treated podocytes. (C) Western blot was carried out to evaluate the levels of Bcl-2 and Cleaved caspase-3 in treated podocytes. * P
Figure Legend Snippet: Effect of SOCS2 overexpression on apoptosis in HG-stimulated podocytes Podocytes transfected with pcDNA-SOCS2 or pcDNA were treated with HG for 24 h. (A) Western blot analysis was conducted to measure the levels of SOCS2 in podocytes after transfection for 48 h. (B) Flow cytometry was performed to assess the apoptosis of treated podocytes. (C) Western blot was carried out to evaluate the levels of Bcl-2 and Cleaved caspase-3 in treated podocytes. * P

Techniques Used: Over Expression, Transfection, Western Blot, Flow Cytometry, Cytometry

Effect of PDTC on apoptosis and inflammatory cytokines expressions in HG-stimulated podocytes transfected with pcDNA-SOCS2 Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were preconditioned with or without 50 μM PDTC for 2 h, followed by exposure to 30 mM glucose for 24 h. (A) The levels of p65, p-p65, IκBα, and p-IκBα were detected by western blot analysis. (B) Apoptosis of treated podocytes was examined by flow cytometry. (C) The levels of Bcl-2 and Cleaved caspase-3 in treated podocytes were determined by western blot. The mRNA expressions of IL-6 (D) , IL-1β (E) and MCP-1 (F) in treated podocytes were determined by qRT-PCR. * P
Figure Legend Snippet: Effect of PDTC on apoptosis and inflammatory cytokines expressions in HG-stimulated podocytes transfected with pcDNA-SOCS2 Non-transfected or transfected (pcDNA or pcDNA-SOCS2) podocytes were preconditioned with or without 50 μM PDTC for 2 h, followed by exposure to 30 mM glucose for 24 h. (A) The levels of p65, p-p65, IκBα, and p-IκBα were detected by western blot analysis. (B) Apoptosis of treated podocytes was examined by flow cytometry. (C) The levels of Bcl-2 and Cleaved caspase-3 in treated podocytes were determined by western blot. The mRNA expressions of IL-6 (D) , IL-1β (E) and MCP-1 (F) in treated podocytes were determined by qRT-PCR. * P

Techniques Used: Transfection, Western Blot, Flow Cytometry, Cytometry, Quantitative RT-PCR

11) Product Images from "Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5"

Article Title: Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2018.59.10.1181

IRF5 attenuates the effect of miR-373 on HCV replication in JFH1-infected Huh 7.5 cells. (A) The effect of IRF5 on miR-373-mediated HCV RNA expression was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (B) The effect of IRF5 inhibition on anti-miR-373-regulated HCV RNA expression was measured in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. (C) The effect of IRF5 on miR-373-mediated protein expression of NS3 and NS5A was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (D) The effect of IRF5 interference on anti-miR-373-mediated regulation of NS3 and NS5A protein levels was evaluated in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. Data represent the mean±standard deviation from at least three independent experiments. * p
Figure Legend Snippet: IRF5 attenuates the effect of miR-373 on HCV replication in JFH1-infected Huh 7.5 cells. (A) The effect of IRF5 on miR-373-mediated HCV RNA expression was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (B) The effect of IRF5 inhibition on anti-miR-373-regulated HCV RNA expression was measured in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. (C) The effect of IRF5 on miR-373-mediated protein expression of NS3 and NS5A was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (D) The effect of IRF5 interference on anti-miR-373-mediated regulation of NS3 and NS5A protein levels was evaluated in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. Data represent the mean±standard deviation from at least three independent experiments. * p

Techniques Used: Infection, RNA Expression, Transfection, Inhibition, Expressing, Standard Deviation

12) Product Images from "A Superoxide-Mediated Mitogen-Activated Protein Kinase Phosphatase-1 Degradation and c-Jun NH2"

Article Title: A Superoxide-Mediated Mitogen-Activated Protein Kinase Phosphatase-1 Degradation and c-Jun NH2

Journal: Molecular Pharmacology

doi: 10.1124/mol.111.076653

Overexpression of a degradation-resistant MKP-1 mutant protects cancer cells from luteolin (Lu)-induced cell death. A, top, A549 cells were transfected with pcDNA-MKP-1EE. Twenty-four hours after transfection, cells were treated with luteolin (40 μM) for the indicated times. MKP-1 was detected by Western blot. β-Tubulin was detected as an input control. Bottom, A549 cells treated with luteolin (40 μM) for the indicated times. MKP-1 was detected by Western blot. β-Tubulin was detected as an input control. B, A549 cells were cotransfected with HA-JNK1 and pcDNA-MKP-1EE or empty vector (pcDNA3.1B). Twenty-four hours after transfection, the cells were treated with or without luteolin (40 μM) for the indicated times. Phospho (p)-JNK1, HA-JNK1, and MKP-1EE proteins were detected by Western blot with anti-phospho-JNK, -HA and -Xpress (Xp), respectively. C, A549 cells were cotransfected in duplicate with pRSV-lacz and pcDNA-MKP-1EE or empty vector (pcDNA3.1B). Twenty-four hours after transfection, the cells were treated with or without luteolin (40 μM) for 36 h and then were fixed and stained with X-gal. Top, cells were photographed under a light microscope (100×). Bottom, quantification of cell survival. Blue cells from nine randomly selected fields were counted. The total cell counts in nontreated groups were regarded as 100%. The percentages of cell counts in the luteolin-treated group related to the control groups show cell survival rates after luteolin challenge. *, p
Figure Legend Snippet: Overexpression of a degradation-resistant MKP-1 mutant protects cancer cells from luteolin (Lu)-induced cell death. A, top, A549 cells were transfected with pcDNA-MKP-1EE. Twenty-four hours after transfection, cells were treated with luteolin (40 μM) for the indicated times. MKP-1 was detected by Western blot. β-Tubulin was detected as an input control. Bottom, A549 cells treated with luteolin (40 μM) for the indicated times. MKP-1 was detected by Western blot. β-Tubulin was detected as an input control. B, A549 cells were cotransfected with HA-JNK1 and pcDNA-MKP-1EE or empty vector (pcDNA3.1B). Twenty-four hours after transfection, the cells were treated with or without luteolin (40 μM) for the indicated times. Phospho (p)-JNK1, HA-JNK1, and MKP-1EE proteins were detected by Western blot with anti-phospho-JNK, -HA and -Xpress (Xp), respectively. C, A549 cells were cotransfected in duplicate with pRSV-lacz and pcDNA-MKP-1EE or empty vector (pcDNA3.1B). Twenty-four hours after transfection, the cells were treated with or without luteolin (40 μM) for 36 h and then were fixed and stained with X-gal. Top, cells were photographed under a light microscope (100×). Bottom, quantification of cell survival. Blue cells from nine randomly selected fields were counted. The total cell counts in nontreated groups were regarded as 100%. The percentages of cell counts in the luteolin-treated group related to the control groups show cell survival rates after luteolin challenge. *, p

Techniques Used: Over Expression, Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Staining, Light Microscopy

13) Product Images from "Histone Deacetylases Regulate Gonadotropin-Releasing Hormone I Gene Expression via Modulating Otx2-Driven Transcriptional Activity"

Article Title: Histone Deacetylases Regulate Gonadotropin-Releasing Hormone I Gene Expression via Modulating Otx2-Driven Transcriptional Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039770

Otx2-mediated transcription activation of Gnrh1 gene modulated by HDACs. (A) Accumulation of Otx2 in nuclei was repressed by HDACIs in GT1–7 cells. The affinity of Otx2 to the proximal Gnrh1 gene promoter was suppressed by HDACIs determined by ChIP (B) and EMSA analysis (C). EMSA was performed with GT1–7 nuclear protein extracts using the DNA probes containing the Otx2-binding sites in the mouse Gnrh1 promoter. A supershifted protein complex ( SS ) was observed on both probes after addition of an Otx2 antibody. A 200-fold excess of unlabeled probes didn't eliminate binding of Otx2 to both probes (complex 2). Complex 1, 3 and 4–6 might non-specific binding. (D) Western blot analysis of protein extracts obtained from GT1–7 cells transfected with pcDNA™ 3.1/ myc -His C empty vector or Otx2-myc-His/pcDNA3.1 expression vector. (E) HDACIs-induced dowenregulation of Gnrh1 mRNA was partly rescued by overexpression of Otx2. The value for empty vector transfected control group without drug treatment was set as 1.0. (n = 3; * p
Figure Legend Snippet: Otx2-mediated transcription activation of Gnrh1 gene modulated by HDACs. (A) Accumulation of Otx2 in nuclei was repressed by HDACIs in GT1–7 cells. The affinity of Otx2 to the proximal Gnrh1 gene promoter was suppressed by HDACIs determined by ChIP (B) and EMSA analysis (C). EMSA was performed with GT1–7 nuclear protein extracts using the DNA probes containing the Otx2-binding sites in the mouse Gnrh1 promoter. A supershifted protein complex ( SS ) was observed on both probes after addition of an Otx2 antibody. A 200-fold excess of unlabeled probes didn't eliminate binding of Otx2 to both probes (complex 2). Complex 1, 3 and 4–6 might non-specific binding. (D) Western blot analysis of protein extracts obtained from GT1–7 cells transfected with pcDNA™ 3.1/ myc -His C empty vector or Otx2-myc-His/pcDNA3.1 expression vector. (E) HDACIs-induced dowenregulation of Gnrh1 mRNA was partly rescued by overexpression of Otx2. The value for empty vector transfected control group without drug treatment was set as 1.0. (n = 3; * p

Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Over Expression

14) Product Images from "A novel approach to biomarker discovery in head and neck cancer using an autoantibody signature"

Article Title: A novel approach to biomarker discovery in head and neck cancer using an autoantibody signature

Journal: Oncogene

doi: 10.1038/onc.2012.532

L23 promotes proliferation and invasion. UM-SCC-11A cells, with low endogenous L23, were transfected with pcDNA (PCD) or pcL23. ( a ) Whole-cell lysates were immunoblotted with L23 and actin. Signal intensity was quantified, normalized to actin and expressed
Figure Legend Snippet: L23 promotes proliferation and invasion. UM-SCC-11A cells, with low endogenous L23, were transfected with pcDNA (PCD) or pcL23. ( a ) Whole-cell lysates were immunoblotted with L23 and actin. Signal intensity was quantified, normalized to actin and expressed

Techniques Used: Transfection

15) Product Images from "The role of miR-100 in regulating apoptosis of breast cancer cells"

Article Title: The role of miR-100 in regulating apoptosis of breast cancer cells

Journal: Scientific Reports

doi: 10.1038/srep11650

The miR-100-mediated signalling pathway for apoptosis of breast cancer cells. ( A ) The influence of miR-100 on p27 expression. SK-BR-3 cells were transfected with AMO-miR-100 or AMO-miR-100-scrambled and at 48 h aft er transfection the cells were subjected to real-time PCR (left) and western blot (right). ( B ) The role of p27 in regulating apoptosis of breast cancer cells. SK-BR-3 cells were transfected with pcDNA-p27 to overexpress p27 and at 36 h after transfection the cells were subjected to western blot analysis. The antibodies were indicated on the right. ( C ) The effects of miR-100 on the cell cycle of breast cancer cells. SK-BR-3 cells were transfected with AMOs and stained 48 h later with PI. The cell cycle was analysed by flow cytometry. ( D ) Detections of key effector proteins involved in the cell cycle. SK-BR-3 cells were transfected with AMO-miR-100 or AMO-miR-100-scrambled. Forty-eight hours later the proteins were analysed by western blot. β-actin was used as a control. The antibodies used were indicated on the right. ( E ) The roles of MTMR3 in the cell cycle regulation and apoptosis in breast cancer cells. The expression of MTMR3 was silenced, overexpressed and rescued in SK-BR-3 cells. The mutated nucleotides were underlined. The expression of p27 was evaluated by western blot. The proportion of cells in cell cycle arrest at the G2/M phase was analysed and caspase 3/7 activity was determined. Lane headings showed the treatments used. Non-treated cells were used as a control. ( F ) The effect of MTMR3 on breast cancer cell apoptosis induced by miR-100 inhibition. SK-BR-3 cells were transfected with AMO-miR-100 and MTMR3-siRNA and the expression of MTMR3 and the apoptotic proteins Bax and Bcl-2 were detected by western blot. ( G ) The expression level of p27 in breast cancer tissues and normal tissues. In all panels, plotted data points referred to the means ± standard deviations of triplicate assays and asterisks represented statistically significant differences (* p
Figure Legend Snippet: The miR-100-mediated signalling pathway for apoptosis of breast cancer cells. ( A ) The influence of miR-100 on p27 expression. SK-BR-3 cells were transfected with AMO-miR-100 or AMO-miR-100-scrambled and at 48 h aft er transfection the cells were subjected to real-time PCR (left) and western blot (right). ( B ) The role of p27 in regulating apoptosis of breast cancer cells. SK-BR-3 cells were transfected with pcDNA-p27 to overexpress p27 and at 36 h after transfection the cells were subjected to western blot analysis. The antibodies were indicated on the right. ( C ) The effects of miR-100 on the cell cycle of breast cancer cells. SK-BR-3 cells were transfected with AMOs and stained 48 h later with PI. The cell cycle was analysed by flow cytometry. ( D ) Detections of key effector proteins involved in the cell cycle. SK-BR-3 cells were transfected with AMO-miR-100 or AMO-miR-100-scrambled. Forty-eight hours later the proteins were analysed by western blot. β-actin was used as a control. The antibodies used were indicated on the right. ( E ) The roles of MTMR3 in the cell cycle regulation and apoptosis in breast cancer cells. The expression of MTMR3 was silenced, overexpressed and rescued in SK-BR-3 cells. The mutated nucleotides were underlined. The expression of p27 was evaluated by western blot. The proportion of cells in cell cycle arrest at the G2/M phase was analysed and caspase 3/7 activity was determined. Lane headings showed the treatments used. Non-treated cells were used as a control. ( F ) The effect of MTMR3 on breast cancer cell apoptosis induced by miR-100 inhibition. SK-BR-3 cells were transfected with AMO-miR-100 and MTMR3-siRNA and the expression of MTMR3 and the apoptotic proteins Bax and Bcl-2 were detected by western blot. ( G ) The expression level of p27 in breast cancer tissues and normal tissues. In all panels, plotted data points referred to the means ± standard deviations of triplicate assays and asterisks represented statistically significant differences (* p

Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Staining, Flow Cytometry, Cytometry, Activity Assay, Inhibition

16) Product Images from "ENST00000430471 Promotes Development and Metastasis of Colorectal Cancer by Regulating the Expression of YBX-1"

Article Title: ENST00000430471 Promotes Development and Metastasis of Colorectal Cancer by Regulating the Expression of YBX-1

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S264308

YBX1 overexpression partly reverses the effects of ENST00000430471 knockdown in colorectal cancer cells. ( A ) The mRNA expression level of YBX-1 was determined using reverse transcription-quantitative PCR following transfection of pcDNA-YBX-1 and pcDNA into HCT116 and SW620. ( B ) YBX-1 mRNA expression level, ( C ) YBX-1 protein expression level, and ( D ) Proliferation ability were determined in HCT116 and SW480 cells co-transfected with si0471#2, along with pcDNA-YBX-1 or pcDNA, using transcription-quantitative PCR, Western blot, Cell Counting Kit-8, respectively. Data are presented as mean ± SD. *P
Figure Legend Snippet: YBX1 overexpression partly reverses the effects of ENST00000430471 knockdown in colorectal cancer cells. ( A ) The mRNA expression level of YBX-1 was determined using reverse transcription-quantitative PCR following transfection of pcDNA-YBX-1 and pcDNA into HCT116 and SW620. ( B ) YBX-1 mRNA expression level, ( C ) YBX-1 protein expression level, and ( D ) Proliferation ability were determined in HCT116 and SW480 cells co-transfected with si0471#2, along with pcDNA-YBX-1 or pcDNA, using transcription-quantitative PCR, Western blot, Cell Counting Kit-8, respectively. Data are presented as mean ± SD. *P

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

17) Product Images from "Long non-coding RNA NNT-AS1 knockdown represses the progression of gastric cancer via modulating the miR-142-5p/SOX4/Wnt/β-catenin signaling pathway"

Article Title: Long non-coding RNA NNT-AS1 knockdown represses the progression of gastric cancer via modulating the miR-142-5p/SOX4/Wnt/β-catenin signaling pathway

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2020.11158

NNT-AS1 knockdown blocks the Wnt/β-catenin signaling pathway via the miR-142-5p/SOX4 axis. Protein expression levels of β-catenin, c-Myc, Bcl-2 and E-cadherin in (A) HGC-27 and (B) AGS cells transfected with sh-NC, sh-NNT-AS1, sh-NNT-AS1 + anti-NC, sh-NNT-AS1 + anti-miR-142-5p, sh-NNT-AS1 + pcDNA or sh-NNT-AS1 + SOX4. *P
Figure Legend Snippet: NNT-AS1 knockdown blocks the Wnt/β-catenin signaling pathway via the miR-142-5p/SOX4 axis. Protein expression levels of β-catenin, c-Myc, Bcl-2 and E-cadherin in (A) HGC-27 and (B) AGS cells transfected with sh-NC, sh-NNT-AS1, sh-NNT-AS1 + anti-NC, sh-NNT-AS1 + anti-miR-142-5p, sh-NNT-AS1 + pcDNA or sh-NNT-AS1 + SOX4. *P

Techniques Used: Expressing, Transfection

SOX4 overexpression reverses NNT-AS1 knockdown-mediated effects on GC cell proliferation, apoptosis, migration and invasion. (A-D) AGS and HGC-27 cells were transfected with sh-NNT-AS1, sh-NC, pcDNA, or NNT-AS1. The mRNA expression levels of SOX4 in (A) HGC-27 and (B) AGS cells. Protein expression levels of SOX4 in (C) HGC-27 and (D) AGS cells. *P
Figure Legend Snippet: SOX4 overexpression reverses NNT-AS1 knockdown-mediated effects on GC cell proliferation, apoptosis, migration and invasion. (A-D) AGS and HGC-27 cells were transfected with sh-NNT-AS1, sh-NC, pcDNA, or NNT-AS1. The mRNA expression levels of SOX4 in (A) HGC-27 and (B) AGS cells. Protein expression levels of SOX4 in (C) HGC-27 and (D) AGS cells. *P

Techniques Used: Over Expression, Migration, Transfection, Expressing

18) Product Images from "Long non-coding RNA HNF1A-AS1 promotes proliferation and suppresses apoptosis of bladder cancer cells through upregulating Bcl-2"

Article Title: Long non-coding RNA HNF1A-AS1 promotes proliferation and suppresses apoptosis of bladder cancer cells through upregulating Bcl-2

Journal: Oncotarget

doi: 10.18632/oncotarget.20795

Effects of corresponding siRNA or pcDNA on HNF1A-AS1 expression level The relative expression level was determined using qRT-PCR. ( A ) The HNF1A-AS1 specific siRNA significantly down-regulated the expression level of HNF1A-AS1 in 5637 and T24 cells. ( B ) The HNF1A-AS1 specific pcDNA3.1 significantly up-regulated the expression level of HNF1A-AS1 in SW780 and UM-UC-3 cells. Data are indicated as mean ± SD.
Figure Legend Snippet: Effects of corresponding siRNA or pcDNA on HNF1A-AS1 expression level The relative expression level was determined using qRT-PCR. ( A ) The HNF1A-AS1 specific siRNA significantly down-regulated the expression level of HNF1A-AS1 in 5637 and T24 cells. ( B ) The HNF1A-AS1 specific pcDNA3.1 significantly up-regulated the expression level of HNF1A-AS1 in SW780 and UM-UC-3 cells. Data are indicated as mean ± SD.

Techniques Used: Expressing, Quantitative RT-PCR

19) Product Images from "CutA Divalent Cation Tolerance Homolog (Escherichia coli) (CUTA) Regulates \u03b2-Cleavage of \u03b2-Amyloid Precursor Protein (APP) through Interacting with \u03b2-Site APP Cleaving Protein 1 (BACE1)"

Article Title: CutA Divalent Cation Tolerance Homolog (Escherichia coli) (CUTA) Regulates \u03b2-Cleavage of \u03b2-Amyloid Precursor Protein (APP) through Interacting with \u03b2-Site APP Cleaving Protein 1 (BACE1)

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.330209

The H component of CUTA interacts with BACE1. A , CUTA with an HA tag at the C terminus (CUTA-HA) was co-transfected with APP, BACE1, or NCT, all of which were Myc-tagged at the C terminus into HEK 293T cells. Cells co-transfected with pcDNA, and the indicated
Figure Legend Snippet: The H component of CUTA interacts with BACE1. A , CUTA with an HA tag at the C terminus (CUTA-HA) was co-transfected with APP, BACE1, or NCT, all of which were Myc-tagged at the C terminus into HEK 293T cells. Cells co-transfected with pcDNA, and the indicated

Techniques Used: Transfection

The heavy component of CUTA containing the N terminus is associated with the membrane, whereas the light component of CUTA is located in the cytosol. Cells were transfected with pcDNA, full-length CUTA, CUTA with N-terminal amino acids 1–42 truncated
Figure Legend Snippet: The heavy component of CUTA containing the N terminus is associated with the membrane, whereas the light component of CUTA is located in the cytosol. Cells were transfected with pcDNA, full-length CUTA, CUTA with N-terminal amino acids 1–42 truncated

Techniques Used: Transfection

Only BACE1-interacting CUTA forms regulate β-processing of APP and Aβ secretion. HEK-Swe cells were transfected with control pcDNA, CUTA-Myc, CUTA(M63L), or CUTA-(63–198). A , secreted Aβ and sAPPβ in conditioned
Figure Legend Snippet: Only BACE1-interacting CUTA forms regulate β-processing of APP and Aβ secretion. HEK-Swe cells were transfected with control pcDNA, CUTA-Myc, CUTA(M63L), or CUTA-(63–198). A , secreted Aβ and sAPPβ in conditioned

Techniques Used: Transfection

Overexpression of CUTA reduces β-processing of APP. A , HEK-Swe cells were transfected with control pcDNA and CUTA-Myc. The sAPPα and sAPPβ in conditioned media were Western-blotted with respective antibodies. Aβ secreted
Figure Legend Snippet: Overexpression of CUTA reduces β-processing of APP. A , HEK-Swe cells were transfected with control pcDNA and CUTA-Myc. The sAPPα and sAPPβ in conditioned media were Western-blotted with respective antibodies. Aβ secreted

Techniques Used: Over Expression, Transfection, Western Blot

20) Product Images from "Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1"

Article Title: Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

Journal: Iranian Journal of Biotechnology

doi: 10.15171/ijb.1350

4.2. Sequencing of pcDNA/TIM-1
Figure Legend Snippet: 4.2. Sequencing of pcDNA/TIM-1

Techniques Used: Sequencing

21) Product Images from "A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC"

Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0030218

PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002
Figure Legend Snippet: PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

Techniques Used: Irradiation, Expressing, Plasmid Preparation, Transfection, Positive Control, shRNA, Real-time Polymerase Chain Reaction, Incubation

22) Product Images from "Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization *"

Article Title: Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.042630

Overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. A , Western analysis of the conditioned medium of MDA-MB-231 cells transfected with control vector pcDNA or full-length human CCN3 (SP-NH25). B , growth curve
Figure Legend Snippet: Overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. A , Western analysis of the conditioned medium of MDA-MB-231 cells transfected with control vector pcDNA or full-length human CCN3 (SP-NH25). B , growth curve

Techniques Used: Over Expression, Western Blot, Multiple Displacement Amplification, Transfection, Plasmid Preparation

23) Product Images from "Multivalent Human Papillomavirus L1 DNA Vaccination Utilizing Electroporation"

Article Title: Multivalent Human Papillomavirus L1 DNA Vaccination Utilizing Electroporation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060507

Neutralizing antibody titer and antibody response of sera from mice vaccinated with L1 or L2×8 delivered as protein or a DNA vaccine via electroporation. Balb/c mice were vaccinated three times at two week intervals with PBS, 10 µg L2×8 DNA vaccine i.m. with electroporation three times, or twice utilizing 10 µg HPV16 L1 DNA vaccine i.m. with electroporation followed by a single boost with 25 ug L2×8 protein in alum s.c., 10 µg HPV16 L1 DNA vaccine i.m. with electroporation three times, three times with 10 µg L2×8 DNA vaccine i.m., or 25 ug L2×8 protein in alum s.c., or Gardasil s.c.,,. Serum samples were collected two weeks after the third vaccination, and were tested for in vitro HPV16 neutralization titer (A) and antibody response to HPV16 L2 (B) as measured by ELISA. (C) To assess relative levels of expression, 293TT cells were transfected with no plasmid, HPV16 L1+L2 DNA in pShell, full length HPV16 L2 DNA in p16L2h, bacterial codon optimized L2×8 DNA in pcDNA, or human codon optimized L2×8 in pcDNA. 293TT cells were lysed two days after transfection. Western blotting was performed with lysate samples using a monoclonal antibody to HPV16 L2 17–36.
Figure Legend Snippet: Neutralizing antibody titer and antibody response of sera from mice vaccinated with L1 or L2×8 delivered as protein or a DNA vaccine via electroporation. Balb/c mice were vaccinated three times at two week intervals with PBS, 10 µg L2×8 DNA vaccine i.m. with electroporation three times, or twice utilizing 10 µg HPV16 L1 DNA vaccine i.m. with electroporation followed by a single boost with 25 ug L2×8 protein in alum s.c., 10 µg HPV16 L1 DNA vaccine i.m. with electroporation three times, three times with 10 µg L2×8 DNA vaccine i.m., or 25 ug L2×8 protein in alum s.c., or Gardasil s.c.,,. Serum samples were collected two weeks after the third vaccination, and were tested for in vitro HPV16 neutralization titer (A) and antibody response to HPV16 L2 (B) as measured by ELISA. (C) To assess relative levels of expression, 293TT cells were transfected with no plasmid, HPV16 L1+L2 DNA in pShell, full length HPV16 L2 DNA in p16L2h, bacterial codon optimized L2×8 DNA in pcDNA, or human codon optimized L2×8 in pcDNA. 293TT cells were lysed two days after transfection. Western blotting was performed with lysate samples using a monoclonal antibody to HPV16 L2 17–36.

Techniques Used: Mouse Assay, Electroporation, In Vitro, Neutralization, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Western Blot

24) Product Images from "Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition"

Article Title: Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

Journal: Mobile DNA

doi: 10.1186/s13100-016-0079-3

MKK3b 2E and pcDNA-MKK6 2E increase Rluc luminescence. a Mean Fluc ( left ) and Rluc ( right ) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX015 or pYX017 in the presence of pcDNA-MKK3b 2E (M3) or pcDNA-MKK6 2E (M6). Averages were derived from data shown in ( b ) by first averaging technical replicates for each biological sample ( n = 2), then using this value to average biological replicates; error bars represent SEM of biological samples, n = 3. b Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) obtained from lysates transfected with pYX015 or pYX017 and the indicated pcDNA expression vectors; technical replicates are side-by-side; biological replicates are indicated with subscripts. c Wells show effects on cell growth in response to expression of pcDNA-MKK3b 2E (M3) or pcDNA-MKK6 2E (M6)
Figure Legend Snippet: MKK3b 2E and pcDNA-MKK6 2E increase Rluc luminescence. a Mean Fluc ( left ) and Rluc ( right ) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX015 or pYX017 in the presence of pcDNA-MKK3b 2E (M3) or pcDNA-MKK6 2E (M6). Averages were derived from data shown in ( b ) by first averaging technical replicates for each biological sample ( n = 2), then using this value to average biological replicates; error bars represent SEM of biological samples, n = 3. b Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) obtained from lysates transfected with pYX015 or pYX017 and the indicated pcDNA expression vectors; technical replicates are side-by-side; biological replicates are indicated with subscripts. c Wells show effects on cell growth in response to expression of pcDNA-MKK3b 2E (M3) or pcDNA-MKK6 2E (M6)

Techniques Used: Transfection, Plasmid Preparation, Derivative Assay, Expressing

p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc ( left ) and Rluc ( right ) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in ( c ) by first averaging technical replicates for each biological sample ( n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate averages in ( b ); technical replicates are side-by-side; biological replicates are indicated with subscripts
Figure Legend Snippet: p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc ( left ) and Rluc ( right ) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in ( c ) by first averaging technical replicates for each biological sample ( n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate averages in ( b ); technical replicates are side-by-side; biological replicates are indicated with subscripts

Techniques Used: Transfection, Expressing, Plasmid Preparation, Derivative Assay

Effects of p38δ on two different L1 reporter assays. a Top rows show duplicate wells of Giemsa-stained G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT), p38δ-F324S (FS) or p38δ-D176A (DA). Bottom row shows the effect of each pcDNA expression vector on cell growth. The right panel indicates fluorescence intensities obtained from cotransfection of EGFP with each indicated p38δ construct or empty vector; results from duplicate wells are shown. b Relative Fluc/Rluc luminescence ratios obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX015 or pYX017 in the presence of indicated pcDNA mammalian expression vectors. Three biological replicates are shown for each experimental condition; error bars represent the SEM from two technical replicates (defined as two distinct samples taken from each biological sample). The graph at right shows the average of three biological replicates shown separately in the left panel; error bars indicate the SEM, n = 3 biological replicates. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate the Fluc/Rluc ratios from pYX017 in ( b ); technical replicates are side-by-side; biological replicates are indicated in subscript. d Mean Fluc and Rluc luminescence values were derived by first averaging the technical replicates for each biological sample ( n = 2), and then averaging the resulting values of each biological replicate; error bars represent the SEM of biological replicates, n = 3
Figure Legend Snippet: Effects of p38δ on two different L1 reporter assays. a Top rows show duplicate wells of Giemsa-stained G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT), p38δ-F324S (FS) or p38δ-D176A (DA). Bottom row shows the effect of each pcDNA expression vector on cell growth. The right panel indicates fluorescence intensities obtained from cotransfection of EGFP with each indicated p38δ construct or empty vector; results from duplicate wells are shown. b Relative Fluc/Rluc luminescence ratios obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX015 or pYX017 in the presence of indicated pcDNA mammalian expression vectors. Three biological replicates are shown for each experimental condition; error bars represent the SEM from two technical replicates (defined as two distinct samples taken from each biological sample). The graph at right shows the average of three biological replicates shown separately in the left panel; error bars indicate the SEM, n = 3 biological replicates. c Individual luminescence values are shown for Fluc ( blue ) and Rluc ( red ) used to calculate the Fluc/Rluc ratios from pYX017 in ( b ); technical replicates are side-by-side; biological replicates are indicated in subscript. d Mean Fluc and Rluc luminescence values were derived by first averaging the technical replicates for each biological sample ( n = 2), and then averaging the resulting values of each biological replicate; error bars represent the SEM of biological replicates, n = 3

Techniques Used: Staining, Transfection, Expressing, Plasmid Preparation, Fluorescence, Cotransfection, Construct, Derivative Assay

25) Product Images from "Human Neutrophil Peptide 1 as immunotherapeutic agent against Leishmania infected BALB/c mice"

Article Title: Human Neutrophil Peptide 1 as immunotherapeutic agent against Leishmania infected BALB/c mice

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006123

Footpad swelling measurement and determination of L. major parasite quantification in lymph node of treated and control groups. Footpad swelling measurement, started one week after challenge, was fulfilled weekly by metric capilar (B) Real time PCR was performed in 30 ng of DNA (n = 5). The G1-G5 represents the results from folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), AmB (G4) and non-treated group (G5), respectively. The stars indicate significant difference and n.s displays not significant.
Figure Legend Snippet: Footpad swelling measurement and determination of L. major parasite quantification in lymph node of treated and control groups. Footpad swelling measurement, started one week after challenge, was fulfilled weekly by metric capilar (B) Real time PCR was performed in 30 ng of DNA (n = 5). The G1-G5 represents the results from folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), AmB (G4) and non-treated group (G5), respectively. The stars indicate significant difference and n.s displays not significant.

Techniques Used: Real-time Polymerase Chain Reaction

26) Product Images from "Sequential signals toward podosome formation in NIH-src cells"

Article Title: Sequential signals toward podosome formation in NIH-src cells

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200801042

Podosomes assemble at the proximal part of focal adhesions followed by PtdIns(3,4)P2 accumulation. (A) NIH-src cells with reduced Tks5/FISH expression expressing myc-tagged Tapp1PH were stained with rhodamine-phalloidin (red) to visualize F-actin with anti-myc antibody (green) and anti-Tks5/FISH antibody (blue). (B) NIH-src cells with reduced Tks5/FISH expression expressing RFP-2×Tapp1PH were stained with anti-FAK antibody (green) and with anti-Tks5/FISH antibody (blue). Arrowheads indicate the polarization of the plasma membrane at FA-related adhesions. The regions outlined by boxes are examples of FA-related adhesions magnified twofold. Bars, 20 μm. (C) NIH3T3 cells were transfected with GFP-vinculin and RFP-2×Tapp1PH on the previous day of transfection with pcDNA-SrcY530F plasmid. The cells were cultured in fresh medium 3 h after the transfection and the observations were started. The region outlined by a box is an example of newly formed FA, which was associated with a podosome (magnified and shown with elapsed time points). Arrowheads indicate the accumulation of 2×Tapp1PH and arrows indicate a developing podosome. Bars, 20 μm.
Figure Legend Snippet: Podosomes assemble at the proximal part of focal adhesions followed by PtdIns(3,4)P2 accumulation. (A) NIH-src cells with reduced Tks5/FISH expression expressing myc-tagged Tapp1PH were stained with rhodamine-phalloidin (red) to visualize F-actin with anti-myc antibody (green) and anti-Tks5/FISH antibody (blue). (B) NIH-src cells with reduced Tks5/FISH expression expressing RFP-2×Tapp1PH were stained with anti-FAK antibody (green) and with anti-Tks5/FISH antibody (blue). Arrowheads indicate the polarization of the plasma membrane at FA-related adhesions. The regions outlined by boxes are examples of FA-related adhesions magnified twofold. Bars, 20 μm. (C) NIH3T3 cells were transfected with GFP-vinculin and RFP-2×Tapp1PH on the previous day of transfection with pcDNA-SrcY530F plasmid. The cells were cultured in fresh medium 3 h after the transfection and the observations were started. The region outlined by a box is an example of newly formed FA, which was associated with a podosome (magnified and shown with elapsed time points). Arrowheads indicate the accumulation of 2×Tapp1PH and arrows indicate a developing podosome. Bars, 20 μm.

Techniques Used: Fluorescence In Situ Hybridization, Expressing, Staining, Transfection, Plasmid Preparation, Cell Culture

27) Product Images from "Knockdown of lncRNA TUG1 Enhances Radiosensitivity of Prostate Cancer via the TUG1/miR-139-5p/SMC1A Axis"

Article Title: Knockdown of lncRNA TUG1 Enhances Radiosensitivity of Prostate Cancer via the TUG1/miR-139-5p/SMC1A Axis

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S236860

The expression of miR-139-5p was downregulated and negatively correlated with TUG1 in PCa samples and cell lines. ( A ) The presumed binding sites of miR-139-5p with TUG1 were predicted. ( B and C ) The luciferase activity in PC-3 and DU145 cells co-transfected with miR-139-5p mimics or mimics-NC and TUG1-WT or TUG1-MUT. ( D ) The expression of miR-139-5p was detected by qRT-PCR in PCa tissue samples. ( E ) The expression of miR-139-5p was negatively correlated with TUG1 in PCa samples by qRT-PCR. ( F ) The expression of miR-139-5p was detected by qRT-PCR. ( G ) The expression of miR-139-5p in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, pcDNA and pcDNA-TUG1 was measured by qRT-PCR. * P
Figure Legend Snippet: The expression of miR-139-5p was downregulated and negatively correlated with TUG1 in PCa samples and cell lines. ( A ) The presumed binding sites of miR-139-5p with TUG1 were predicted. ( B and C ) The luciferase activity in PC-3 and DU145 cells co-transfected with miR-139-5p mimics or mimics-NC and TUG1-WT or TUG1-MUT. ( D ) The expression of miR-139-5p was detected by qRT-PCR in PCa tissue samples. ( E ) The expression of miR-139-5p was negatively correlated with TUG1 in PCa samples by qRT-PCR. ( F ) The expression of miR-139-5p was detected by qRT-PCR. ( G ) The expression of miR-139-5p in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, pcDNA and pcDNA-TUG1 was measured by qRT-PCR. * P

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

Restoration of HMGB1 expression reversed the improved radiosensitivity induced by TUG1 knockdown in PCa cells. ( A and B ) SMC1A mRNA and protein expression were detected by qRT-PCR and Weston blot in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ inhibitor NC or si-TUG1#1+ miR-139-5p inhibitor at 24 hrs. ( C ) SMC1A mRNA expression was positively correlated with TUG1 by qRT-PCR. ( D and E ) The expression of SMC1A was measured by qRT-PCR. ( F and G ) MTT assay detected the viability of PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ pcDNA-SMC1A at the indicated time points (0 hrs, 24 hrs, 48 hrs and 72 hrs) after 4 Gy of radiation. ( H and I ) Flow cytometry determined cell apoptosis rate of PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ SMC1A at 24 hrs after 4 Gy radiation. ( J and K ) The expression level of apoptosis-related proteins (Bcl-2, Bax and cleaved-casp-3) was tested by Weston blot in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ SMC1A at 24 hrs after 4 Gy radiation. ( L and M ) The survival fraction was established by Kaplan–Meier and log-rank in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ SMC1A and subjected to 0, 2, 4, 6 and 8 Gy of radiation. * P
Figure Legend Snippet: Restoration of HMGB1 expression reversed the improved radiosensitivity induced by TUG1 knockdown in PCa cells. ( A and B ) SMC1A mRNA and protein expression were detected by qRT-PCR and Weston blot in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ inhibitor NC or si-TUG1#1+ miR-139-5p inhibitor at 24 hrs. ( C ) SMC1A mRNA expression was positively correlated with TUG1 by qRT-PCR. ( D and E ) The expression of SMC1A was measured by qRT-PCR. ( F and G ) MTT assay detected the viability of PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ pcDNA-SMC1A at the indicated time points (0 hrs, 24 hrs, 48 hrs and 72 hrs) after 4 Gy of radiation. ( H and I ) Flow cytometry determined cell apoptosis rate of PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ SMC1A at 24 hrs after 4 Gy radiation. ( J and K ) The expression level of apoptosis-related proteins (Bcl-2, Bax and cleaved-casp-3) was tested by Weston blot in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ SMC1A at 24 hrs after 4 Gy radiation. ( L and M ) The survival fraction was established by Kaplan–Meier and log-rank in PC-3 and DU145 cells transfected with si-NC, si-TUG1#1, si-TUG1#1+ pcDNA or si-TUG1#1+ SMC1A and subjected to 0, 2, 4, 6 and 8 Gy of radiation. * P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, MTT Assay, Flow Cytometry

28) Product Images from "Sirtuin1 (Sirt1) Promotes Cortical Bone Formation by Preventing β-Catenin Sequestration by FoxO Transcription Factors in Osteoblast Progenitors *"

Article Title: Sirtuin1 (Sirt1) Promotes Cortical Bone Formation by Preventing β-Catenin Sequestration by FoxO Transcription Factors in Osteoblast Progenitors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.561803

Sirt1 inhibits FoxO activity. A , FoxO3 expression was induced in the OPF-iFoxO3 cell line by withdrawal of doxycycline, and cells were transfected with a FoxO-luc reporter construct and a Sirt1 expression plasmid, as indicated. Twenty-four hours later, vehicle ( veh ) or SRT2104 was added to the cultures for another 24 h. RLU , relative luminescence units. B , ST2 cells transfected with an empty vector control ( pcDNA ), FoxO1, or FoxO3 expression plasmids and co-transfected with a Sirt1 plasmid followed by treatment as in A. C and D , GFP + cell cultures derived from neonatal calvaria of FoxO1,3,4 ( C ) and Sirt1 ( D ) conditional deletion mouse models. E , ST2 cells transfected with FoxO-luc and with empty vector, wild-type ( WT ) FoxO1, acetylation mimic ( KQ ), or acetylation mutant (KR). †, p
Figure Legend Snippet: Sirt1 inhibits FoxO activity. A , FoxO3 expression was induced in the OPF-iFoxO3 cell line by withdrawal of doxycycline, and cells were transfected with a FoxO-luc reporter construct and a Sirt1 expression plasmid, as indicated. Twenty-four hours later, vehicle ( veh ) or SRT2104 was added to the cultures for another 24 h. RLU , relative luminescence units. B , ST2 cells transfected with an empty vector control ( pcDNA ), FoxO1, or FoxO3 expression plasmids and co-transfected with a Sirt1 plasmid followed by treatment as in A. C and D , GFP + cell cultures derived from neonatal calvaria of FoxO1,3,4 ( C ) and Sirt1 ( D ) conditional deletion mouse models. E , ST2 cells transfected with FoxO-luc and with empty vector, wild-type ( WT ) FoxO1, acetylation mimic ( KQ ), or acetylation mutant (KR). †, p

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

29) Product Images from "A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation"

Article Title: A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

Journal: Scientific Reports

doi: 10.1038/s41598-019-50702-2

Differential engagement of PLK1 PBD substrates via the Tyr pocket. ( A ) FRAP analysis of interphase centrosomes in live cells. GFP-PLK1 Wt/AAD/AM at the centrosome was photobleached and its recovery monitored at 0.2 s intervals. The median t 1/2 time taken for 50% maximal recovery for GFP-PLK1 Wt/AAD/AM were found to be 0.7 s, 0.4 s and 0.3 s respectively. The arrowhead indicates the point of photobleaching. An inset shows data points for the first 5 seconds including photobleaching and recovery time of GFP signal. ( B ) HeLa cells expressing GFP-PLK1 Wt/AAD/AM were synchronized in mitosis by double thymidine block and released as shown in the experimental schedule. The cell lysates were immunoprecipitated using GFP-Trap® beads to pull down GFP-PLK1 Wt/AAD/AM and analysed by immunoblotting. The data presented is representative of two independent experiments. ( C ) HeLa cells expressing GFP-PLK1 Wt/AAD/AM were transfected with PBIP1 Wt -V5 or pcDNA™3.1/V5-HisA vector (or V5 vector) and harvested 24 h later. PBIP1 Wt -V5/ V5 was pulled down from the lysates using V5 antibody and immunoprecipitates were analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments. ( D ) HeLa GFP-PLK1 Wt overexpressing cells were transfected with either PBIP1 Wt/F71A/T78A -V5 or V5 vector and harvested 24 h later. PBIP1 Wt/F71A/T78A -V5 was pulled down from the lysates using V5 antibody and analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments.
Figure Legend Snippet: Differential engagement of PLK1 PBD substrates via the Tyr pocket. ( A ) FRAP analysis of interphase centrosomes in live cells. GFP-PLK1 Wt/AAD/AM at the centrosome was photobleached and its recovery monitored at 0.2 s intervals. The median t 1/2 time taken for 50% maximal recovery for GFP-PLK1 Wt/AAD/AM were found to be 0.7 s, 0.4 s and 0.3 s respectively. The arrowhead indicates the point of photobleaching. An inset shows data points for the first 5 seconds including photobleaching and recovery time of GFP signal. ( B ) HeLa cells expressing GFP-PLK1 Wt/AAD/AM were synchronized in mitosis by double thymidine block and released as shown in the experimental schedule. The cell lysates were immunoprecipitated using GFP-Trap® beads to pull down GFP-PLK1 Wt/AAD/AM and analysed by immunoblotting. The data presented is representative of two independent experiments. ( C ) HeLa cells expressing GFP-PLK1 Wt/AAD/AM were transfected with PBIP1 Wt -V5 or pcDNA™3.1/V5-HisA vector (or V5 vector) and harvested 24 h later. PBIP1 Wt -V5/ V5 was pulled down from the lysates using V5 antibody and immunoprecipitates were analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments. ( D ) HeLa GFP-PLK1 Wt overexpressing cells were transfected with either PBIP1 Wt/F71A/T78A -V5 or V5 vector and harvested 24 h later. PBIP1 Wt/F71A/T78A -V5 was pulled down from the lysates using V5 antibody and analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments.

Techniques Used: Expressing, Blocking Assay, Immunoprecipitation, Transfection, Plasmid Preparation

30) Product Images from "GroEL1, from Chlamydia pneumoniae, Induces Vascular Adhesion Molecule 1 Expression by p37AUF1 in Endothelial Cells and Hypercholesterolemic Rabbit"

Article Title: GroEL1, from Chlamydia pneumoniae, Induces Vascular Adhesion Molecule 1 Expression by p37AUF1 in Endothelial Cells and Hypercholesterolemic Rabbit

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042808

The 5′ UTR flanking sequence of VCAM-1 mRNA conferred P37 AUF1 -responsiveness in the BAECs. (A) The BAECs were transfected with the 4His-A-AUF1-p37 plasmid (p37 AUF1 ), 4His-A-AUF1-p40 plasmid (p40 AUF1 ), 4His-A-AUF1-p42 plasmid (p42 AUF1 ), or 4His-A-AUF1-p45 plasmid (p45 AUF1 ). The level of VCAM-1 mRNA were analyzed using real-time PCR after transfectiuon for 24 hours. (B) The VCAM-1 mRNA stability was analyzed using an actinomycin D chase experiment in the AUF1-transfected BAECs. (C) Schematic representation of the various plasmids containing the luciferase and UTR of the VCAM-1 mRNA. Control plasmid: pcDNA™ 3.1 plasmid; construct A, CMV-Luciferase plasmid; construct B, CMV-Luciferase-VCAM1 5′UTR (sense) plasmid; construct C, CMV-Luciferase-VCAM1 5′UTR (antisense) plasmid; construct D, CMV-Luciferase-VCAM1 3′UTR (sense) plasmid; construct E, CMV-Luciferase-VCAM1 3′UTR (antisense) plasmid. (D), BAECs were co-transfected with the CMV-Luciferase-VCAM1 UTR plasmid, the β-galactosidase reporter plasmid, and the 4His-A-AUF1 plasmid. Uniform transfection efficiencies were confirmed using a β-galactosidase reporter plasmid. The luciferase activity was quantified by luminometry. Data are expressed as relative luciferase units, presented as the mean ± SEM and represent the results of three independent experiments (* P
Figure Legend Snippet: The 5′ UTR flanking sequence of VCAM-1 mRNA conferred P37 AUF1 -responsiveness in the BAECs. (A) The BAECs were transfected with the 4His-A-AUF1-p37 plasmid (p37 AUF1 ), 4His-A-AUF1-p40 plasmid (p40 AUF1 ), 4His-A-AUF1-p42 plasmid (p42 AUF1 ), or 4His-A-AUF1-p45 plasmid (p45 AUF1 ). The level of VCAM-1 mRNA were analyzed using real-time PCR after transfectiuon for 24 hours. (B) The VCAM-1 mRNA stability was analyzed using an actinomycin D chase experiment in the AUF1-transfected BAECs. (C) Schematic representation of the various plasmids containing the luciferase and UTR of the VCAM-1 mRNA. Control plasmid: pcDNA™ 3.1 plasmid; construct A, CMV-Luciferase plasmid; construct B, CMV-Luciferase-VCAM1 5′UTR (sense) plasmid; construct C, CMV-Luciferase-VCAM1 5′UTR (antisense) plasmid; construct D, CMV-Luciferase-VCAM1 3′UTR (sense) plasmid; construct E, CMV-Luciferase-VCAM1 3′UTR (antisense) plasmid. (D), BAECs were co-transfected with the CMV-Luciferase-VCAM1 UTR plasmid, the β-galactosidase reporter plasmid, and the 4His-A-AUF1 plasmid. Uniform transfection efficiencies were confirmed using a β-galactosidase reporter plasmid. The luciferase activity was quantified by luminometry. Data are expressed as relative luciferase units, presented as the mean ± SEM and represent the results of three independent experiments (* P

Techniques Used: Sequencing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Luciferase, Construct, Activity Assay

31) Product Images from "Protection of a Single Dose West Nile Virus Recombinant Subviral Particle Vaccine against Lineage 1 or 2 Strains and Analysis of the Cross-Reactivity with Usutu Virus"

Article Title: Protection of a Single Dose West Nile Virus Recombinant Subviral Particle Vaccine against Lineage 1 or 2 Strains and Analysis of the Cross-Reactivity with Usutu Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0108056

Expression of WNV proteins encoded by plasmid pcDNA-WNV. (A) Immunofluorescence analysis of E protein expression. HeLa cells were transfected with pcDNA or pcDNA-WNV and, 24 h later, fixed and processed for immunofluorescence using a monoclonal anti-E and a secondary antibody coupled to Alexa Fluor 594. Nuclei were stained with DAPI. Scale bar 20 µm. (B) Analysis of WNV protein expression in the culture medium from 293T or HeLa cells transfected with pcDNA-WNV by enzyme-linked immunodot assay. Both cell lines were transfected with pcDNA or pcDNA-WNV and, 24, 48 or 72 h later, the culture medium was adsorbed onto a nitrocellulose membrane and detected using a monoclonal anti-E or a polyclonal anti-M and the corresponding secondary antibodies coupled to horseradish peroxidase. (C) Western blot analysis of WNV proteins released to the culture medium after transfection with pcDNA-WNV. HeLa or 293T cells were transfected with plasmid pcDNA-WNV and the viral proteins in the culture medium were purified by ultracentrifugation through a 20% sucrose cushion. Proteins were blotted with the antibodies used in panel (B). A control lane containing purified WNV from WNV-infected Vero cells was included. (D) Expression of E protein in the culture medium of HeLa cells stably transfected with pcDNA-WNV (HeLa3-WNV). The E protein content in the culture medium from different passages of HeLa3-WNV cells was determined by an enzyme-linked immunodot assay using a monoclonal antibody against E protein. (E) Analysis by ELISA of the reactivity of WNV antigens released to culture medium of HeLa3-WNV cells. ELISA plates were coated with two-fold serial dilutions of culture medium from HeLa3-WNV cells and incubated either with a monoclonal anti-E or an irrelevant isotype-matched control antibody. Samples were incubated with appropriate secondary antibodies and developed using O-phenylene diamine dihydrochloride. ELISA results were expressed as the absorbance at 492 nm (A492). (F, G) Analysis of the reactivity of viral proteins secreted by HeLa3-WNV with pooled sera from experimentally infected mice (F) or birds (G). ELISA plates were coated with a fixed dilution of HeLa3-WNV culture medium and incubated with two-fold serial dilutions of sera from WNV-infected mice or red-legged partridges and the ELISA was performed as described in (E). Pooled sera from control uninfected animals were included as negative controls.
Figure Legend Snippet: Expression of WNV proteins encoded by plasmid pcDNA-WNV. (A) Immunofluorescence analysis of E protein expression. HeLa cells were transfected with pcDNA or pcDNA-WNV and, 24 h later, fixed and processed for immunofluorescence using a monoclonal anti-E and a secondary antibody coupled to Alexa Fluor 594. Nuclei were stained with DAPI. Scale bar 20 µm. (B) Analysis of WNV protein expression in the culture medium from 293T or HeLa cells transfected with pcDNA-WNV by enzyme-linked immunodot assay. Both cell lines were transfected with pcDNA or pcDNA-WNV and, 24, 48 or 72 h later, the culture medium was adsorbed onto a nitrocellulose membrane and detected using a monoclonal anti-E or a polyclonal anti-M and the corresponding secondary antibodies coupled to horseradish peroxidase. (C) Western blot analysis of WNV proteins released to the culture medium after transfection with pcDNA-WNV. HeLa or 293T cells were transfected with plasmid pcDNA-WNV and the viral proteins in the culture medium were purified by ultracentrifugation through a 20% sucrose cushion. Proteins were blotted with the antibodies used in panel (B). A control lane containing purified WNV from WNV-infected Vero cells was included. (D) Expression of E protein in the culture medium of HeLa cells stably transfected with pcDNA-WNV (HeLa3-WNV). The E protein content in the culture medium from different passages of HeLa3-WNV cells was determined by an enzyme-linked immunodot assay using a monoclonal antibody against E protein. (E) Analysis by ELISA of the reactivity of WNV antigens released to culture medium of HeLa3-WNV cells. ELISA plates were coated with two-fold serial dilutions of culture medium from HeLa3-WNV cells and incubated either with a monoclonal anti-E or an irrelevant isotype-matched control antibody. Samples were incubated with appropriate secondary antibodies and developed using O-phenylene diamine dihydrochloride. ELISA results were expressed as the absorbance at 492 nm (A492). (F, G) Analysis of the reactivity of viral proteins secreted by HeLa3-WNV with pooled sera from experimentally infected mice (F) or birds (G). ELISA plates were coated with a fixed dilution of HeLa3-WNV culture medium and incubated with two-fold serial dilutions of sera from WNV-infected mice or red-legged partridges and the ELISA was performed as described in (E). Pooled sera from control uninfected animals were included as negative controls.

Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence, Transfection, Staining, Western Blot, Purification, Infection, Stable Transfection, Enzyme-linked Immunosorbent Assay, Incubation, Mouse Assay

32) Product Images from "Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer"

Article Title: Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer

Journal: Journal of Oncology

doi: 10.1155/2018/3812581

(a) RKO cells stable-transfected with pcDNA (IRS-4) or pcDNA were untreated or treated with wortmannin (200 nM) during 18 h and several proteins were analysed by western blot. (b) RKO cells were starved overnight with serum-free medium and stimulated with IGF-1 (25 nM) during 30 min, then harvested, and lysed. Immunoprecipitation with anti-IRS-4 antibody was carried out and tyrosine phosphorylation was analysed by immunoblot. (c) Immunoprecipitations with anti-IRS-4 antibody and (d) anti-IRS-1 antibody was performed in RKO cells after IGF-1 treatment (25 nM) during 30 min, then the association with α p85 was studied by immunoblot. (e) RKO cells with or without IGF-1 stimulation (25 nM) during 30 min were lysed and immunoprecipitated with anti-BRK antibody and its interaction with IRS-4 was studied by western blot. (f) RKO cells stable-transfected with pcDNA (IRS-4) or pcDNA and treated with wortmannin (200 nM) during 18 h were immunoprecipitated with anti-BRK antibody and its association with IRS-4, IGF-1 receptor and phosphorylated IGF-1 receptor were studied by western blot. Negative control (C-) was assessed replacing the lysis extract for sample buffer. Results shown are representative for two or three independent experiments. H.C. = heavy chain of the immunoprecipitation antibody. SB = sample buffer.
Figure Legend Snippet: (a) RKO cells stable-transfected with pcDNA (IRS-4) or pcDNA were untreated or treated with wortmannin (200 nM) during 18 h and several proteins were analysed by western blot. (b) RKO cells were starved overnight with serum-free medium and stimulated with IGF-1 (25 nM) during 30 min, then harvested, and lysed. Immunoprecipitation with anti-IRS-4 antibody was carried out and tyrosine phosphorylation was analysed by immunoblot. (c) Immunoprecipitations with anti-IRS-4 antibody and (d) anti-IRS-1 antibody was performed in RKO cells after IGF-1 treatment (25 nM) during 30 min, then the association with α p85 was studied by immunoblot. (e) RKO cells with or without IGF-1 stimulation (25 nM) during 30 min were lysed and immunoprecipitated with anti-BRK antibody and its interaction with IRS-4 was studied by western blot. (f) RKO cells stable-transfected with pcDNA (IRS-4) or pcDNA and treated with wortmannin (200 nM) during 18 h were immunoprecipitated with anti-BRK antibody and its association with IRS-4, IGF-1 receptor and phosphorylated IGF-1 receptor were studied by western blot. Negative control (C-) was assessed replacing the lysis extract for sample buffer. Results shown are representative for two or three independent experiments. H.C. = heavy chain of the immunoprecipitation antibody. SB = sample buffer.

Techniques Used: Transfection, Western Blot, Immunoprecipitation, Negative Control, Lysis

(a) Western blot analyses of IRS-4, IRS-1, and ERK levels in RKO and HepG2 cell lines. (b) IRS-1 and IRS-4 mRNA expression levels in RKO and HepG2 cell lines were detected using qPCR; graphs depict the calculated ratios of IRS-1/IRS-4 to 18S for each sample. (c) RKO and HepG2 cells were transiently transfected with pcDNA (IRS-4) or pcDNA (negative control); cells were lysed and several proteins were analysed by western blot. A representative experiment of three performed is shown.
Figure Legend Snippet: (a) Western blot analyses of IRS-4, IRS-1, and ERK levels in RKO and HepG2 cell lines. (b) IRS-1 and IRS-4 mRNA expression levels in RKO and HepG2 cell lines were detected using qPCR; graphs depict the calculated ratios of IRS-1/IRS-4 to 18S for each sample. (c) RKO and HepG2 cells were transiently transfected with pcDNA (IRS-4) or pcDNA (negative control); cells were lysed and several proteins were analysed by western blot. A representative experiment of three performed is shown.

Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control

33) Product Images from "Nuclear Translocation of Acinetobacter baumannii Transposase Induces DNA Methylation of CpG Regions in the Promoters of E-cadherin Gene"

Article Title: Nuclear Translocation of Acinetobacter baumannii Transposase Induces DNA Methylation of CpG Regions in the Promoters of E-cadherin Gene

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038974

A. baumannii transposase targets in the nucleus of host cells via NLSs. COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp 1–362 and Tnp 1–230 , were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp 1–37 and Tnp 1–224 , were located in the cytoplasm.
Figure Legend Snippet: A. baumannii transposase targets in the nucleus of host cells via NLSs. COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp 1–362 and Tnp 1–230 , were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp 1–37 and Tnp 1–224 , were located in the cytoplasm.

Techniques Used: Transfection, Plasmid Preparation, Construct, Clone Assay, Incubation, Microscopy

Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene. (A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods . Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp 1–37 ; 7, A549 cells transfected with plasmid constructs of Tnp 1–224 ; 8, A549 cells transfected with plasmid constructs of Tnp 1–230 ; 9, A549 cells transfected with plasmid constructs of Tnp 1–362 . (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods . Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p
Figure Legend Snippet: Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene. (A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods . Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp 1–37 ; 7, A549 cells transfected with plasmid constructs of Tnp 1–224 ; 8, A549 cells transfected with plasmid constructs of Tnp 1–230 ; 9, A549 cells transfected with plasmid constructs of Tnp 1–362 . (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods . Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p

Techniques Used: DNA Methylation Assay, Expressing, Transfection, Clone Assay, Incubation, Purification, Methylation, Polymerase Chain Reaction, Marker, Plasmid Preparation, Construct, Quantitative RT-PCR

34) Product Images from "Human resistin, a proinflammatory cytokine, shows chaperone-like activity"

Article Title: Human resistin, a proinflammatory cytokine, shows chaperone-like activity

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1306145110

Resistin expression causes an inhibition of apoptosis of HeLa cells. HeLa cells were transiently transfected with pCDNA plasmid vector, recombinant pCDNAhRes plasmid construct, or mock transfected (UT) HeLa cells. Apoptosis was induced by treating cells
Figure Legend Snippet: Resistin expression causes an inhibition of apoptosis of HeLa cells. HeLa cells were transiently transfected with pCDNA plasmid vector, recombinant pCDNAhRes plasmid construct, or mock transfected (UT) HeLa cells. Apoptosis was induced by treating cells

Techniques Used: Expressing, Inhibition, Transfection, Plasmid Preparation, Recombinant, Construct

35) Product Images from "Long non-coding RNA NEAT1 modifies cell proliferation, colony formation, apoptosis, migration and invasion via the miR-4500/BZW1 axis in ovarian cancer"

Article Title: Long non-coding RNA NEAT1 modifies cell proliferation, colony formation, apoptosis, migration and invasion via the miR-4500/BZW1 axis in ovarian cancer

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2020.11408

BZW1 overexpression reverses miR-4500 mimic-mediated effects on OC cell behavior. (A) Transfection efficiency of BZW1 overexpression. CAOV3 and ES-2 cells were transfected with miR-NC + pcDNA, miR-4500 + pcDNA or miR-4500 + BZW1. BZW1 protein expression levels in (B) CAOV3 and (C) ES-2 cells. The CCK-8 assay was performed to assess (D) CAOV3 and (E) ES-2 cell proliferation at 72 h. (F) Colony formation was evaluated by performing a colony formation assay. (G) OC cell apoptosis was determined via flow cytometry. Transwell assays were performed to investigate the function of miR-4500 and BZW1 in (H) CAOV3 and (I) ES-2 cell migration and invasion. ECAR in (J) CAOV3 and (K) ES-2 cells was determined using an XF96 metabolic flux analyzer. *P
Figure Legend Snippet: BZW1 overexpression reverses miR-4500 mimic-mediated effects on OC cell behavior. (A) Transfection efficiency of BZW1 overexpression. CAOV3 and ES-2 cells were transfected with miR-NC + pcDNA, miR-4500 + pcDNA or miR-4500 + BZW1. BZW1 protein expression levels in (B) CAOV3 and (C) ES-2 cells. The CCK-8 assay was performed to assess (D) CAOV3 and (E) ES-2 cell proliferation at 72 h. (F) Colony formation was evaluated by performing a colony formation assay. (G) OC cell apoptosis was determined via flow cytometry. Transwell assays were performed to investigate the function of miR-4500 and BZW1 in (H) CAOV3 and (I) ES-2 cell migration and invasion. ECAR in (J) CAOV3 and (K) ES-2 cells was determined using an XF96 metabolic flux analyzer. *P

Techniques Used: Over Expression, Transfection, Expressing, CCK-8 Assay, Colony Assay, Flow Cytometry, Migration

36) Product Images from "Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5"

Article Title: Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2018.59.10.1181

IRF5 attenuates the effect of miR-373 on HCV replication in JFH1-infected Huh 7.5 cells. (A) The effect of IRF5 on miR-373-mediated HCV RNA expression was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (B) The effect of IRF5 inhibition on anti-miR-373-regulated HCV RNA expression was measured in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. (C) The effect of IRF5 on miR-373-mediated protein expression of NS3 and NS5A was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (D) The effect of IRF5 interference on anti-miR-373-mediated regulation of NS3 and NS5A protein levels was evaluated in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. Data represent the mean±standard deviation from at least three independent experiments. * p
Figure Legend Snippet: IRF5 attenuates the effect of miR-373 on HCV replication in JFH1-infected Huh 7.5 cells. (A) The effect of IRF5 on miR-373-mediated HCV RNA expression was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (B) The effect of IRF5 inhibition on anti-miR-373-regulated HCV RNA expression was measured in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. (C) The effect of IRF5 on miR-373-mediated protein expression of NS3 and NS5A was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (D) The effect of IRF5 interference on anti-miR-373-mediated regulation of NS3 and NS5A protein levels was evaluated in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. Data represent the mean±standard deviation from at least three independent experiments. * p

Techniques Used: Infection, RNA Expression, Transfection, Inhibition, Expressing, Standard Deviation

37) Product Images from "Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells"

Article Title: Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells

Journal: Journal of pineal research

doi: 10.1111/j.1600-079X.2010.00767.x

Effect of Per2 overexpression on apoptosis. A) Effect of Per2 over-expression on cleaved PARP protein levels: Following lipofectamine mediated transfection of hPer2 or pCDNA in 22Rν1, DU145 and PC3 cells, cleaved PARP protein levels were detected
Figure Legend Snippet: Effect of Per2 overexpression on apoptosis. A) Effect of Per2 over-expression on cleaved PARP protein levels: Following lipofectamine mediated transfection of hPer2 or pCDNA in 22Rν1, DU145 and PC3 cells, cleaved PARP protein levels were detected

Techniques Used: Over Expression, Transfection

Overexpression of Per2 in PCa cells. A) Effect of overexpression on Per2 protein levels: Following Lipofectamine 2000 mediated transfections of hPer2 or pCDNA in 22Rν1, DU145 and PC3 cells, Per2 protein levels were detected by Western blot analysis.
Figure Legend Snippet: Overexpression of Per2 in PCa cells. A) Effect of overexpression on Per2 protein levels: Following Lipofectamine 2000 mediated transfections of hPer2 or pCDNA in 22Rν1, DU145 and PC3 cells, Per2 protein levels were detected by Western blot analysis.

Techniques Used: Over Expression, Transfection, Western Blot

38) Product Images from "Role of RACK1 in the differential proliferative effects of neuropeptide Y1–36 and peptide YY1–36 in SHR vs. WKY preglomerular vascular smooth muscle cells"

Article Title: Role of RACK1 in the differential proliferative effects of neuropeptide Y1–36 and peptide YY1–36 in SHR vs. WKY preglomerular vascular smooth muscle cells

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00646.2012

The cDNA fragment encoding rat RACK1 was obtained by PCR using rat PGVSMC cDNA as a template with primer set: sense, 5′-ctaagctatccggtgccatc-3′ and antisense, 5′-gcgggtaccaatagtcacctg-3′. The RACK1 cDNA was then cloned into pcDNA 3.1/V5-His-TOPO vector by using pcDNA3.1/V5-His TOPO TA expression kit (Life Technologies) as per the manufacturer's instructions. The clone was verified by DNA sequencing. Western blot depicts expression of V5-tagged RACK1 in WKY PGVSMCs treated with RACK1-expressing plasmid vs. and empty (control) plasmid.
Figure Legend Snippet: The cDNA fragment encoding rat RACK1 was obtained by PCR using rat PGVSMC cDNA as a template with primer set: sense, 5′-ctaagctatccggtgccatc-3′ and antisense, 5′-gcgggtaccaatagtcacctg-3′. The RACK1 cDNA was then cloned into pcDNA 3.1/V5-His-TOPO vector by using pcDNA3.1/V5-His TOPO TA expression kit (Life Technologies) as per the manufacturer's instructions. The clone was verified by DNA sequencing. Western blot depicts expression of V5-tagged RACK1 in WKY PGVSMCs treated with RACK1-expressing plasmid vs. and empty (control) plasmid.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Expressing, DNA Sequencing, Western Blot

39) Product Images from "HuD Promotes BDNF Expression in Brain Neurons via Selective Stabilization of the BDNF Long 3?UTR mRNA"

Article Title: HuD Promotes BDNF Expression in Brain Neurons via Selective Stabilization of the BDNF Long 3?UTR mRNA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055718

HuD selectively enhances expression of the luciferase reporter that harbors the BDNF long 3′UTR in an ARE-dependent manner. CAD cells were cotransfected by 200 ng of reporter construct together with either pcDNA-HuD (HuD) or the pcDNA parental vector (PC). 20 ng of pRL-CMV Renilla luciferase construct was included in each transfection. Twenty-four hours after transfection, cells were harvested and subjected to dual luciferase reporter assay or mRNA extraction followed by DNAase-treatment and RT-qPCR. (A) myc-HuD enhances expression of the luciferase reporter that carries the BDNF long 3′UTR but not the short 3′UTR. (B) myc-HuD reduces decay of the BDNF long 3′UTR reporter mRNA in co-transfected CAD cells in which transcription is inhibited by actinomycin D. (C) Loss of the ARE in the BDNF long 3′UTR abolished the repose to HuD-dependent enhancement of reporter expression. (D) HuD regulates reporter mRNA expression mediated by the ARE in the BDNF long 3′UTR. * indicates P
Figure Legend Snippet: HuD selectively enhances expression of the luciferase reporter that harbors the BDNF long 3′UTR in an ARE-dependent manner. CAD cells were cotransfected by 200 ng of reporter construct together with either pcDNA-HuD (HuD) or the pcDNA parental vector (PC). 20 ng of pRL-CMV Renilla luciferase construct was included in each transfection. Twenty-four hours after transfection, cells were harvested and subjected to dual luciferase reporter assay or mRNA extraction followed by DNAase-treatment and RT-qPCR. (A) myc-HuD enhances expression of the luciferase reporter that carries the BDNF long 3′UTR but not the short 3′UTR. (B) myc-HuD reduces decay of the BDNF long 3′UTR reporter mRNA in co-transfected CAD cells in which transcription is inhibited by actinomycin D. (C) Loss of the ARE in the BDNF long 3′UTR abolished the repose to HuD-dependent enhancement of reporter expression. (D) HuD regulates reporter mRNA expression mediated by the ARE in the BDNF long 3′UTR. * indicates P

Techniques Used: Expressing, Luciferase, Construct, Plasmid Preparation, Transfection, Reporter Assay, Quantitative RT-PCR

40) Product Images from "Comparative transcriptome analysis reveals the role of p53 signalling pathway during red‐spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells. Comparative transcriptome analysis reveals the role of p53 signalling pathway during red‐spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells"

Article Title: Comparative transcriptome analysis reveals the role of p53 signalling pathway during red‐spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells. Comparative transcriptome analysis reveals the role of p53 signalling pathway during red‐spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells

Journal: Journal of Fish Diseases

doi: 10.1111/jfd.12960

Effects of Ljp53 on RGNNV replication and zebrafish IFN1 promoter activity. (a) qRT‐PCR detection of RDRP in LJB cells transfected with pcDNA‐Ljp53 or control plasmid at 48 hr post‐RGNNV infection. (b) qRT‐PCR detection of RDRP in LJB cells treated with DMSO (control) or pifithrin‐α. (c) HEK293T cells were analysed at 48 hr after transfected with the pRL‐TK, pcDNA3.1(+) or pcDNA‐Ljp53 plasmids together with zebrafish IFN1 promoter‐driven reporter plasmid, and the luciferase activity was determined. Asterisks indicate significant differences between groups (** p
Figure Legend Snippet: Effects of Ljp53 on RGNNV replication and zebrafish IFN1 promoter activity. (a) qRT‐PCR detection of RDRP in LJB cells transfected with pcDNA‐Ljp53 or control plasmid at 48 hr post‐RGNNV infection. (b) qRT‐PCR detection of RDRP in LJB cells treated with DMSO (control) or pifithrin‐α. (c) HEK293T cells were analysed at 48 hr after transfected with the pRL‐TK, pcDNA3.1(+) or pcDNA‐Ljp53 plasmids together with zebrafish IFN1 promoter‐driven reporter plasmid, and the luciferase activity was determined. Asterisks indicate significant differences between groups (** p

Techniques Used: Activity Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Infection, Luciferase

Related Articles

Clone Assay:

Article Title: Intranasal Immunization with a Lentiviral Vector Coding for SARS-CoV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models
Article Snippet: .. Recombinant SCoV-2 proteins Codon-optimized nucleotide fragments encoding a stabilized foldon-trimerized version of the SARS-CoV-2 S ectodomain (a.a. 1 to 1208), the S1 monomer (a.a. 16 to 681) and the RBD subdomain (amino acid 331 to 519) both preceded by a murine IgK leader peptide and followed by an 8xHis Tag were synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Proteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described ( ).

Article Title: SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors.
Article Snippet: .. ELISA tri-S. A codon-optimized nucleotide fragment encoding a stabilized version of the SARS-CoV-2 S ectodomain (amino acid 1 to 1208) followed by a foldon trimerization motif and tags (8xHisTag, StrepTag, and AviTag) was synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Trimeric S (tri-S) glycoproteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described 30.

Article Title: Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)
Article Snippet: .. The full-length zebrafish pxr cDNA was cloned into the expression vector pcDNA 3.1 Zeo (+) (Invitrogen) and transiently transfected into COS-7 cells along with a reporter construct (XREM-TK-Luc) bearing a CYP3A enhancer module (a gift from John Moore), and a transfection control construct (pRL-TK) (Promega). .. Transfected DNA amounts were 100 ng of either zebrafish Pxr or human full length PXR (a gift from Steven Kliewer, used for comparison and as a positive control), 50 ng of XREM-TK-Luc and 3 ng of pRL-TK.

Transfection:

Article Title: Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)
Article Snippet: .. The full-length zebrafish pxr cDNA was cloned into the expression vector pcDNA 3.1 Zeo (+) (Invitrogen) and transiently transfected into COS-7 cells along with a reporter construct (XREM-TK-Luc) bearing a CYP3A enhancer module (a gift from John Moore), and a transfection control construct (pRL-TK) (Promega). .. Transfected DNA amounts were 100 ng of either zebrafish Pxr or human full length PXR (a gift from Steven Kliewer, used for comparison and as a positive control), 50 ng of XREM-TK-Luc and 3 ng of pRL-TK.

Amplification:

Article Title: Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly
Article Snippet: .. For mammalian cellular expression, the same C191 coding region was amplified by PCR and inserted into the NheI and EcoRI sites of the pCDNA 3.1Zeo vector (Invitrogen). .. The prolactin plasmid was obtained from V. Lingappa, University of California-San Francisco.

Sequencing:

Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function
Article Snippet: .. Recombinant Constructs of THY1 For expression of wild type (WT) human Thy-1, the complete cDNA of human THY1 (AAH65559.1) was ligated into the mammalian expression vector pcDNA3.1/Zeo (+) (Invitrogen V860-20) with the Kozak sequence GCCGCC just upstream of the start codon. ..

Article Title: Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization
Article Snippet: .. A clone for mammalian expression of CD81 large extracellular loop (LEL), containing N-terminal tPA signal sequence and C-terminal twin Strep tag, was provided by Joe Grove (University College London). .. CD81-LEL was expressed through transiently transfection in Expi293F cells (ThermoFisher) and purified from supernatant with a Gravity Flow Strep-Tactin Superflow high capacity column (IBA Lifesciences).

Article Title: Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation
Article Snippet: .. The bicistronic plasmid termed pIRF contains the coding sequence for the firefly luciferase under the control of the cytomegalovirus promoter followed by HCV genomic sequence (HCV nucleotides 1–371) and the coding sequences for the Renilla luciferase in the pcDNA3.1 Zeo vector (Invitrogen). ..

Construct:

Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function
Article Snippet: .. Recombinant Constructs of THY1 For expression of wild type (WT) human Thy-1, the complete cDNA of human THY1 (AAH65559.1) was ligated into the mammalian expression vector pcDNA3.1/Zeo (+) (Invitrogen V860-20) with the Kozak sequence GCCGCC just upstream of the start codon. ..

Article Title: Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)
Article Snippet: .. The full-length zebrafish pxr cDNA was cloned into the expression vector pcDNA 3.1 Zeo (+) (Invitrogen) and transiently transfected into COS-7 cells along with a reporter construct (XREM-TK-Luc) bearing a CYP3A enhancer module (a gift from John Moore), and a transfection control construct (pRL-TK) (Promega). .. Transfected DNA amounts were 100 ng of either zebrafish Pxr or human full length PXR (a gift from Steven Kliewer, used for comparison and as a positive control), 50 ng of XREM-TK-Luc and 3 ng of pRL-TK.

Enzyme-linked Immunosorbent Assay:

Article Title: SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors.
Article Snippet: .. ELISA tri-S. A codon-optimized nucleotide fragment encoding a stabilized version of the SARS-CoV-2 S ectodomain (amino acid 1 to 1208) followed by a foldon trimerization motif and tags (8xHisTag, StrepTag, and AviTag) was synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Trimeric S (tri-S) glycoproteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described 30.

Polymerase Chain Reaction:

Article Title: Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly
Article Snippet: .. For mammalian cellular expression, the same C191 coding region was amplified by PCR and inserted into the NheI and EcoRI sites of the pCDNA 3.1Zeo vector (Invitrogen). .. The prolactin plasmid was obtained from V. Lingappa, University of California-San Francisco.

Luciferase:

Article Title: Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation
Article Snippet: .. The bicistronic plasmid termed pIRF contains the coding sequence for the firefly luciferase under the control of the cytomegalovirus promoter followed by HCV genomic sequence (HCV nucleotides 1–371) and the coding sequences for the Renilla luciferase in the pcDNA3.1 Zeo vector (Invitrogen). ..

Expressing:

Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function
Article Snippet: .. Recombinant Constructs of THY1 For expression of wild type (WT) human Thy-1, the complete cDNA of human THY1 (AAH65559.1) was ligated into the mammalian expression vector pcDNA3.1/Zeo (+) (Invitrogen V860-20) with the Kozak sequence GCCGCC just upstream of the start codon. ..

Article Title: Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly
Article Snippet: .. For mammalian cellular expression, the same C191 coding region was amplified by PCR and inserted into the NheI and EcoRI sites of the pCDNA 3.1Zeo vector (Invitrogen). .. The prolactin plasmid was obtained from V. Lingappa, University of California-San Francisco.

Article Title: Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization
Article Snippet: .. A clone for mammalian expression of CD81 large extracellular loop (LEL), containing N-terminal tPA signal sequence and C-terminal twin Strep tag, was provided by Joe Grove (University College London). .. CD81-LEL was expressed through transiently transfection in Expi293F cells (ThermoFisher) and purified from supernatant with a Gravity Flow Strep-Tactin Superflow high capacity column (IBA Lifesciences).

Article Title: Intranasal Immunization with a Lentiviral Vector Coding for SARS-CoV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models
Article Snippet: .. Recombinant SCoV-2 proteins Codon-optimized nucleotide fragments encoding a stabilized foldon-trimerized version of the SARS-CoV-2 S ectodomain (a.a. 1 to 1208), the S1 monomer (a.a. 16 to 681) and the RBD subdomain (amino acid 331 to 519) both preceded by a murine IgK leader peptide and followed by an 8xHis Tag were synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Proteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described ( ).

Article Title: SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors.
Article Snippet: .. ELISA tri-S. A codon-optimized nucleotide fragment encoding a stabilized version of the SARS-CoV-2 S ectodomain (amino acid 1 to 1208) followed by a foldon trimerization motif and tags (8xHisTag, StrepTag, and AviTag) was synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Trimeric S (tri-S) glycoproteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described 30.

Article Title: Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)
Article Snippet: .. The full-length zebrafish pxr cDNA was cloned into the expression vector pcDNA 3.1 Zeo (+) (Invitrogen) and transiently transfected into COS-7 cells along with a reporter construct (XREM-TK-Luc) bearing a CYP3A enhancer module (a gift from John Moore), and a transfection control construct (pRL-TK) (Promega). .. Transfected DNA amounts were 100 ng of either zebrafish Pxr or human full length PXR (a gift from Steven Kliewer, used for comparison and as a positive control), 50 ng of XREM-TK-Luc and 3 ng of pRL-TK.

Strep-tag:

Article Title: Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization
Article Snippet: .. A clone for mammalian expression of CD81 large extracellular loop (LEL), containing N-terminal tPA signal sequence and C-terminal twin Strep tag, was provided by Joe Grove (University College London). .. CD81-LEL was expressed through transiently transfection in Expi293F cells (ThermoFisher) and purified from supernatant with a Gravity Flow Strep-Tactin Superflow high capacity column (IBA Lifesciences).

Chloramphenicol Acetyltransferase Assay:

Article Title: Stable inhibition of mmu-miR-466h-5p improves apoptosis resistance and protein production in CHO cells
Article Snippet: .. No.617379) using EcoRI and NotI and inserted directionally into the pcDNA3.1+ zeo vector (Life Technologies, Cat.No. ..

Recombinant:

Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function
Article Snippet: .. Recombinant Constructs of THY1 For expression of wild type (WT) human Thy-1, the complete cDNA of human THY1 (AAH65559.1) was ligated into the mammalian expression vector pcDNA3.1/Zeo (+) (Invitrogen V860-20) with the Kozak sequence GCCGCC just upstream of the start codon. ..

Article Title: Intranasal Immunization with a Lentiviral Vector Coding for SARS-CoV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models
Article Snippet: .. Recombinant SCoV-2 proteins Codon-optimized nucleotide fragments encoding a stabilized foldon-trimerized version of the SARS-CoV-2 S ectodomain (a.a. 1 to 1208), the S1 monomer (a.a. 16 to 681) and the RBD subdomain (amino acid 331 to 519) both preceded by a murine IgK leader peptide and followed by an 8xHis Tag were synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Proteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described ( ).

Plasmid Preparation:

Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function
Article Snippet: .. Recombinant Constructs of THY1 For expression of wild type (WT) human Thy-1, the complete cDNA of human THY1 (AAH65559.1) was ligated into the mammalian expression vector pcDNA3.1/Zeo (+) (Invitrogen V860-20) with the Kozak sequence GCCGCC just upstream of the start codon. ..

Article Title: Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly
Article Snippet: .. For mammalian cellular expression, the same C191 coding region was amplified by PCR and inserted into the NheI and EcoRI sites of the pCDNA 3.1Zeo vector (Invitrogen). .. The prolactin plasmid was obtained from V. Lingappa, University of California-San Francisco.

Article Title: Intranasal Immunization with a Lentiviral Vector Coding for SARS-CoV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models
Article Snippet: .. Recombinant SCoV-2 proteins Codon-optimized nucleotide fragments encoding a stabilized foldon-trimerized version of the SARS-CoV-2 S ectodomain (a.a. 1 to 1208), the S1 monomer (a.a. 16 to 681) and the RBD subdomain (amino acid 331 to 519) both preceded by a murine IgK leader peptide and followed by an 8xHis Tag were synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Proteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described ( ).

Article Title: Stable inhibition of mmu-miR-466h-5p improves apoptosis resistance and protein production in CHO cells
Article Snippet: .. No.617379) using EcoRI and NotI and inserted directionally into the pcDNA3.1+ zeo vector (Life Technologies, Cat.No. ..

Article Title: Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation
Article Snippet: .. The bicistronic plasmid termed pIRF contains the coding sequence for the firefly luciferase under the control of the cytomegalovirus promoter followed by HCV genomic sequence (HCV nucleotides 1–371) and the coding sequences for the Renilla luciferase in the pcDNA3.1 Zeo vector (Invitrogen). ..

Article Title: SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors.
Article Snippet: .. ELISA tri-S. A codon-optimized nucleotide fragment encoding a stabilized version of the SARS-CoV-2 S ectodomain (amino acid 1 to 1208) followed by a foldon trimerization motif and tags (8xHisTag, StrepTag, and AviTag) was synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). .. Trimeric S (tri-S) glycoproteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described 30.

Article Title: Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)
Article Snippet: .. The full-length zebrafish pxr cDNA was cloned into the expression vector pcDNA 3.1 Zeo (+) (Invitrogen) and transiently transfected into COS-7 cells along with a reporter construct (XREM-TK-Luc) bearing a CYP3A enhancer module (a gift from John Moore), and a transfection control construct (pRL-TK) (Promega). .. Transfected DNA amounts were 100 ng of either zebrafish Pxr or human full length PXR (a gift from Steven Kliewer, used for comparison and as a positive control), 50 ng of XREM-TK-Luc and 3 ng of pRL-TK.

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    Thermo Fisher nf1 nanoluc pcdna expression plasmid
    AMG-510 prevents KRAS-G12C from interacting with <t>NF1</t> and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and <t>NF1-NanoLuc</t> with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.
    Nf1 Nanoluc Pcdna Expression Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna myc vector
    Expression of <t>YES1</t> attenuated the inhibition of cell growth by miR-133 in TNBC cells. A, Cells were transfected with the overexpression plasmid of <t>pcDNA-3Myc-YES1,</t> and the ectopic expression of YES1 was confirmed by Western blot with anti-Myc antibody. B and C, Inhibition of miR-133 in the proliferation of TNBC cells was reversed with YES1 overexpression. D, Restoration of YES1 attenuated the suppressive role of miR-133 in the colony formation of TNBC cells. ns indicates no significance; TNBC, triple-negative breast cancer.
    Pcdna Myc Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna trub1
    <t>TruB1</t> selectively promotes the expression of let-7 family miRNAs (A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. MiRNAs significantly suppressed are enclosed by the red square. (B). Northern blotting for let-7a and RNU6B in HEK293FT cells with TruB1 KD or ctrl (siRNA). (C). Hybridization intensities of (B) were quantified and normalized to ctrl. (D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. (E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. All experiments were performed in triplicate. Error bars show SD; n=3.
    Pcdna Trub1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna kcnq1ot1
    Effect of <t>KCNQ1OT1</t> on β-catenin in mMSCs differentiation. a The mRNA and protein expression levels of β-catenin in mMSCs transfected with <t>pcDNA-KCNQ1OT1,</t> sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. b ALP activity in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. c The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 and siRNA-β-catenin before the addition of PMMA particles. d Osteoblastic differentiation in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. e The protein and mRNA levels of β-catenin in KCNQ1OT1 pulled down pellet using IgG antibody as negative control by RNA pull-down assay. *P
    Pcdna Kcnq1ot1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AMG-510 prevents KRAS-G12C from interacting with NF1 and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and NF1-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.

    Journal: bioRxiv

    Article Title: Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

    doi: 10.1101/845263

    Figure Lengend Snippet: AMG-510 prevents KRAS-G12C from interacting with NF1 and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and NF1-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.

    Article Snippet: Twenty-four hours after seeding, cells were co-transfected with either a constant concentration of 0.1 μg of NF1-NanoLuc pcDNA expression plasmid or CRAF RBD-NanoLuc pcDNA expression plasmid with increasing concentrations of GFP-tagged KRAS (WT or Mutant) with 0.25 μl of Lipofectamine 2000 per well following the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: MTT Assay, Transfection, Bioluminescence Resonance Energy Transfer, Co-Immunoprecipitation Assay, Standard Deviation

    KRAS-G12C binds less well to NF1 and to CRAF. ( A ) HEK293T cells were transfected with increasing amount of KRAS-GFP expression plasmid and a constant concentration of NF1-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with eight biological replicates, and were repeated three times. ( B ) Average BRET ratio from the three experiments at a RAS:NF1 ratio of 4:1. ( C ) HEK293T cells were transfected with either WT-KRAS-GFP, G12V-KRAS-GFP, G12C-KRAS GFP or NF1-FLAG expression plasmids. The NF1 co-IP mixing assay was performed and IP product and input lysate were probed by western blot for NF1, KRAS, and GFP. Results are representative of three independent experiments. ( D ) Average intensity of NF1 co-IP from the three independent experiments. ( E ) Isoelectric focused whole cell lysates from SW48 G12C, SW48 G12V and SW48 WT isogenic cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( F ) Average proportions of KRAS, NRAS, and HRAS bound to RBD and in whole cell lysates from three separate IEF experiments. ( G ) Isoelectric focused whole cell lysates from heterozygous mutant KRAS G12C SW837 and homozygous mutant KRAS G12C SW1463 cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( H ) Average proportions of KRAS, NRAS, and HRAS bound to and in whole cell lysates from three separate IEF experiments. ( I ) HEK293T cells were transfected with increasing concentrations of KRAS-GFP expression plasmids and a constant concentration of CRAF-RBD-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with three experimental replicates, each containing eight biological replicates. ( J ) Average BRET ratio from the three experiments at a RAS:RBD ratio of 2:1. ( K ) SW48 Isogenic cells were harvested and prepared for RAF-RBD pulldown assay, one set was not exposed to γ-S-GTP and the other was incubated in γ-S-GTP for 20 minutes. IP-product and input were probed by western blot for KRAS and GAPDH. Results are representative of three independent experiments. ( L ) Average quantity of KRAS WT, KRAS G12C, and KRAS G12C pulled down by RAF-RBD from three independent experiments. All experiments were performed three times. Treatment groups were analyzed by one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons and P-values are indicated. Error bars in all panels represent standard deviation.

    Journal: bioRxiv

    Article Title: Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

    doi: 10.1101/845263

    Figure Lengend Snippet: KRAS-G12C binds less well to NF1 and to CRAF. ( A ) HEK293T cells were transfected with increasing amount of KRAS-GFP expression plasmid and a constant concentration of NF1-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with eight biological replicates, and were repeated three times. ( B ) Average BRET ratio from the three experiments at a RAS:NF1 ratio of 4:1. ( C ) HEK293T cells were transfected with either WT-KRAS-GFP, G12V-KRAS-GFP, G12C-KRAS GFP or NF1-FLAG expression plasmids. The NF1 co-IP mixing assay was performed and IP product and input lysate were probed by western blot for NF1, KRAS, and GFP. Results are representative of three independent experiments. ( D ) Average intensity of NF1 co-IP from the three independent experiments. ( E ) Isoelectric focused whole cell lysates from SW48 G12C, SW48 G12V and SW48 WT isogenic cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( F ) Average proportions of KRAS, NRAS, and HRAS bound to RBD and in whole cell lysates from three separate IEF experiments. ( G ) Isoelectric focused whole cell lysates from heterozygous mutant KRAS G12C SW837 and homozygous mutant KRAS G12C SW1463 cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( H ) Average proportions of KRAS, NRAS, and HRAS bound to and in whole cell lysates from three separate IEF experiments. ( I ) HEK293T cells were transfected with increasing concentrations of KRAS-GFP expression plasmids and a constant concentration of CRAF-RBD-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with three experimental replicates, each containing eight biological replicates. ( J ) Average BRET ratio from the three experiments at a RAS:RBD ratio of 2:1. ( K ) SW48 Isogenic cells were harvested and prepared for RAF-RBD pulldown assay, one set was not exposed to γ-S-GTP and the other was incubated in γ-S-GTP for 20 minutes. IP-product and input were probed by western blot for KRAS and GAPDH. Results are representative of three independent experiments. ( L ) Average quantity of KRAS WT, KRAS G12C, and KRAS G12C pulled down by RAF-RBD from three independent experiments. All experiments were performed three times. Treatment groups were analyzed by one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons and P-values are indicated. Error bars in all panels represent standard deviation.

    Article Snippet: Twenty-four hours after seeding, cells were co-transfected with either a constant concentration of 0.1 μg of NF1-NanoLuc pcDNA expression plasmid or CRAF RBD-NanoLuc pcDNA expression plasmid with increasing concentrations of GFP-tagged KRAS (WT or Mutant) with 0.25 μl of Lipofectamine 2000 per well following the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: Transfection, Expressing, Plasmid Preparation, Concentration Assay, Bioluminescence Resonance Energy Transfer, Co-Immunoprecipitation Assay, Western Blot, Electrofocusing, Mutagenesis, Incubation, Standard Deviation

    Expression of YES1 attenuated the inhibition of cell growth by miR-133 in TNBC cells. A, Cells were transfected with the overexpression plasmid of pcDNA-3Myc-YES1, and the ectopic expression of YES1 was confirmed by Western blot with anti-Myc antibody. B and C, Inhibition of miR-133 in the proliferation of TNBC cells was reversed with YES1 overexpression. D, Restoration of YES1 attenuated the suppressive role of miR-133 in the colony formation of TNBC cells. ns indicates no significance; TNBC, triple-negative breast cancer.

    Journal: Technology in Cancer Research & Treatment

    Article Title: MiR-133 Targets YES1 and Inhibits the Growth of Triple-Negative Breast Cancer Cells

    doi: 10.1177/1533033820927011

    Figure Lengend Snippet: Expression of YES1 attenuated the inhibition of cell growth by miR-133 in TNBC cells. A, Cells were transfected with the overexpression plasmid of pcDNA-3Myc-YES1, and the ectopic expression of YES1 was confirmed by Western blot with anti-Myc antibody. B and C, Inhibition of miR-133 in the proliferation of TNBC cells was reversed with YES1 overexpression. D, Restoration of YES1 attenuated the suppressive role of miR-133 in the colony formation of TNBC cells. ns indicates no significance; TNBC, triple-negative breast cancer.

    Article Snippet: The expression plasmid of pcDNA-Myc-YES1 was constructed by amplifying the complementary DNA (cDNA) fragment of YES1 via polymerase chain reaction (PCR) and inserted into the backbone of pcDNA-Myc vector.

    Techniques: Expressing, Inhibition, Transfection, Over Expression, Plasmid Preparation, Western Blot

    TruB1 selectively promotes the expression of let-7 family miRNAs (A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. MiRNAs significantly suppressed are enclosed by the red square. (B). Northern blotting for let-7a and RNU6B in HEK293FT cells with TruB1 KD or ctrl (siRNA). (C). Hybridization intensities of (B) were quantified and normalized to ctrl. (D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. (E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 selectively promotes the expression of let-7 family miRNAs (A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. MiRNAs significantly suppressed are enclosed by the red square. (B). Northern blotting for let-7a and RNU6B in HEK293FT cells with TruB1 KD or ctrl (siRNA). (C). Hybridization intensities of (B) were quantified and normalized to ctrl. (D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. (E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Expressing, Northern Blot, Hybridization, Quantitative RT-PCR

    TruB1 promotes the microprocessing of primary let-7 and enhances binding of the microprocesser to primary let-7 (A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). (A). RI intensities of (B) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. (C). RIP assay of pri-let-7a1 and DGCR-8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR-8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). (D). RIP assay of pri-let-7a1 and TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 promotes the microprocessing of primary let-7 and enhances binding of the microprocesser to primary let-7 (A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). (A). RI intensities of (B) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. (C). RIP assay of pri-let-7a1 and DGCR-8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR-8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). (D). RIP assay of pri-let-7a1 and TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Binding Assay, In Vitro, Transfection, Over Expression, Western Blot, Quantitative RT-PCR

    TruB1 promotes let-7 processing independently of its enzymatic activity. (A). Amino acid sequences encoding for the enzyme activity and RNA binding ability in E.coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom). (B). In vitro enzyme activity assay. 32 p-UTP-labelled tRNA phe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent UTP, and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate. (C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. (D). Northern blotting for let-7a and RNU6B in HEK-293 cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment. (E). Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). (F). Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32 p-UTP-labelled tRNA phe , pri-let-7a1 or pri-miR10a were treated with recombinant TruB1. Upper bands represent UTP, lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate. (G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNA phe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows. All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 promotes let-7 processing independently of its enzymatic activity. (A). Amino acid sequences encoding for the enzyme activity and RNA binding ability in E.coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom). (B). In vitro enzyme activity assay. 32 p-UTP-labelled tRNA phe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent UTP, and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate. (C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. (D). Northern blotting for let-7a and RNU6B in HEK-293 cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment. (E). Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). (F). Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32 p-UTP-labelled tRNA phe , pri-let-7a1 or pri-miR10a were treated with recombinant TruB1. Upper bands represent UTP, lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate. (G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNA phe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows. All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Activity Assay, RNA Binding Assay, Mutagenesis, In Vitro, Enzyme Activity Assay, Recombinant, Thin Layer Chromatography, Expressing, Infection, Quantitative RT-PCR, Northern Blot, Hybridization, Purification, Sequencing

    TruB1 binds to tRNA and primary-let-7 (A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. (B). EMSA of 32 p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes. (C). Design of Flag-labelled TruB1 knock-in (KI) cells. HiBIT and 3 x Flag sequences were inserted into the N-terminus of the TruB1 gene in HEK-293FT cells. (D) Sequencing clusters obtained from HITS-CLIP experiment. Left are for let-7, and right for tRNAs. (E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array. (F). The proportions of different types of mapped transcripts from the HITS-CLIP experiment. (G). Sequence motifs from reads mapped to tRNAs from the HITS-CLIP experiment. (H). TruB-modified site in tRNAs. Location of pseudouridine is colored in red. (I). Sequence of the terminal loop in pri-let-7a1. Lin28B binding site is colored in red.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 binds to tRNA and primary-let-7 (A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. (B). EMSA of 32 p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes. (C). Design of Flag-labelled TruB1 knock-in (KI) cells. HiBIT and 3 x Flag sequences were inserted into the N-terminus of the TruB1 gene in HEK-293FT cells. (D) Sequencing clusters obtained from HITS-CLIP experiment. Left are for let-7, and right for tRNAs. (E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array. (F). The proportions of different types of mapped transcripts from the HITS-CLIP experiment. (G). Sequence motifs from reads mapped to tRNAs from the HITS-CLIP experiment. (H). TruB-modified site in tRNAs. Location of pseudouridine is colored in red. (I). Sequence of the terminal loop in pri-let-7a1. Lin28B binding site is colored in red.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Quantitative RT-PCR, Infection, Expressing, Recombinant, Knock-In, Sequencing, Cross-linking Immunoprecipitation, Modification, Binding Assay

    TruB1 suppresses cell growth by regulating let-7 (A). Design of luciferase reporter vector comprising the let-7 target region of KRAS mRNA (KRAS reporter). (B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. (C). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2’-O-methylated antisense inhibitor). (D). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. (E). Schematic model for TruB1-dependent induction of let-7. All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 suppresses cell growth by regulating let-7 (A). Design of luciferase reporter vector comprising the let-7 target region of KRAS mRNA (KRAS reporter). (B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. (C). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2’-O-methylated antisense inhibitor). (D). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. (E). Schematic model for TruB1-dependent induction of let-7. All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Luciferase, Plasmid Preparation, Activity Assay, Infection, Expressing, Quantitative RT-PCR, Methylation, Glo Assay

    Effect of KCNQ1OT1 on β-catenin in mMSCs differentiation. a The mRNA and protein expression levels of β-catenin in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. b ALP activity in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. c The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 and siRNA-β-catenin before the addition of PMMA particles. d Osteoblastic differentiation in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. e The protein and mRNA levels of β-catenin in KCNQ1OT1 pulled down pellet using IgG antibody as negative control by RNA pull-down assay. *P

    Journal: Cell & Bioscience

    Article Title: LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/β-catenin activation

    doi: 10.1186/s13578-018-0216-4

    Figure Lengend Snippet: Effect of KCNQ1OT1 on β-catenin in mMSCs differentiation. a The mRNA and protein expression levels of β-catenin in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. b ALP activity in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. c The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 and siRNA-β-catenin before the addition of PMMA particles. d Osteoblastic differentiation in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. e The protein and mRNA levels of β-catenin in KCNQ1OT1 pulled down pellet using IgG antibody as negative control by RNA pull-down assay. *P

    Article Snippet: Briefly, mMSCs were seeded in antibiotics-free medium in six-well plates at a density of 5 × 105 /mL followed by transfection with pcDNA-KCNQ1OT1 or co-transfection with pcDNA-KCNQ1OT1 and siRNA-β-catenin (Thermo Fisher Scientific, Waltham, MA, USA) along with corresponding negative control (pcDNA) using Lipofectamine 2000 (Invitrogen) and then incubated at 37 °C with 5% CO2 for 24 h. MMSCs were used to detect the protein levels of β-catenin using western blot, ALP activity and the mRNA expression of Runx2, Osterix and OCN using qRT-PCR as well as mMSCs differentiation using ARS staining.

    Techniques: Expressing, Transfection, ALP Assay, Activity Assay, Negative Control, Pull Down Assay

    Effect of KCNQ1OT1 in mMSCs stimulated with PMMA particles. a ALP activity in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. b The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. c Osteoblastic differentiation in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles assessed by ARS staining. d The relative expression levels of KCNQ1OT1 in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA. *P

    Journal: Cell & Bioscience

    Article Title: LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/β-catenin activation

    doi: 10.1186/s13578-018-0216-4

    Figure Lengend Snippet: Effect of KCNQ1OT1 in mMSCs stimulated with PMMA particles. a ALP activity in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. b The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. c Osteoblastic differentiation in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles assessed by ARS staining. d The relative expression levels of KCNQ1OT1 in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA. *P

    Article Snippet: Briefly, mMSCs were seeded in antibiotics-free medium in six-well plates at a density of 5 × 105 /mL followed by transfection with pcDNA-KCNQ1OT1 or co-transfection with pcDNA-KCNQ1OT1 and siRNA-β-catenin (Thermo Fisher Scientific, Waltham, MA, USA) along with corresponding negative control (pcDNA) using Lipofectamine 2000 (Invitrogen) and then incubated at 37 °C with 5% CO2 for 24 h. MMSCs were used to detect the protein levels of β-catenin using western blot, ALP activity and the mRNA expression of Runx2, Osterix and OCN using qRT-PCR as well as mMSCs differentiation using ARS staining.

    Techniques: ALP Assay, Activity Assay, Transfection, Expressing, Staining