Structured Review

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RT-qPCR validation of the expression levels of randomly selected DE <t>circRNAs</t> (listed in Table I ) in exosomes from patients with GC and HC. <t>GAPDH</t> was used as internal standard. Expression level of each circRNA in sample HC190128-1 was set as 1. Data were expressed as the means ± standard deviation of the mean (n=5). **P
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1) Product Images from "Circular RNA profiling in plasma exosomes from patients with gastric cancer"

Article Title: Circular RNA profiling in plasma exosomes from patients with gastric cancer

Journal: Oncology Letters

doi: 10.3892/ol.2020.11800

RT-qPCR validation of the expression levels of randomly selected DE circRNAs (listed in Table I ) in exosomes from patients with GC and HC. GAPDH was used as internal standard. Expression level of each circRNA in sample HC190128-1 was set as 1. Data were expressed as the means ± standard deviation of the mean (n=5). **P
Figure Legend Snippet: RT-qPCR validation of the expression levels of randomly selected DE circRNAs (listed in Table I ) in exosomes from patients with GC and HC. GAPDH was used as internal standard. Expression level of each circRNA in sample HC190128-1 was set as 1. Data were expressed as the means ± standard deviation of the mean (n=5). **P

Techniques Used: Quantitative RT-PCR, Expressing, Standard Deviation

2) Product Images from "Exosomal secretion of α-synuclein as protective mechanism after upstream blockage of macroautophagy"

Article Title: Exosomal secretion of α-synuclein as protective mechanism after upstream blockage of macroautophagy

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0816-2

ATG5 siRNA protects against α-Syn-induced toxicity. The effect of ATG5 siRNA on ATG5 expression, autophagosome formation, and α-Syn-induced toxicity was analyzed. a qRT-PCR for ATG5 in cells overexpressing α-Syn, in cells overexpressing α-Syn and transfected with control siRNA against GAPDH , cells overexpressing the control protein GFP, and cells overexpressing α-Syn and transfected with ATG5 siRNA, showing the silencing efficacy of ATG5 siRNA on the mRNA level. The dashed line shows ATG5 levels in naïve (untransduced, untransfected) cells as reference. b Representative Western blot for ATG5 protein in naïve control cells (Ctrl) and cells in the conditions reported in a . β-actin was used as loading control. c Quantification of ATG5 protein, normalized to β-actin, from Western blots as shown in b , showing the silencing efficacy of ATG5 siRNA on the protein level. d Representative Western blot for the autophagosome marker LC3B in naïve cells (Ctrl), and cells transfected with ATG5 siRNA, with or without chloroquine (Chl) treatment to block autophagosome–lysosome fusion. e Quantification of LC3B-II protein, normalized to β-actin, from individual Western blots as shown in d , showing that Chl increases LC3B-II in naïve cells more than in ATG5 siRNA treated cells. f Quantification of lactate dehydrogenase (LDH) released into the culture medium as measure for toxicity. Data are expressed as percentage of α-Syn. ATG5 silencing significantly reduced α-Syn-induced toxicity. Data in a , c , e , f are mean ± standard error from n ≥ 3 biological replicates. * p
Figure Legend Snippet: ATG5 siRNA protects against α-Syn-induced toxicity. The effect of ATG5 siRNA on ATG5 expression, autophagosome formation, and α-Syn-induced toxicity was analyzed. a qRT-PCR for ATG5 in cells overexpressing α-Syn, in cells overexpressing α-Syn and transfected with control siRNA against GAPDH , cells overexpressing the control protein GFP, and cells overexpressing α-Syn and transfected with ATG5 siRNA, showing the silencing efficacy of ATG5 siRNA on the mRNA level. The dashed line shows ATG5 levels in naïve (untransduced, untransfected) cells as reference. b Representative Western blot for ATG5 protein in naïve control cells (Ctrl) and cells in the conditions reported in a . β-actin was used as loading control. c Quantification of ATG5 protein, normalized to β-actin, from Western blots as shown in b , showing the silencing efficacy of ATG5 siRNA on the protein level. d Representative Western blot for the autophagosome marker LC3B in naïve cells (Ctrl), and cells transfected with ATG5 siRNA, with or without chloroquine (Chl) treatment to block autophagosome–lysosome fusion. e Quantification of LC3B-II protein, normalized to β-actin, from individual Western blots as shown in d , showing that Chl increases LC3B-II in naïve cells more than in ATG5 siRNA treated cells. f Quantification of lactate dehydrogenase (LDH) released into the culture medium as measure for toxicity. Data are expressed as percentage of α-Syn. ATG5 silencing significantly reduced α-Syn-induced toxicity. Data in a , c , e , f are mean ± standard error from n ≥ 3 biological replicates. * p

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Marker, Blocking Assay

3) Product Images from "Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells"

Article Title: Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells

Journal: Journal of Molecular Signaling

doi: 10.1186/1750-2187-5-9

Effect of G17 on Snail expression in gastric cancer cells . (A) AGSE cells were treated as in 1A or (B) 1B above and subjected to Western Blot analysis utilizing antibodies against Snail and GAPDH (as control). (C) Western Blot analysis of cell extracts with the indicated antibodies, treated with 100 nM G17 for 1 hour, following an overnight pretreatment with 100 nM YM 022. (D) Subconfluent AGSE cells were transiently transfected with Snail-luciferase vector (Snail-luc) along with β-Gal vector (for normalization of transfection). Forty-eight hours after transfection, cells were treated overnight in the presence (+) or absence (-) of 100 nM G17, and luciferase and β-Gal assays were performed. The RLU/β-Gal values were represented as percent control, considering the untreated samples as 100%. Each transfection was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments.
Figure Legend Snippet: Effect of G17 on Snail expression in gastric cancer cells . (A) AGSE cells were treated as in 1A or (B) 1B above and subjected to Western Blot analysis utilizing antibodies against Snail and GAPDH (as control). (C) Western Blot analysis of cell extracts with the indicated antibodies, treated with 100 nM G17 for 1 hour, following an overnight pretreatment with 100 nM YM 022. (D) Subconfluent AGSE cells were transiently transfected with Snail-luciferase vector (Snail-luc) along with β-Gal vector (for normalization of transfection). Forty-eight hours after transfection, cells were treated overnight in the presence (+) or absence (-) of 100 nM G17, and luciferase and β-Gal assays were performed. The RLU/β-Gal values were represented as percent control, considering the untreated samples as 100%. Each transfection was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments.

Techniques Used: Expressing, Western Blot, Transfection, Luciferase, Plasmid Preparation

Effect of GSK3β inhibition on G17-induced Snail expression and β-catenin nuclear translocation . (A) AGSE cells were treated with (+) or without (-) 100 nM G17, following an overnight pretreatment with either none (lanes 1, 2), 5 μM (lanes 3, 4) or 10 μM (lanes 5, 6) AR-A014418. Western Blot analysis was then performed with the antibodies indicated. (B) Luciferase (with Snail-luc) and β-Gal assays were performed as in 2D following a 1 hour pretreatment with AR-A014418. (C) AGSE cells were co-transfected with Snail-luc and β-Gal vectors along with either Empty vector (lanes 1, 2), GSK3β-S9A mutant vector (lanes 3, 4) or GSK3β-K/A mutant vector (lanes 5, 6). Luciferase and β-Gal assays were performed after G17 treatment as in 2D. Each transfection (3B, 3C) was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments. (D) Upper Panel : Confluent AGSE cells were treated with G17 for 8 hours after an overnight pretreatment with none (lanes 1, 2), or AR-A014418 (lanes 3, 4) or SP600125 (lanes 5, 6). At the end of treatment, nuclear protein was isolated and subjected to Western Blot analysis with antibodies against β-catenin, GAPDH (cytoplasmic protein) or Lamin A/C (nuclear protein). Lower Panel : Cells were pretreated as in the upper panel, followed by 1 hour G17 treatment and Western Blot analysis.
Figure Legend Snippet: Effect of GSK3β inhibition on G17-induced Snail expression and β-catenin nuclear translocation . (A) AGSE cells were treated with (+) or without (-) 100 nM G17, following an overnight pretreatment with either none (lanes 1, 2), 5 μM (lanes 3, 4) or 10 μM (lanes 5, 6) AR-A014418. Western Blot analysis was then performed with the antibodies indicated. (B) Luciferase (with Snail-luc) and β-Gal assays were performed as in 2D following a 1 hour pretreatment with AR-A014418. (C) AGSE cells were co-transfected with Snail-luc and β-Gal vectors along with either Empty vector (lanes 1, 2), GSK3β-S9A mutant vector (lanes 3, 4) or GSK3β-K/A mutant vector (lanes 5, 6). Luciferase and β-Gal assays were performed after G17 treatment as in 2D. Each transfection (3B, 3C) was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments. (D) Upper Panel : Confluent AGSE cells were treated with G17 for 8 hours after an overnight pretreatment with none (lanes 1, 2), or AR-A014418 (lanes 3, 4) or SP600125 (lanes 5, 6). At the end of treatment, nuclear protein was isolated and subjected to Western Blot analysis with antibodies against β-catenin, GAPDH (cytoplasmic protein) or Lamin A/C (nuclear protein). Lower Panel : Cells were pretreated as in the upper panel, followed by 1 hour G17 treatment and Western Blot analysis.

Techniques Used: Inhibition, Expressing, Translocation Assay, Western Blot, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Isolation

Effect of overexpression of GSK3β on G17-induced migration . (A) . Subconfluent AGSE cells were transiently transfected with Empty Vector, GSK3β-WT, GSK3β-KA mutant or GSK3β-S9A mutant vectors. The cells were wounded linearly 48 hours post-transfection and, after an overnight recovery following wounding, they were treated with G17 and pictures obtained at the indicated times. (B) AGSE cells were transfected as in 4A followed by G17 treatment and wound healing assay. The distance of migration of the wounded edges for each time point were measured at several places and the average distance was represented by bar diagrams as
Figure Legend Snippet: Effect of overexpression of GSK3β on G17-induced migration . (A) . Subconfluent AGSE cells were transiently transfected with Empty Vector, GSK3β-WT, GSK3β-KA mutant or GSK3β-S9A mutant vectors. The cells were wounded linearly 48 hours post-transfection and, after an overnight recovery following wounding, they were treated with G17 and pictures obtained at the indicated times. (B) AGSE cells were transfected as in 4A followed by G17 treatment and wound healing assay. The distance of migration of the wounded edges for each time point were measured at several places and the average distance was represented by bar diagrams as "Average Gap". (C) AGSE cells transfected in A and treated with G17 were analyzed for protein expression. Western Blot analysis was performed with an HA.11 antibody to detect ectopic HA-tagged GSK3β proteins and with β-catenin and GAPDH antibodies to detect the corresponding endogenous proteins.

Techniques Used: Over Expression, Migration, Transfection, Plasmid Preparation, Mutagenesis, Wound Healing Assay, Expressing, Western Blot

4) Product Images from "Nuclear Factor-κB Increases Intracellular Calcium by Upregulation of Na+-Ca2+ Exchanger 1 in Cerulein-Induced Acute Pancreatitis"

Article Title: Nuclear Factor-κB Increases Intracellular Calcium by Upregulation of Na+-Ca2+ Exchanger 1 in Cerulein-Induced Acute Pancreatitis

Journal: Pancreas

doi: 10.1097/MPA.0000000000001465

Potential NF-κB binding sites in the NCX1 promoter region and cerulein-induced increase in NF-κB expression. A, Structure of NCX1 gene. B, Expression of pP65 protein measured by Western blot after 100 nM cerulein treatment in AR42J cells. GAPDH was used as the internal control. C, Expression of p65 protein (brown) in cerulein-induced AP rat model was measured by immunohistochemical staining. n = 5 rats per group in this experiment.
Figure Legend Snippet: Potential NF-κB binding sites in the NCX1 promoter region and cerulein-induced increase in NF-κB expression. A, Structure of NCX1 gene. B, Expression of pP65 protein measured by Western blot after 100 nM cerulein treatment in AR42J cells. GAPDH was used as the internal control. C, Expression of p65 protein (brown) in cerulein-induced AP rat model was measured by immunohistochemical staining. n = 5 rats per group in this experiment.

Techniques Used: Binding Assay, Expressing, Western Blot, Immunohistochemistry, Staining

5) Product Images from "NKX2-5 Regulates the Expression of ?-Catenin and GATA4 in Ventricular Myocytes"

Article Title: NKX2-5 Regulates the Expression of ?-Catenin and GATA4 in Ventricular Myocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005698

The level of Gata4 and β-catenin RNA in Nkx2-5 +/- heterozygous and wild type hearts. Quantitative RT-PCR analysis of Nkx2-5, Gata4, and β-catenin in the hearts of 11.5 dpc wild type (WT) and Nkx2-5 +/- (HET) embryos revealed augmentation of both Gata4 and β-catenin and reduction in Nkx2-5 RNA levels. The values (mean±SEM) for each gene were normalized against GAPDH. N = 9 for WT and N = 11 for HET hearts.
Figure Legend Snippet: The level of Gata4 and β-catenin RNA in Nkx2-5 +/- heterozygous and wild type hearts. Quantitative RT-PCR analysis of Nkx2-5, Gata4, and β-catenin in the hearts of 11.5 dpc wild type (WT) and Nkx2-5 +/- (HET) embryos revealed augmentation of both Gata4 and β-catenin and reduction in Nkx2-5 RNA levels. The values (mean±SEM) for each gene were normalized against GAPDH. N = 9 for WT and N = 11 for HET hearts.

Techniques Used: Quantitative RT-PCR

6) Product Images from "The effects of repeated Toll-like receptors 2 and 4 stimulation in COPD alveolar macrophages"

Article Title: The effects of repeated Toll-like receptors 2 and 4 stimulation in COPD alveolar macrophages

Journal: International Journal of Chronic Obstructive Pulmonary Disease

doi: 10.2147/COPD.S97071

The effects of LPS, Pam3CSK4, and UPLPS on TLR2 and TLR4 expression. COPD alveolar macrophages were left untreated or stimulated with LPS (1 μg/mL; n=6) ( A ), Pam3CSK4 (0.1 μg/mL) ( B ), or UPLPS (0.1 μg/mL; n=5 different donors) ( C ) for 4, 6, 24, and 48 hours. TLR2 and TLR4 gene expression was measured by qPCR and fold change was normalized to GAPDH . Paired t -tests were carried out to compare fold induction to unstimulated time-matched controls. *,**Indicates significantly increased above unstimulated time-matched control ( p
Figure Legend Snippet: The effects of LPS, Pam3CSK4, and UPLPS on TLR2 and TLR4 expression. COPD alveolar macrophages were left untreated or stimulated with LPS (1 μg/mL; n=6) ( A ), Pam3CSK4 (0.1 μg/mL) ( B ), or UPLPS (0.1 μg/mL; n=5 different donors) ( C ) for 4, 6, 24, and 48 hours. TLR2 and TLR4 gene expression was measured by qPCR and fold change was normalized to GAPDH . Paired t -tests were carried out to compare fold induction to unstimulated time-matched controls. *,**Indicates significantly increased above unstimulated time-matched control ( p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

LPS tolerance results in the differential desensitization of TNFα and CXCL8 gene expression. COPD alveolar macrophages were cultured in media or LPS (1 μg/mL) for 24 hours before washing and restimulating with LPS (1 μg/mL) for further 4 or 24 hours as indicated. Cells were harvested in TRIzol and TNFα ( A ) and CXCL8 ( B ) gene expression was measured by qPCR and normalized to GAPDH levels (n=6). Data show mean ± SEM fold induction compared to non-stimulated time-matched controls. Paired t -tests were carried out to compare each condition to ML. ** p
Figure Legend Snippet: LPS tolerance results in the differential desensitization of TNFα and CXCL8 gene expression. COPD alveolar macrophages were cultured in media or LPS (1 μg/mL) for 24 hours before washing and restimulating with LPS (1 μg/mL) for further 4 or 24 hours as indicated. Cells were harvested in TRIzol and TNFα ( A ) and CXCL8 ( B ) gene expression was measured by qPCR and normalized to GAPDH levels (n=6). Data show mean ± SEM fold induction compared to non-stimulated time-matched controls. Paired t -tests were carried out to compare each condition to ML. ** p

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

7) Product Images from "Double Negative (CD3+4−8−) TCR?? Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes"

Article Title: Double Negative (CD3+4−8−) TCR?? Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011427

Cytokines and transcriptional events in NOD DNCD3 splenic cells. The DN splenic cells and mature, splenic CD4 + T-cells from a pool (n = 10) of 14 day-old female mice were negatively-selected on mouse CD4/CD8 tandem columns at 95% purity according to FACS analysis. Cells (1×10 6 ) were stimulated or not for 1, 3, or 5 days with a mixture of soluble CD3/CD28 Abs (2.5 µg each), or with CD3/CD28 mAbs in Th1 or Th2 conditioned medium, as described. The one-day stimulation assay was used to measure the IL-2 secretion in the culture medium. Panel A , DN splenic cell cultures stimulated under Th1 ( left panel ) and Th2 ( right panel ) conditions, then stained 3 days later with CD4 Ab-APC and CD8 Ab-PerCP, and analyzed by FACS. Shown is the mean values (%) of CD4 − 8 + cytotoxic, CD4 + 8 + double positive, and CD4 + 8 − single positive T-cells from duplicate cultures ± SD. Panel B , mean values of cytokines measured in duplicate wells by ELISA in the same DNCD3 cell cultures (pg/mL ± SD) described in panel A . Panel C , mRNA extracted 3 days after stimulation of aliquot DNCD3 cell cultures like in panels A B , and amplified in RT-PCR using specific primers for IL-10. In the upper panel, lane 1, molecular markers; lane 2, non-stimulated cells after 1 day of culturing in medium alone; lane 3, CD3/CD28 stimulated cultures; lane 4, cell cultures stimulated in Th1 conditioned medium; and lane 5, cell cultures stimulated in Th2 conditioned medium. Lower panel shows the corresponding GAPDH mRNA amplicons for the corresponding samples analyzed in the upper panel for IL-10 mRNA expression. Panel D , mRNA extracted 3 days after stimulation from aliquot DNCD3 cell cultures like in panels A B , and amplified in RT-PCR using specific primers for the major Th1 and Th2 transcription factors ( lane 1 , molecular markers, lane 2 , STAT6; lane 3 , GATA-3; lane 4 , cMAF; lane 5 , NF-ATc; lane 6 , STAT4, and lane 7 , T-bet). Lower panel shows the corresponding GAPDH mRNA amplicons for each transcription factor. Each panel shows one of two representative experiments.
Figure Legend Snippet: Cytokines and transcriptional events in NOD DNCD3 splenic cells. The DN splenic cells and mature, splenic CD4 + T-cells from a pool (n = 10) of 14 day-old female mice were negatively-selected on mouse CD4/CD8 tandem columns at 95% purity according to FACS analysis. Cells (1×10 6 ) were stimulated or not for 1, 3, or 5 days with a mixture of soluble CD3/CD28 Abs (2.5 µg each), or with CD3/CD28 mAbs in Th1 or Th2 conditioned medium, as described. The one-day stimulation assay was used to measure the IL-2 secretion in the culture medium. Panel A , DN splenic cell cultures stimulated under Th1 ( left panel ) and Th2 ( right panel ) conditions, then stained 3 days later with CD4 Ab-APC and CD8 Ab-PerCP, and analyzed by FACS. Shown is the mean values (%) of CD4 − 8 + cytotoxic, CD4 + 8 + double positive, and CD4 + 8 − single positive T-cells from duplicate cultures ± SD. Panel B , mean values of cytokines measured in duplicate wells by ELISA in the same DNCD3 cell cultures (pg/mL ± SD) described in panel A . Panel C , mRNA extracted 3 days after stimulation of aliquot DNCD3 cell cultures like in panels A B , and amplified in RT-PCR using specific primers for IL-10. In the upper panel, lane 1, molecular markers; lane 2, non-stimulated cells after 1 day of culturing in medium alone; lane 3, CD3/CD28 stimulated cultures; lane 4, cell cultures stimulated in Th1 conditioned medium; and lane 5, cell cultures stimulated in Th2 conditioned medium. Lower panel shows the corresponding GAPDH mRNA amplicons for the corresponding samples analyzed in the upper panel for IL-10 mRNA expression. Panel D , mRNA extracted 3 days after stimulation from aliquot DNCD3 cell cultures like in panels A B , and amplified in RT-PCR using specific primers for the major Th1 and Th2 transcription factors ( lane 1 , molecular markers, lane 2 , STAT6; lane 3 , GATA-3; lane 4 , cMAF; lane 5 , NF-ATc; lane 6 , STAT4, and lane 7 , T-bet). Lower panel shows the corresponding GAPDH mRNA amplicons for each transcription factor. Each panel shows one of two representative experiments.

Techniques Used: Mouse Assay, FACS, Staining, Enzyme-linked Immunosorbent Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing

8) Product Images from "NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizureDecision letterAuthor response"

Article Title: NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizureDecision letterAuthor response

Journal: eLife

doi: 10.7554/eLife.00178.031

Dlg3 mRNA isoforms in Nova KO brain. Northern blot analysis of Dlg3 mRNA in WT and Nova DKO brain. ( A ) Gapdh probe was used as a normalizing control. Panel to right: Quantitation of relative RNA intensity (WT/DKO) was plotted as a relative ratio of Dlg3 mRNA/GAPDH in WT/DKO; error bars represent standard deviation (p
Figure Legend Snippet: Dlg3 mRNA isoforms in Nova KO brain. Northern blot analysis of Dlg3 mRNA in WT and Nova DKO brain. ( A ) Gapdh probe was used as a normalizing control. Panel to right: Quantitation of relative RNA intensity (WT/DKO) was plotted as a relative ratio of Dlg3 mRNA/GAPDH in WT/DKO; error bars represent standard deviation (p

Techniques Used: Northern Blot, Quantitation Assay, Standard Deviation

9) Product Images from "HTLV-1 Tax transgenic mice develop spontaneous osteolytic bone metastases prevented by osteoclast inhibition"

Article Title: HTLV-1 Tax transgenic mice develop spontaneous osteolytic bone metastases prevented by osteoclast inhibition

Journal: Blood

doi: 10.1182/blood-2005-04-1730

Isolated Tax tumor cells express transcripts for osteoclast-activating factors. (A) Single-cell suspensions of 3 Tax + tumors were sorted by FACS according to FcγR II/III (CD16/CD32) FITC-negative, -low, and -high intensity. Expression of the Tax gene was detected in CD16/CD32-high but not in -negative cell populations by RT-PCR. GAPDH was used as an internal control. (B) Cytospin and Wright-Giemsa-Grünwald staining was performed on sorted cells. CD16/CD32 FITC-negative cells were normal lymphocytes/stromal cells. CD16/CD32 FITC-high cells were tumor cells, whereas CD16/CD32 FITC-low cells were neutrophils (× 20). (C) CD16/CD32 FITC-high cells were immunofluorescently stained with Tax antibody (green, × 20). (D) RT-PCR of selected osteoclast-activating factors in CD16/CD32 FITC-negative (N) and FITC-high (H) cells was performed on the mRNA of purified cell types isolated from whole tumors. RANKL was expressed in CD16/CD32 lymphocytes/stromal cells; IL-6, IL-1α, and M-CSF were expressed in Tax tumor cells; IL-1β, TGFβ, PTHrP, and TNFα were expressed in both CD16/CD32-negative and -high cell populations.
Figure Legend Snippet: Isolated Tax tumor cells express transcripts for osteoclast-activating factors. (A) Single-cell suspensions of 3 Tax + tumors were sorted by FACS according to FcγR II/III (CD16/CD32) FITC-negative, -low, and -high intensity. Expression of the Tax gene was detected in CD16/CD32-high but not in -negative cell populations by RT-PCR. GAPDH was used as an internal control. (B) Cytospin and Wright-Giemsa-Grünwald staining was performed on sorted cells. CD16/CD32 FITC-negative cells were normal lymphocytes/stromal cells. CD16/CD32 FITC-high cells were tumor cells, whereas CD16/CD32 FITC-low cells were neutrophils (× 20). (C) CD16/CD32 FITC-high cells were immunofluorescently stained with Tax antibody (green, × 20). (D) RT-PCR of selected osteoclast-activating factors in CD16/CD32 FITC-negative (N) and FITC-high (H) cells was performed on the mRNA of purified cell types isolated from whole tumors. RANKL was expressed in CD16/CD32 lymphocytes/stromal cells; IL-6, IL-1α, and M-CSF were expressed in Tax tumor cells; IL-1β, TGFβ, PTHrP, and TNFα were expressed in both CD16/CD32-negative and -high cell populations.

Techniques Used: Isolation, FACS, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Purification

Tax induces IL-6 expression in tumor cells. (A-B) Real-time RT-PCR of IL-6 and RANKL normalized to GAPDH from 7 CD16/CD32-fractionated Tax tumors from 7 mice. RANKL was expressed in normal lymphocytes/stromal cells, whereas IL-6 was expressed in Tax tumor cells. All data are depicted as the mean ± SEM. (C) The preosteoblast cell line ST-2 was transduced with MSCV-Tax-GFP or MSCV-GFP. Controls were nontreated ST-2 cells and ST-2 cells treated with vitamin D for 3 days. Left panel pictures were taken under phase contrast microscopy; right panel pictures were taken under FITC channel of conventional fluorescent microscopy. (D) MSCV-Tax transduction in ST-2 cells induces IL-6 and TNFα expression but not RANKL. RT-PCR of selected osteoclast-activating factors was performed on cDNAs from transduced ST-2 cells, vitamin D–treated ST-2 cells, and nontreated ST-2 cells. (E) Serum was obtained retroorbitally, and IL-6 levels were determined by ELISA. Results for Tax + (n = 17) and wild-type (n = 19) mice are shown as picogram per milliliter. Tax + mice have increased serum IL-6 levels ( P
Figure Legend Snippet: Tax induces IL-6 expression in tumor cells. (A-B) Real-time RT-PCR of IL-6 and RANKL normalized to GAPDH from 7 CD16/CD32-fractionated Tax tumors from 7 mice. RANKL was expressed in normal lymphocytes/stromal cells, whereas IL-6 was expressed in Tax tumor cells. All data are depicted as the mean ± SEM. (C) The preosteoblast cell line ST-2 was transduced with MSCV-Tax-GFP or MSCV-GFP. Controls were nontreated ST-2 cells and ST-2 cells treated with vitamin D for 3 days. Left panel pictures were taken under phase contrast microscopy; right panel pictures were taken under FITC channel of conventional fluorescent microscopy. (D) MSCV-Tax transduction in ST-2 cells induces IL-6 and TNFα expression but not RANKL. RT-PCR of selected osteoclast-activating factors was performed on cDNAs from transduced ST-2 cells, vitamin D–treated ST-2 cells, and nontreated ST-2 cells. (E) Serum was obtained retroorbitally, and IL-6 levels were determined by ELISA. Results for Tax + (n = 17) and wild-type (n = 19) mice are shown as picogram per milliliter. Tax + mice have increased serum IL-6 levels ( P

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Transduction, Microscopy, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

10) Product Images from "Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions"

Article Title: Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

Journal: Nature Communications

doi: 10.1038/ncomms15440

Chromosome 1 alterations predispose to JAK1 deficiency. ( a ) Clinical history of patient Ma-Mel-61. Vertical line, time axis; left, therapeutic regimens; right, metastases development; arrows indicate cell lines established from metastases Ma-Mel-61a, Ma-Mel-61b, Ma-Mel-61c, Ma-Mel-61e, Ma-Mel-61g and Ma-Mel-61h; grey box, stage IV disease. ( b ) Surface expression of CD54, HLA class I, and PD-L1 on IFNγ-treated (48 h) Ma-Mel-61b and Ma-Mel-61g cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( c ) Mutation defined by targeted sequencing on DNA from Ma-Mel-61g cells. Plots of aligned sequencing reads in the location where the JAK1 c.1798G > T, p.G600W mutation was identified. Arrow highlights mutation site in Ma-Mel-61g or corresponding wild-type site (WT) in Ma-Mel-61b cells. Number of sequencing reads notated on the left; %, frequency of mutation in reads. ( d ) Lysates from Ma-Mel-61b and Ma-Mel-61g cells analysed by western blot for JAK1 expression; GAPDH, loading control. Representative data from n =3 independent experiments. ( e ) Lysates from IFNγ-treated (48 h) melanoma cells analysed by western blot for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n =3 independent experiments. ( f ) Lysates from IFNγ-treated (48 h) JAK1- transfected Ma-Mel-61g cells analysed for expression of the indicated proteins. Representative data from n =3 independent experiments. ( g ) IFNγ release by autologous CD8 + T cells in the presence of melanoma cells, measured by ELISpot assay. Means and s.e.m. (error bars) from n =4 independent measurement. Statistical significant differences defined by paired Student's t -test are indicated, * P
Figure Legend Snippet: Chromosome 1 alterations predispose to JAK1 deficiency. ( a ) Clinical history of patient Ma-Mel-61. Vertical line, time axis; left, therapeutic regimens; right, metastases development; arrows indicate cell lines established from metastases Ma-Mel-61a, Ma-Mel-61b, Ma-Mel-61c, Ma-Mel-61e, Ma-Mel-61g and Ma-Mel-61h; grey box, stage IV disease. ( b ) Surface expression of CD54, HLA class I, and PD-L1 on IFNγ-treated (48 h) Ma-Mel-61b and Ma-Mel-61g cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( c ) Mutation defined by targeted sequencing on DNA from Ma-Mel-61g cells. Plots of aligned sequencing reads in the location where the JAK1 c.1798G > T, p.G600W mutation was identified. Arrow highlights mutation site in Ma-Mel-61g or corresponding wild-type site (WT) in Ma-Mel-61b cells. Number of sequencing reads notated on the left; %, frequency of mutation in reads. ( d ) Lysates from Ma-Mel-61b and Ma-Mel-61g cells analysed by western blot for JAK1 expression; GAPDH, loading control. Representative data from n =3 independent experiments. ( e ) Lysates from IFNγ-treated (48 h) melanoma cells analysed by western blot for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n =3 independent experiments. ( f ) Lysates from IFNγ-treated (48 h) JAK1- transfected Ma-Mel-61g cells analysed for expression of the indicated proteins. Representative data from n =3 independent experiments. ( g ) IFNγ release by autologous CD8 + T cells in the presence of melanoma cells, measured by ELISpot assay. Means and s.e.m. (error bars) from n =4 independent measurement. Statistical significant differences defined by paired Student's t -test are indicated, * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Mutagenesis, Sequencing, Western Blot, Transfection, Enzyme-linked Immunospot

JAK2 deficiency blocks HLA class I upregulation by IFNγ. ( a ) Clinical history of patient Ma-Mel-54. Vertical line, time axis; left, therapeutic regimens; right, primary tumour (PT)/metastases development; arrows indicate cell lines established from metastases Ma-Mel-54a and Ma-Mel-54b; grey box, stage IV disease. ( b ) Mutations defined by targeted sequencing on DNA from melanoma cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK2 c.2876A > C, p.Q959P mutation was identified. WT sequences shown on the bottom, arrows highlight mutation sites. Number of sequencing reads notated on the left; %, frequency of mutations in reads. ( c ) Lysates from Ma-Mel-54a, Ma-Mel-54b and control cells (ctrl) analysed by western blot for JAK2 expression. ( d ) Lysates from IFNγ-treated (48 h) Ma-Mel-54a and Ma-Mel-54b cells analysed for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains. ( c , d ) GAPDH, loading control. Representative data from n =2 independent experiments. ( e ) Expression of CD54, HLA class I and PD-L1 on IFNγ-treated (48 h) melanoma cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( f ) SNP results given as allelic distribution of chromosome 9p shown for DNA obtained from Ma-Mel-54a, Ma-Mel-54b and autologous peripheral blood cells as normal control (germline). Loss of one chromosomal allele in region 9p24.3–p13.2 (Chr.9:203,861-37,578,327; hg19) present in both cell lines. Dashed line indicates JAK2 location at Chr.9p24.1. ( g ) Lysates from IFNγ-treated (48 h) Ma-Mel-54a-JAK2 transfectants analysed by western blot for expression of indicated proteins. ( h ) Real-time cell proliferation in the presence or absence of IFNγ. Bold grey vertical lines indicate addition of IFNγ. ( g , h ) Representative data from n =2 independent experiments. ( i ) Ma-Mel-54a and Ma-Mel-54b cells analysed for specific mRNA expression by quantitative reverse transcription–PCR. ( j ) Ma-Mel-54a-JAK2 transfectants analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence or absence of IFNγ (48 h). ( i , j ) Relative expression levels given as means (+s.e.m.) from n =2 independent experiments. ( k ) HLA class I expression on IFNγ-treated (48 h) Ma-Mel-54a cells and Ma-Mel-54a-JAK2 transfectants, measured by flow cytometry. Representative data from n =2 independent experiments.
Figure Legend Snippet: JAK2 deficiency blocks HLA class I upregulation by IFNγ. ( a ) Clinical history of patient Ma-Mel-54. Vertical line, time axis; left, therapeutic regimens; right, primary tumour (PT)/metastases development; arrows indicate cell lines established from metastases Ma-Mel-54a and Ma-Mel-54b; grey box, stage IV disease. ( b ) Mutations defined by targeted sequencing on DNA from melanoma cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK2 c.2876A > C, p.Q959P mutation was identified. WT sequences shown on the bottom, arrows highlight mutation sites. Number of sequencing reads notated on the left; %, frequency of mutations in reads. ( c ) Lysates from Ma-Mel-54a, Ma-Mel-54b and control cells (ctrl) analysed by western blot for JAK2 expression. ( d ) Lysates from IFNγ-treated (48 h) Ma-Mel-54a and Ma-Mel-54b cells analysed for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains. ( c , d ) GAPDH, loading control. Representative data from n =2 independent experiments. ( e ) Expression of CD54, HLA class I and PD-L1 on IFNγ-treated (48 h) melanoma cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( f ) SNP results given as allelic distribution of chromosome 9p shown for DNA obtained from Ma-Mel-54a, Ma-Mel-54b and autologous peripheral blood cells as normal control (germline). Loss of one chromosomal allele in region 9p24.3–p13.2 (Chr.9:203,861-37,578,327; hg19) present in both cell lines. Dashed line indicates JAK2 location at Chr.9p24.1. ( g ) Lysates from IFNγ-treated (48 h) Ma-Mel-54a-JAK2 transfectants analysed by western blot for expression of indicated proteins. ( h ) Real-time cell proliferation in the presence or absence of IFNγ. Bold grey vertical lines indicate addition of IFNγ. ( g , h ) Representative data from n =2 independent experiments. ( i ) Ma-Mel-54a and Ma-Mel-54b cells analysed for specific mRNA expression by quantitative reverse transcription–PCR. ( j ) Ma-Mel-54a-JAK2 transfectants analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence or absence of IFNγ (48 h). ( i , j ) Relative expression levels given as means (+s.e.m.) from n =2 independent experiments. ( k ) HLA class I expression on IFNγ-treated (48 h) Ma-Mel-54a cells and Ma-Mel-54a-JAK2 transfectants, measured by flow cytometry. Representative data from n =2 independent experiments.

Techniques Used: Sequencing, Mutagenesis, Western Blot, Expressing, Flow Cytometry, Cytometry, Polymerase Chain Reaction

IFNγ-resistant melanoma evolves into a T-cell-resistant lesion. ( a ) Mutation defined by targeted sequencing on DNA from Ma-Mel-61h cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK1 c.1798G > T, p.G600W mutation was identified. WT sequence shown on the bottom, arrow highlights mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutations in reads. ( b ) Lysates from IFNγ-treated (48 h) Ma-Mel-61b, Ma-Mel-61g and Ma-Mel-61h cells analysed by western blot for expression of IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n =3 independent experiments. ( c ) HLA class I and CD54 surface expression on IFNγ-treated (48 h) Ma-Mel-61g and Ma-Mel-61h cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( d ) Immunohistochemical staining of serial cryostat tissue sections from metastasis Ma-Mel-61g for melanoma marker HMB45 and HLA class I. ( e ) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 or mutant JAK1-G600W, analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence of absence or IFNγ (48 h). Ma-Mel-61b cells served as a control (ctrl). Relative expression levels given as means (+s.e.m.) from n =2 independent experiments. ( f ) HLA class I surface expression on IFNγ-treated (48 h) Ma-Mel-61h-JAK1 and Ma-Mel-61h-JAK1-G600W transfectants, measured by flow cytometry. Representative data from n =2 independent experiments. ( g ) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 (WT) or mutant JAK1-G600W (M), analysed for recognition by autologous CD8 + T cells in the presence or absence of IFNγ (48 h). T-cell activation measured as IFNγ release by ELISpot assay. Representative data from n =2 independent experiments.
Figure Legend Snippet: IFNγ-resistant melanoma evolves into a T-cell-resistant lesion. ( a ) Mutation defined by targeted sequencing on DNA from Ma-Mel-61h cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK1 c.1798G > T, p.G600W mutation was identified. WT sequence shown on the bottom, arrow highlights mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutations in reads. ( b ) Lysates from IFNγ-treated (48 h) Ma-Mel-61b, Ma-Mel-61g and Ma-Mel-61h cells analysed by western blot for expression of IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n =3 independent experiments. ( c ) HLA class I and CD54 surface expression on IFNγ-treated (48 h) Ma-Mel-61g and Ma-Mel-61h cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( d ) Immunohistochemical staining of serial cryostat tissue sections from metastasis Ma-Mel-61g for melanoma marker HMB45 and HLA class I. ( e ) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 or mutant JAK1-G600W, analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence of absence or IFNγ (48 h). Ma-Mel-61b cells served as a control (ctrl). Relative expression levels given as means (+s.e.m.) from n =2 independent experiments. ( f ) HLA class I surface expression on IFNγ-treated (48 h) Ma-Mel-61h-JAK1 and Ma-Mel-61h-JAK1-G600W transfectants, measured by flow cytometry. Representative data from n =2 independent experiments. ( g ) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 (WT) or mutant JAK1-G600W (M), analysed for recognition by autologous CD8 + T cells in the presence or absence of IFNγ (48 h). T-cell activation measured as IFNγ release by ELISpot assay. Representative data from n =2 independent experiments.

Techniques Used: Mutagenesis, Sequencing, Western Blot, Expressing, Flow Cytometry, Cytometry, Immunohistochemistry, Staining, Marker, Transfection, Polymerase Chain Reaction, Activation Assay, Enzyme-linked Immunospot

Protection from cytokine-induced cell death by acquired IFNγ resistance. ( a ) Clinical history of patient Ma-Mel-36. Vertical line, time axis; left, therapeutic regimens; right, primary tumour (PT)/metastases development; arrow indicates cell line established from metastasis Ma-Mel-36; grey box, stage IV disease. ( b ) IFNγ-sensitive Ma-Mel-36_sens and IFNγ-resistant Ma-Mel-36_res cells sorted from IFNγ-treated (48 h) Ma-Mel-36_bulk cells based on their HLA-DR expression profile. Surface expression of indicated proteins measured by flow cytometry. Representative data from n =3 independent experiments. ( c ) JAK1 mutation defined by targeted sequencing on DNA from Ma-Mel-36_bulk and Ma-Mel-36_res cells. Plots of aligned sequencing reads in the location where the JAK1 c.843C > A, p.Y281* mutation was identified, arrows highlight mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutation in reads. ( d ) Melanoma cells analysed by western blot for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains after IFNγ treatment (48 h); GAPDH, loading control. Representative data from n =2 independent experiments. ( e ) Ma-Mel-36_sens (S) and Ma-Mel-36_res (R) cells analysed for protein expression after IFNα and IFNγ treatment (48 h). Representative data from n =2 independent experiments. ( f ) SNP results given as allelic distribution of chromosome 1p shown for DNA obtained from Ma-Mel-36_bulk, Ma-Mel-36_sens, Ma-Mel-36_res and autologous Epstein-Barr virus-transformed B cells as a control. Loss of one chromosomal allele in the region 1p36.3-1p13.1 (Chr.1:854,277-116,804,754; hg19) in all Ma-Mel-36 cell populations. Dashed line indicates JAK1 location at Chr.1p31.3. ( g ) IFNγ release by autologous CD8 + T cells in the presence of IFNγ-treated (24 h) Ma-Mel-36 cell populations measured by ELISpot assay. Mean values (+s.e.m.) from n =2 measurements. ( h ) Real-time proliferation of Ma-Mel-36 cell populations in the presence/absence of IFNγ. Bold grey vertical lines indicate addition of IFNγ. Representative data from n =3 independent experiments. ( i ) IFNγ-induced (7 days) apoptosis in Ma-Mel-36 cell populations determined by AnnexinV/PI staining. Percentage of early (AnnexinV+/PI−) and late apoptotic (AnnexinV+/PI+) cells depicted. Mean values (+s.e.m.) from n =3 independent experiments. Only statistical significant differences defined by paired Student's t -test are indicated, * P
Figure Legend Snippet: Protection from cytokine-induced cell death by acquired IFNγ resistance. ( a ) Clinical history of patient Ma-Mel-36. Vertical line, time axis; left, therapeutic regimens; right, primary tumour (PT)/metastases development; arrow indicates cell line established from metastasis Ma-Mel-36; grey box, stage IV disease. ( b ) IFNγ-sensitive Ma-Mel-36_sens and IFNγ-resistant Ma-Mel-36_res cells sorted from IFNγ-treated (48 h) Ma-Mel-36_bulk cells based on their HLA-DR expression profile. Surface expression of indicated proteins measured by flow cytometry. Representative data from n =3 independent experiments. ( c ) JAK1 mutation defined by targeted sequencing on DNA from Ma-Mel-36_bulk and Ma-Mel-36_res cells. Plots of aligned sequencing reads in the location where the JAK1 c.843C > A, p.Y281* mutation was identified, arrows highlight mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutation in reads. ( d ) Melanoma cells analysed by western blot for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains after IFNγ treatment (48 h); GAPDH, loading control. Representative data from n =2 independent experiments. ( e ) Ma-Mel-36_sens (S) and Ma-Mel-36_res (R) cells analysed for protein expression after IFNα and IFNγ treatment (48 h). Representative data from n =2 independent experiments. ( f ) SNP results given as allelic distribution of chromosome 1p shown for DNA obtained from Ma-Mel-36_bulk, Ma-Mel-36_sens, Ma-Mel-36_res and autologous Epstein-Barr virus-transformed B cells as a control. Loss of one chromosomal allele in the region 1p36.3-1p13.1 (Chr.1:854,277-116,804,754; hg19) in all Ma-Mel-36 cell populations. Dashed line indicates JAK1 location at Chr.1p31.3. ( g ) IFNγ release by autologous CD8 + T cells in the presence of IFNγ-treated (24 h) Ma-Mel-36 cell populations measured by ELISpot assay. Mean values (+s.e.m.) from n =2 measurements. ( h ) Real-time proliferation of Ma-Mel-36 cell populations in the presence/absence of IFNγ. Bold grey vertical lines indicate addition of IFNγ. Representative data from n =3 independent experiments. ( i ) IFNγ-induced (7 days) apoptosis in Ma-Mel-36 cell populations determined by AnnexinV/PI staining. Percentage of early (AnnexinV+/PI−) and late apoptotic (AnnexinV+/PI+) cells depicted. Mean values (+s.e.m.) from n =3 independent experiments. Only statistical significant differences defined by paired Student's t -test are indicated, * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Mutagenesis, Sequencing, Western Blot, Transformation Assay, Enzyme-linked Immunospot, Staining

11) Product Images from "Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening"

Article Title: Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065994

Prednisolone and salbutamol inhibit the HMGB-induced TNFα production. RAW 264.7 cells were pretreated with prednisolone (1 µM) and salbutamol (1 µM) and then exposed to HMGB1 (5 µg/ml) for various time up to 18 hours. A : TNFα secretion measured in the supernatant is plotted versus exposure length. (MEAN±SD values are shown) B : TNFα mRNA expression, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is shown as fold expression values of vehicle treated cells. (CTL: vehicle treated control, HMGB: cells exposed to HMGB1, Pred: cells pretreated with prednisolone and exposed to HMGB1, Salb: cells pretreated with salbutamol and exposed to HMGB1, Pred+Salb: cells pretreated with both prednisolone and salbutamol and exposed to HMGB1. § p
Figure Legend Snippet: Prednisolone and salbutamol inhibit the HMGB-induced TNFα production. RAW 264.7 cells were pretreated with prednisolone (1 µM) and salbutamol (1 µM) and then exposed to HMGB1 (5 µg/ml) for various time up to 18 hours. A : TNFα secretion measured in the supernatant is plotted versus exposure length. (MEAN±SD values are shown) B : TNFα mRNA expression, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is shown as fold expression values of vehicle treated cells. (CTL: vehicle treated control, HMGB: cells exposed to HMGB1, Pred: cells pretreated with prednisolone and exposed to HMGB1, Salb: cells pretreated with salbutamol and exposed to HMGB1, Pred+Salb: cells pretreated with both prednisolone and salbutamol and exposed to HMGB1. § p

Techniques Used: Expressing, CTL Assay

12) Product Images from "Slfn2 Regulates Type I Interferon Responses by Modulating the NF-κB Pathway"

Article Title: Slfn2 Regulates Type I Interferon Responses by Modulating the NF-κB Pathway

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00053-18

Loss of Slfn2 enhances transcription of antiviral ISGs and increases IFN-inducible antiviral responses. (A) Slfn2 WT and KO MEFs were either left untreated or treated with mouse IFN-β for 6 h. qRT-PCR analyses of the relative mRNA expression of the indicated genes after IFN-β stimulation are shown. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over untreated samples, and the graphs represent means and SE of the results of three independent experiments ( Mx1 and Oasl2 ) and four independent experiments ( Cxcl10 , Ifit1 , Ifit3 , Irf7 , Irf9 , and Isg15 ). Statistical analyses were performed using a one-tailed ratio-paired t test (*, P
Figure Legend Snippet: Loss of Slfn2 enhances transcription of antiviral ISGs and increases IFN-inducible antiviral responses. (A) Slfn2 WT and KO MEFs were either left untreated or treated with mouse IFN-β for 6 h. qRT-PCR analyses of the relative mRNA expression of the indicated genes after IFN-β stimulation are shown. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over untreated samples, and the graphs represent means and SE of the results of three independent experiments ( Mx1 and Oasl2 ) and four independent experiments ( Cxcl10 , Ifit1 , Ifit3 , Irf7 , Irf9 , and Isg15 ). Statistical analyses were performed using a one-tailed ratio-paired t test (*, P

Techniques Used: Quantitative RT-PCR, Expressing, One-tailed Test

Loss of Slfn2 enhances transcription of select type I IFN-stimulated genes. (A) Slfn2 KO MEFs were generated using CRISPR/Cas9 technology. (Left) Schematic representation of the three single guide RNA (sgRNA) target sites located in the Slfn2 locus (red rectangles). (Right) Slfn2 mRNA expression in Slfn2 WT and Slfn2 KO MEFs was determined by qRT-PCR using Slfn2 -specific primers, and Gapdh was used for normalization and as an internal control. The graph shows means and standard errors (SE) of the results of three independent experiments. Statistical analyses were performed using a ratio-paired one-tailed t test (**, P
Figure Legend Snippet: Loss of Slfn2 enhances transcription of select type I IFN-stimulated genes. (A) Slfn2 KO MEFs were generated using CRISPR/Cas9 technology. (Left) Schematic representation of the three single guide RNA (sgRNA) target sites located in the Slfn2 locus (red rectangles). (Right) Slfn2 mRNA expression in Slfn2 WT and Slfn2 KO MEFs was determined by qRT-PCR using Slfn2 -specific primers, and Gapdh was used for normalization and as an internal control. The graph shows means and standard errors (SE) of the results of three independent experiments. Statistical analyses were performed using a ratio-paired one-tailed t test (**, P

Techniques Used: Generated, CRISPR, Expressing, Quantitative RT-PCR, One-tailed Test

Higher expression of IFN-β-induced ISGs in Slfn2 KO MEFs is NF-κB dependent. (A to C) Slfn2 WT and Slfn2 KO MEFs were serum starved overnight and then treated with mouse IFN-β, as indicated. Cellular lysates were prepared and fractionated into cytosol and nuclear fractions, and then equal amounts of lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) Slfn2 WT and Slfn2 KO MEFs were transfected with pcDNA-IκBαM vector or pcDNA empty-vector plasmids. Twenty-four hours later, cellular lysates were prepared and fractionated into cytosol and nuclear fractions, and then equal amounts of lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) Slfn2 WT and Slfn2 KO MEFs were transfected with pcDNA-IκBαM vector or pcDNA empty-vector plasmids. Twenty-four hours after transfection, the cells were treated with mouse IFN-β for 6 h. qRT-PCR analyses of the relative mRNA expression of the indicated genes after IFN-β stimulation are shown. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over untreated samples, and the graphs represent means and SE of the results of three independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, followed by Tukey's multiple-comparison test. P values between IFN-β-treated pcDNA- and IκBαM-transfected Slfn2 KO MEFs are shown (*, P
Figure Legend Snippet: Higher expression of IFN-β-induced ISGs in Slfn2 KO MEFs is NF-κB dependent. (A to C) Slfn2 WT and Slfn2 KO MEFs were serum starved overnight and then treated with mouse IFN-β, as indicated. Cellular lysates were prepared and fractionated into cytosol and nuclear fractions, and then equal amounts of lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) Slfn2 WT and Slfn2 KO MEFs were transfected with pcDNA-IκBαM vector or pcDNA empty-vector plasmids. Twenty-four hours later, cellular lysates were prepared and fractionated into cytosol and nuclear fractions, and then equal amounts of lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) Slfn2 WT and Slfn2 KO MEFs were transfected with pcDNA-IκBαM vector or pcDNA empty-vector plasmids. Twenty-four hours after transfection, the cells were treated with mouse IFN-β for 6 h. qRT-PCR analyses of the relative mRNA expression of the indicated genes after IFN-β stimulation are shown. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over untreated samples, and the graphs represent means and SE of the results of three independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, followed by Tukey's multiple-comparison test. P values between IFN-β-treated pcDNA- and IκBαM-transfected Slfn2 KO MEFs are shown (*, P

Techniques Used: Expressing, SDS Page, Transfection, Plasmid Preparation, Quantitative RT-PCR

13) Product Images from "Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response"

Article Title: Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003384

HPV induces expression of UCHL1 in keratinocytes. ( A ) Summary of all differentially expressed genes within the Protein Ubiquitination Pathway. Differentially expressed genes between four uninfected KC and four hrHPV+ KC cultures with adjusted p -value≤0.05 identified 24 hours after poly(I∶C) stimulation by microarray analysis (log2 ratios) are shown. ( B ) UCHL1 microarray gene expression values (log2 intensities) after 0, 4, and 24 hours of poly(I∶C) stimulation in four primary KCs and four hrHPV+ KCs (circles). The box represents the 25 th and 75 th percentiles, the median is indicated with a horizontal line within the box, and the whiskers represent the minimum and maximum. ( C ) UCHL1 expression in HPV16+ human foreskin keratinocytes (HFK; left panel) and HPV16+ human vaginal keratinocytes (HVK; right panel) when compared to uninfected KCs. KCs were either left unstimulated or stimulated with poly(I∶C) for 24 hrs. UCHL1 expression was normalized against GAPDH . ( D ) UCHL1 protein levels in HPV16+ human foreskin keratinocytes (HPV16) and HPV16+ or HPV18+ human vaginal keratinocytes (HVK16 or HVK18, respectively) when compared to non-infected KCs (HFK) as detected by western blotting (WB) in whole cell extracts. β-actin served as loading control. ( E ) UCHL1 expression at the initial stage of HPV16 infection. Primary basal layer human foreskin keratinocytes were infected with native HPV16 (HPV16 infected keratinocytes) or not (Mock). UCHL1 mRNA expression was analyzed by qRT-PCR 2 days after infection. Gene expression was normalized against GAPDH mRNA levels and standardized against the non-infected cells. Similar results were observed in two independent experiments. ( F ) UCHL1 expression in HPV+ KCs transfected with control siRNA (siControl) or siRNA targeting HPV16 E2 (siHPV16 E2). UCHL1 expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against siControl. Similar results were observed in more than 3 independent experiments.
Figure Legend Snippet: HPV induces expression of UCHL1 in keratinocytes. ( A ) Summary of all differentially expressed genes within the Protein Ubiquitination Pathway. Differentially expressed genes between four uninfected KC and four hrHPV+ KC cultures with adjusted p -value≤0.05 identified 24 hours after poly(I∶C) stimulation by microarray analysis (log2 ratios) are shown. ( B ) UCHL1 microarray gene expression values (log2 intensities) after 0, 4, and 24 hours of poly(I∶C) stimulation in four primary KCs and four hrHPV+ KCs (circles). The box represents the 25 th and 75 th percentiles, the median is indicated with a horizontal line within the box, and the whiskers represent the minimum and maximum. ( C ) UCHL1 expression in HPV16+ human foreskin keratinocytes (HFK; left panel) and HPV16+ human vaginal keratinocytes (HVK; right panel) when compared to uninfected KCs. KCs were either left unstimulated or stimulated with poly(I∶C) for 24 hrs. UCHL1 expression was normalized against GAPDH . ( D ) UCHL1 protein levels in HPV16+ human foreskin keratinocytes (HPV16) and HPV16+ or HPV18+ human vaginal keratinocytes (HVK16 or HVK18, respectively) when compared to non-infected KCs (HFK) as detected by western blotting (WB) in whole cell extracts. β-actin served as loading control. ( E ) UCHL1 expression at the initial stage of HPV16 infection. Primary basal layer human foreskin keratinocytes were infected with native HPV16 (HPV16 infected keratinocytes) or not (Mock). UCHL1 mRNA expression was analyzed by qRT-PCR 2 days after infection. Gene expression was normalized against GAPDH mRNA levels and standardized against the non-infected cells. Similar results were observed in two independent experiments. ( F ) UCHL1 expression in HPV+ KCs transfected with control siRNA (siControl) or siRNA targeting HPV16 E2 (siHPV16 E2). UCHL1 expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against siControl. Similar results were observed in more than 3 independent experiments.

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

UCHL1 is responsible for suppressing poly(I∶C) mediated gene activation of IFN-I and proinflammatory cytokines in hrHPV-infected KC. ( A – C ) UCHL1 knock-down effect of poly(I∶C) mediated gene expression of IFN-I and proinflammatory cytokines. HPV16+ keratinocytes were transduced with lentiviral vectors expressing shRNA against control mRNA (TurboGFP; shControl) or targeting mRNA of UCHL1 (shUCHL1). Cells were either left unstimulated, or were stimulated with poly(I∶C) for 3 or 24 hrs. ( A ) UCHL1 mRNA expression was analyzed by qRT-PCR and ( B ) UCHL1 protein levels were analyzed by western blotting in whole cell extracts, β-actin served as loading control. ( C ) MIP3α , RANTES and IFNβ mRNA expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against 0 h of stimulation with poly(I∶C). ( D , E ) UCHL1 overexpression effect on the activation of poly(I∶C) mediated gene expression of IFNβ and proinflammatory cytokines. Uninfected keratinocytes were transfected with a vector harboring the UCHL1 gene, an empty control or only received the transfection agent (TFRO). Cells were either left unstimulated, or were stimulated with poly(I∶C) for 24 hrs. ( D ) UCHL1 protein levels were upregulated in the UCHL1 -transfected cells as detected by western blotting in whole cell extracts, β-actin served as loading control. ( E ) MIP3α and RANTES mRNA expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against the TFRO at 0 h of stimulation with poly(I∶C).
Figure Legend Snippet: UCHL1 is responsible for suppressing poly(I∶C) mediated gene activation of IFN-I and proinflammatory cytokines in hrHPV-infected KC. ( A – C ) UCHL1 knock-down effect of poly(I∶C) mediated gene expression of IFN-I and proinflammatory cytokines. HPV16+ keratinocytes were transduced with lentiviral vectors expressing shRNA against control mRNA (TurboGFP; shControl) or targeting mRNA of UCHL1 (shUCHL1). Cells were either left unstimulated, or were stimulated with poly(I∶C) for 3 or 24 hrs. ( A ) UCHL1 mRNA expression was analyzed by qRT-PCR and ( B ) UCHL1 protein levels were analyzed by western blotting in whole cell extracts, β-actin served as loading control. ( C ) MIP3α , RANTES and IFNβ mRNA expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against 0 h of stimulation with poly(I∶C). ( D , E ) UCHL1 overexpression effect on the activation of poly(I∶C) mediated gene expression of IFNβ and proinflammatory cytokines. Uninfected keratinocytes were transfected with a vector harboring the UCHL1 gene, an empty control or only received the transfection agent (TFRO). Cells were either left unstimulated, or were stimulated with poly(I∶C) for 24 hrs. ( D ) UCHL1 protein levels were upregulated in the UCHL1 -transfected cells as detected by western blotting in whole cell extracts, β-actin served as loading control. ( E ) MIP3α and RANTES mRNA expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against the TFRO at 0 h of stimulation with poly(I∶C).

Techniques Used: Activation Assay, Infection, Expressing, Transduction, shRNA, Quantitative RT-PCR, Western Blot, Over Expression, Transfection, Plasmid Preparation

The presence of high risk human papillomavirus interferes with pattern recognition receptor (PRR) signaling of keratinocytes. ( A ) Cytokine production of non-differentiated uninfected or HPV16+ keratinocytes after stimulation with different indicated PRR stimuli as measured by ELISA. ( B ) TLR9 expression as measured by qRT-PCR on total RNA samples from undifferentiated (und) and terminally differentiated (terminal dif) uninfected KCs, and HPV16 and HPV18 positive KC cultures. ( C ) IFNβ , IL-8 and MIP3α expression levels in unstimulated or CpG ODN-stimulated uninfected KCs, and two different HPV (16 or 18) positive KC cultures as examined by qRT-PCR. KCs were either left undifferentiated (und) or terminally differentiated (terminal dif) after which they were stimulated with CpG (10 µg/ml) for 7 hours. ( B – C ) Gene expression was normalized using GAPDH mRNA expression levels.
Figure Legend Snippet: The presence of high risk human papillomavirus interferes with pattern recognition receptor (PRR) signaling of keratinocytes. ( A ) Cytokine production of non-differentiated uninfected or HPV16+ keratinocytes after stimulation with different indicated PRR stimuli as measured by ELISA. ( B ) TLR9 expression as measured by qRT-PCR on total RNA samples from undifferentiated (und) and terminally differentiated (terminal dif) uninfected KCs, and HPV16 and HPV18 positive KC cultures. ( C ) IFNβ , IL-8 and MIP3α expression levels in unstimulated or CpG ODN-stimulated uninfected KCs, and two different HPV (16 or 18) positive KC cultures as examined by qRT-PCR. KCs were either left undifferentiated (und) or terminally differentiated (terminal dif) after which they were stimulated with CpG (10 µg/ml) for 7 hours. ( B – C ) Gene expression was normalized using GAPDH mRNA expression levels.

Techniques Used: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Expression of human papillomaviral transcripts are required to impair cytokine expression of poly(I∶C) stimulated keratinocytes. ( A , B ) Cytokine expression at the initial stage of HPV16 infection. Primary basal layer human foreskin keratinocytes were infected with native HPV16. ( A ) Viral early gene E6 expression was analyzed 1 and 2 (24 h poly(I∶C)) days after infection by PCR. NC: negative control, PC: positive control, HPV16+ KCs. ( B ) MIP3a , RANTES and IFNβ expression was measured by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against the 0 h poly(I∶C) stimulated non-infected cells. Similar results were observed in two independent experiments. ( C , D ) Poly(I∶C)-induced cytokine expression in HPV+ KCs transfected with control siRNA (siControl) or siRNA targeting HPV16 E2 (siHPV16 E2). E1 , E2 , E6 , E7 ( C ) as well as MIP3a , RANTES , and IFNβ ( D ) expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against no poly(I∶C) siControl. For all three genes the response to poly(I∶C) was significantly higher when HPV16 E2 was suppressed (p
Figure Legend Snippet: Expression of human papillomaviral transcripts are required to impair cytokine expression of poly(I∶C) stimulated keratinocytes. ( A , B ) Cytokine expression at the initial stage of HPV16 infection. Primary basal layer human foreskin keratinocytes were infected with native HPV16. ( A ) Viral early gene E6 expression was analyzed 1 and 2 (24 h poly(I∶C)) days after infection by PCR. NC: negative control, PC: positive control, HPV16+ KCs. ( B ) MIP3a , RANTES and IFNβ expression was measured by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against the 0 h poly(I∶C) stimulated non-infected cells. Similar results were observed in two independent experiments. ( C , D ) Poly(I∶C)-induced cytokine expression in HPV+ KCs transfected with control siRNA (siControl) or siRNA targeting HPV16 E2 (siHPV16 E2). E1 , E2 , E6 , E7 ( C ) as well as MIP3a , RANTES , and IFNβ ( D ) expression was analyzed by qRT-PCR. Gene expression was normalized against GAPDH mRNA levels and standardized against no poly(I∶C) siControl. For all three genes the response to poly(I∶C) was significantly higher when HPV16 E2 was suppressed (p

Techniques Used: Expressing, Infection, Polymerase Chain Reaction, Negative Control, Positive Control, Quantitative RT-PCR, Transfection

Canonical NF-κB signaling is impaired upstream of the transcription factor p65. ( A ) Poly(I∶C) induced cytokine expression in HPV16+ KCs compared to non-infected KCs. MIP3a , RANTES and IFNβ expression was measured by qRT-PCR. Gene expression was normalized using GAPDH mRNA levels and standardized against 0 h of stimulation with poly(I∶C). ( B ) Poly(I∶C) stimulated phosphorylation levels of p65 in HPV16+ KCs compared to non-infected KCs. Total p65 levels and p65 phosphorylation status were determined in whole cell extracts by western blotting. β-actin served as loading control. ( C ) NEMO degradation in HPV16+ KCs compared to non-infected KCs. Monolayer cultures were treated with 100 µM cycloheximide (CHX) and harvested after 0, 3, 6, 9, 12, 18 and 24 hours. Whole cell extracts were analyzed by western blotting using antibodies against NEMO and β-actin (control for protein degradation). ( D ) Poly(I∶C) stimulation-induced phosphorylation levels of IRF3 in hrHPV+ KCs compared to KCs. Total IRF3 levels and IRF3 phosphorylation status were determined in whole cell extracts by western blotting. β-actin served as loading control.
Figure Legend Snippet: Canonical NF-κB signaling is impaired upstream of the transcription factor p65. ( A ) Poly(I∶C) induced cytokine expression in HPV16+ KCs compared to non-infected KCs. MIP3a , RANTES and IFNβ expression was measured by qRT-PCR. Gene expression was normalized using GAPDH mRNA levels and standardized against 0 h of stimulation with poly(I∶C). ( B ) Poly(I∶C) stimulated phosphorylation levels of p65 in HPV16+ KCs compared to non-infected KCs. Total p65 levels and p65 phosphorylation status were determined in whole cell extracts by western blotting. β-actin served as loading control. ( C ) NEMO degradation in HPV16+ KCs compared to non-infected KCs. Monolayer cultures were treated with 100 µM cycloheximide (CHX) and harvested after 0, 3, 6, 9, 12, 18 and 24 hours. Whole cell extracts were analyzed by western blotting using antibodies against NEMO and β-actin (control for protein degradation). ( D ) Poly(I∶C) stimulation-induced phosphorylation levels of IRF3 in hrHPV+ KCs compared to KCs. Total IRF3 levels and IRF3 phosphorylation status were determined in whole cell extracts by western blotting. β-actin served as loading control.

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

14) Product Images from "Transcriptome analysis reveals mucin 4 to be highly associated with periodontitis and identifies pleckstrin as a link to systemic diseases"

Article Title: Transcriptome analysis reveals mucin 4 to be highly associated with periodontitis and identifies pleckstrin as a link to systemic diseases

Journal: Scientific Reports

doi: 10.1038/srep18475

MUC4 and MMP7 mRNA and protein expression in gingival biopsies and gingival cells. ( a ) Boxplot demonstrating the normalized number of transcripts of MUC4 in gingival tissue from patients with periodontitis and healthy controls. ( b ) Representative staining for MUC4 in the gingival epithelium from a patient with periodontitis and ( c ) a healthy control subject. EC = epithelial cells. Scale bars = 50 μm. ( d ) Relative gene expression of MUC4 in gingival epithelial cells treated with lipopolysaccharide (LPS), for 24 hours, compared to control cells (Ctrl) treated with medium only, expressed as fold change normalized to the expression of GAPDH . Data are shown as mean ± S.D. from triplicates (* P
Figure Legend Snippet: MUC4 and MMP7 mRNA and protein expression in gingival biopsies and gingival cells. ( a ) Boxplot demonstrating the normalized number of transcripts of MUC4 in gingival tissue from patients with periodontitis and healthy controls. ( b ) Representative staining for MUC4 in the gingival epithelium from a patient with periodontitis and ( c ) a healthy control subject. EC = epithelial cells. Scale bars = 50 μm. ( d ) Relative gene expression of MUC4 in gingival epithelial cells treated with lipopolysaccharide (LPS), for 24 hours, compared to control cells (Ctrl) treated with medium only, expressed as fold change normalized to the expression of GAPDH . Data are shown as mean ± S.D. from triplicates (* P

Techniques Used: Expressing, Staining

Expression of PLEK in gingival biopsies and gingival fibroblasts. ( a ) Representative PLEK staining of gingival tissue sections from a patient with periodontitis and ( b ) a healthy control subject. EC = epithelial cells, F = fibroblasts, EnC = endothelial cells, IC = immune cells. Scale bars = 50 μm. ( c ) Relative gene expression of PLEK in fibroblast cells stimulated with lipopolysaccharide (LPS) compared to control cells treated with medium only (Ctrl), expressed as fold change normalized to the expression of GAPDH . Results shown are representative of three experiments performed in gingival fibroblasts obtained from three individuals and data presented as mean ± S.D. from triplicates (* P
Figure Legend Snippet: Expression of PLEK in gingival biopsies and gingival fibroblasts. ( a ) Representative PLEK staining of gingival tissue sections from a patient with periodontitis and ( b ) a healthy control subject. EC = epithelial cells, F = fibroblasts, EnC = endothelial cells, IC = immune cells. Scale bars = 50 μm. ( c ) Relative gene expression of PLEK in fibroblast cells stimulated with lipopolysaccharide (LPS) compared to control cells treated with medium only (Ctrl), expressed as fold change normalized to the expression of GAPDH . Results shown are representative of three experiments performed in gingival fibroblasts obtained from three individuals and data presented as mean ± S.D. from triplicates (* P

Techniques Used: Expressing, Staining

15) Product Images from "Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells"

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113530

CTSS increases PAR-2 gene and protein expression after 24 h in human corneal epithelial cells. ( A ) PAR-2 gene expression in HCE-T cells without and with CTSS. PAR-2 gene expression was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells); ( B ) PAR-2 protein expression measured by using the human PAR-2 ELISA assay in HCE-T cell lysates without and with CTSS. PAR-2 protein expression was normalized to total protein in lysates ( n = 4 samples/group and a two-tailed, unpaired Student’s t -test was used to compare treated to untreated cells); ( C ) HCE-T cells treated without and with CTSS for 24 h and fixed and processed using primary and secondary antibodies to detect PAR-2 by indirect immunofluorescence. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . (* p ≤ 0.05, ** p ≤ 0.01 and data are represented as mean ± SEM).
Figure Legend Snippet: CTSS increases PAR-2 gene and protein expression after 24 h in human corneal epithelial cells. ( A ) PAR-2 gene expression in HCE-T cells without and with CTSS. PAR-2 gene expression was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells); ( B ) PAR-2 protein expression measured by using the human PAR-2 ELISA assay in HCE-T cell lysates without and with CTSS. PAR-2 protein expression was normalized to total protein in lysates ( n = 4 samples/group and a two-tailed, unpaired Student’s t -test was used to compare treated to untreated cells); ( C ) HCE-T cells treated without and with CTSS for 24 h and fixed and processed using primary and secondary antibodies to detect PAR-2 by indirect immunofluorescence. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . (* p ≤ 0.05, ** p ≤ 0.01 and data are represented as mean ± SEM).

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Immunofluorescence, Activity Assay

CTSS exposure in human corneal epithelial cells increases cellular CTSS gene and protein expression after 8- and 24-h ( A ) CTSS gene expression in HCE-T cells without and with CTSS. CTSS gene expression was normalized to expression of the endogenous gene, GAPDH . One-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells; ( B ) CTSS protein expression measured by using the human CTSS human ELISA assay in HCE-T cell lysates without and with CTSS treatment at different time points. CTSS protein expression was normalized to total protein in lysates and a one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells; ( C ) Enzymatic CTSS activity after 24 h in HCE-T cells without and with CTSS. Enzymatic CTSS activity was normalized to total protein in lysates and a two-tailed, unpaired Student’s t -test was used to compare treated to untreated cells. ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, data are represented as mean ± SEM). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods .
Figure Legend Snippet: CTSS exposure in human corneal epithelial cells increases cellular CTSS gene and protein expression after 8- and 24-h ( A ) CTSS gene expression in HCE-T cells without and with CTSS. CTSS gene expression was normalized to expression of the endogenous gene, GAPDH . One-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells; ( B ) CTSS protein expression measured by using the human CTSS human ELISA assay in HCE-T cell lysates without and with CTSS treatment at different time points. CTSS protein expression was normalized to total protein in lysates and a one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells; ( C ) Enzymatic CTSS activity after 24 h in HCE-T cells without and with CTSS. Enzymatic CTSS activity was normalized to total protein in lysates and a two-tailed, unpaired Student’s t -test was used to compare treated to untreated cells. ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, data are represented as mean ± SEM). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods .

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test

CTSS activity is required for early induction of pro-inflammatory cytokines in human corneal epithelial cells. ( A ) IL-8 gene expression in HCE-T cells without (untreated), with heat-inactivated CTSS, and with active CTSS; ( B ) IL-6 gene expression in HCE-T cells without (untreated), with heat-inactivated CTSS, and with active CTSS. IL-8 and IL-6 gene expression were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, data are represented as mean ± SEM, and one-way ANOVA with Tukey’s multiple comparison was used to compare cells within different CTSS treatments. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Heat inactivation was by heating at 90 °C for 30 min.
Figure Legend Snippet: CTSS activity is required for early induction of pro-inflammatory cytokines in human corneal epithelial cells. ( A ) IL-8 gene expression in HCE-T cells without (untreated), with heat-inactivated CTSS, and with active CTSS; ( B ) IL-6 gene expression in HCE-T cells without (untreated), with heat-inactivated CTSS, and with active CTSS. IL-8 and IL-6 gene expression were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, data are represented as mean ± SEM, and one-way ANOVA with Tukey’s multiple comparison was used to compare cells within different CTSS treatments. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Heat inactivation was by heating at 90 °C for 30 min.

Techniques Used: Activity Assay, Expressing

CTSS increases IL-1β , IL-8 , IL-6 , and TNF-α gene expression after 2- and 4-hours of treatment in a human corneal epithelial cell line (HCE-T cells) ( A ) IL-1β gene expression without and with CTSS treatment in HCE-T cells; ( B ) IL-8 gene expression without and with CTSS treatment in HCE-T cells; ( C ) IL-6 gene expression without and with CTSS treatment in HCE-T cells; ( D ) TNF-α gene expression without and with CTSS treatment in HCE-T cells. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).
Figure Legend Snippet: CTSS increases IL-1β , IL-8 , IL-6 , and TNF-α gene expression after 2- and 4-hours of treatment in a human corneal epithelial cell line (HCE-T cells) ( A ) IL-1β gene expression without and with CTSS treatment in HCE-T cells; ( B ) IL-8 gene expression without and with CTSS treatment in HCE-T cells; ( C ) IL-6 gene expression without and with CTSS treatment in HCE-T cells; ( D ) TNF-α gene expression without and with CTSS treatment in HCE-T cells. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

Techniques Used: Expressing, Activity Assay

CTSS increases MMP-9 gene expression after 24 h in human corneal epithelial cells. MMP-9 gene expression in HCE-T cells without and with CTSS. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . MMP-9 gene expression was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, ** p ≤ 0.01, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).
Figure Legend Snippet: CTSS increases MMP-9 gene expression after 24 h in human corneal epithelial cells. MMP-9 gene expression in HCE-T cells without and with CTSS. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . MMP-9 gene expression was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, ** p ≤ 0.01, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

Techniques Used: Expressing, Activity Assay

Gene expression of pro-inflammatory cytokines ( IL-8 , IL-6 , TNF-α , and IL-1β ) after 15 min, 1 h, and 2 h of CTSS treatment in human corneal epithelial cells (HCE-T cells). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).
Figure Legend Snippet: Gene expression of pro-inflammatory cytokines ( IL-8 , IL-6 , TNF-α , and IL-1β ) after 15 min, 1 h, and 2 h of CTSS treatment in human corneal epithelial cells (HCE-T cells). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

Techniques Used: Expressing, Activity Assay

PAR-2 gene and protein expression after 48 h of PAR-2 or scrambled siRNA transfection in human corneal epithelial cells. ( A ) PAR-2 gene expression in HCE-T cells transfected with PAR-2 or scrambled siRNA. PAR-2 gene expression was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group; ( B ) PAR-2 bands measured by Western Blotting in lysates from human corneal epithelial cells transfected with PAR-2 or scrambled siRNA; ( C ) PAR-2 band intensity in human corneal epithelial cells transfected with PAR-2 or scrambled siRNA. The intensity signal of PAR-2 band was normalized to the band intensity of GAPDH and designated as 100% for scrambled siRNA-treated cells ( n = 5 samples/group); ( D ) PAR-2 protein expression in HCE-T cells transfected with PAR-2 or scrambled siRNA as determined by ELISA. PAR-2 protein expression was normalized to total protein in lysates ( n = 3 samples in PAR-2 siRNA transfected and n = 2 in scrambled siRNA transfected, * p ≤ 0.05, *** p ≤ 0.001 data are represented as mean ± SEM, and a two-tailed, unpaired Student’s t -test was used to compare PAR-2 siRNA transfected to scrambled siRNA transfected cells).
Figure Legend Snippet: PAR-2 gene and protein expression after 48 h of PAR-2 or scrambled siRNA transfection in human corneal epithelial cells. ( A ) PAR-2 gene expression in HCE-T cells transfected with PAR-2 or scrambled siRNA. PAR-2 gene expression was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group; ( B ) PAR-2 bands measured by Western Blotting in lysates from human corneal epithelial cells transfected with PAR-2 or scrambled siRNA; ( C ) PAR-2 band intensity in human corneal epithelial cells transfected with PAR-2 or scrambled siRNA. The intensity signal of PAR-2 band was normalized to the band intensity of GAPDH and designated as 100% for scrambled siRNA-treated cells ( n = 5 samples/group); ( D ) PAR-2 protein expression in HCE-T cells transfected with PAR-2 or scrambled siRNA as determined by ELISA. PAR-2 protein expression was normalized to total protein in lysates ( n = 3 samples in PAR-2 siRNA transfected and n = 2 in scrambled siRNA transfected, * p ≤ 0.05, *** p ≤ 0.001 data are represented as mean ± SEM, and a two-tailed, unpaired Student’s t -test was used to compare PAR-2 siRNA transfected to scrambled siRNA transfected cells).

Techniques Used: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

16) Product Images from "cAMP attenuates TGF-β’s profibrotic responses in osteoarthritic synoviocytes: involvement of hyaluronan and PRG4"

Article Title: cAMP attenuates TGF-β’s profibrotic responses in osteoarthritic synoviocytes: involvement of hyaluronan and PRG4

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00041.2018

Impact of forskolin (FsK; 10 μM) treatment on basal and transforming growth factor (TGF)-β1-induced proteoglycan-4 (PRG4) gene expression and production by osteoarthritic (OA) synoviocytes and efficacy of human synoviocyte PRG4 (100 μg/ml) and hyaluronan (HA) (100 μg/ml) in modulating TGF-β1-induced expression and production of α-smooth muscle actin and collagen type I in OA synoviocytes. Data are means ± SD of experiments utilizing OA synoviocytes from 4 patients. A : TGF-β1 increased PRG4 expression, and FsK treatment enhanced TGF-β1’s effect. B : FsK treatment enhanced TGF-β1-linked PRG4 production by OA synoviocytes. C : PRG4 and/or HA treatments reduced TGF-β1-induced α-smooth muscle actin gene ( ACTA2 ) expression in OA synoviocytes. D : PRG4 treatment reduced TGF-β1-induced collagen type I gene ( COL1A1) expression in OA synoviocytes. E : Western blot of α-smooth muscle action (α-SMA) (predicted mol mass: 42 kDa) in control and TGF-β1-, TGF-β1 + PRG4 (100 μg/ml)- and TGF-β1 + HA (100 μg/ml)-treated OA synoviocytes. GAPDH (predicted mol mass: 40 kDa) was used as loading control. F : semiquantitative densitometry analysis of α-SMA normalized to GAPDH and expressed as ratio to control in cell extracts of control and TGF-β1-, TGF-β1 + PRG4-, and TGF-β1 + HA-treated OA synoviocytes. PRG4 and HA treatments reduced TGF-β1-linked increase in α-SMA in OA synoviocytes. G : procollagen type I content in cell extracts of control and TGF-β1-, TGF-β1 + PRG4-, and TGF-β1 + HA-treated OA synoviocytes. Data were normalized to total protein content. PRG4 treatment reduced TGF-β1-linked increase in procollagen type I content in OA synoviocytes. * P
Figure Legend Snippet: Impact of forskolin (FsK; 10 μM) treatment on basal and transforming growth factor (TGF)-β1-induced proteoglycan-4 (PRG4) gene expression and production by osteoarthritic (OA) synoviocytes and efficacy of human synoviocyte PRG4 (100 μg/ml) and hyaluronan (HA) (100 μg/ml) in modulating TGF-β1-induced expression and production of α-smooth muscle actin and collagen type I in OA synoviocytes. Data are means ± SD of experiments utilizing OA synoviocytes from 4 patients. A : TGF-β1 increased PRG4 expression, and FsK treatment enhanced TGF-β1’s effect. B : FsK treatment enhanced TGF-β1-linked PRG4 production by OA synoviocytes. C : PRG4 and/or HA treatments reduced TGF-β1-induced α-smooth muscle actin gene ( ACTA2 ) expression in OA synoviocytes. D : PRG4 treatment reduced TGF-β1-induced collagen type I gene ( COL1A1) expression in OA synoviocytes. E : Western blot of α-smooth muscle action (α-SMA) (predicted mol mass: 42 kDa) in control and TGF-β1-, TGF-β1 + PRG4 (100 μg/ml)- and TGF-β1 + HA (100 μg/ml)-treated OA synoviocytes. GAPDH (predicted mol mass: 40 kDa) was used as loading control. F : semiquantitative densitometry analysis of α-SMA normalized to GAPDH and expressed as ratio to control in cell extracts of control and TGF-β1-, TGF-β1 + PRG4-, and TGF-β1 + HA-treated OA synoviocytes. PRG4 and HA treatments reduced TGF-β1-linked increase in α-SMA in OA synoviocytes. G : procollagen type I content in cell extracts of control and TGF-β1-, TGF-β1 + PRG4-, and TGF-β1 + HA-treated OA synoviocytes. Data were normalized to total protein content. PRG4 treatment reduced TGF-β1-linked increase in procollagen type I content in OA synoviocytes. * P

Techniques Used: Expressing, Western Blot

Impact of forskolin (FsK) treatment on intracellular cAMP levels, basal and transforming growth factor(TGF)-β1-induced α-smooth muscle actin ( ACTA2 ), collagen I ( COL1A1 ), tissue inhibitor of metalloproteinase 1 ( TIMP-1 ), and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 ( PLOD2 ) gene expression and α-smooth muscle action (α-SMA) and procollagen type I production in osteoarthritic (OA) synoviocytes. TGF-β1 (1 ng/ml) stimulation of OA fibroblast-like synoviocytes was performed for 24 h in all experiments except α-SMA immunocytostaining (stimulation was performed with 1 ng/ml TGF-β1 for 48 h). Data are means ± SD of experiments utilizing OA synoviocytes from different patients. A : representative dynamic change in intracellular cAMP levels in OA synoviocytes after treatment with FsK (0.01, 0.1, 1, and 10 μM). FsK treatment at 0.1, 1, and 10 μM resulted in detectable cAMP levels in OA synoviocytes. cAMP signal was detected with a cAMP-specific sensor. ΔF/F 0 , ratio of fluorescence intensity reduction at each time point to fluorescence intensity at baseline. B : cAMP levels were elevated in FsK (10 μM)-treated OA synoviocytes ( n = 3 patients). C : FsK treatment at 1 and 10 μM reduced TGF-β1-induced ACTA2 expression ( n = 4 patients). D : FsK treatment at 10 μM reduced TGF-β1-induced COL1A1 expression ( n = 4 patients). E : FsK treatment at 10 μM reduced TGF-β1-induced TIMP-1 expression ( n = 4 patients). F : FsK treatment (10 μM) did not alter TGF-β1-induced PLOD2 expression ( n = 4 patients). G : Western blot of α-SMA (predicted mol mass: 42 kDa) in control and TGF-β1-, TGF-β1 + FsK-, and FsK-treated OA synoviocytes. GAPDH (predicted mol mass: 40 kDa) was used as loading control. H : semiquantitative densitometry analysis of α-SMA normalized to GAPDH and expressed as ratio to control in cell extracts of control and TGF-β1-, TGF-β1 + FsK-, and FsK-treated OA synoviocytes. FsK (10 μM) treatment reduced TGF-β1-linked increase in α-SMA in OA synoviocytes ( n = 6 patients). I : procollagen type I content in cell extracts of control and TGF-β1-, TGF-β1 + FsK-, and FsK-treated OA synoviocytes. Data were normalized to total protein content. FsK (10 μM) treatment reduced TGF-β1-linked increase in procollagen type I content in OA synoviocytes ( n = 3 patients). J : FsK (10 μM) treatment reduced α-SMA staining and myofibroblast-like phenotype in TGF-β1-stimulated OA synoviocytes. * P
Figure Legend Snippet: Impact of forskolin (FsK) treatment on intracellular cAMP levels, basal and transforming growth factor(TGF)-β1-induced α-smooth muscle actin ( ACTA2 ), collagen I ( COL1A1 ), tissue inhibitor of metalloproteinase 1 ( TIMP-1 ), and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 ( PLOD2 ) gene expression and α-smooth muscle action (α-SMA) and procollagen type I production in osteoarthritic (OA) synoviocytes. TGF-β1 (1 ng/ml) stimulation of OA fibroblast-like synoviocytes was performed for 24 h in all experiments except α-SMA immunocytostaining (stimulation was performed with 1 ng/ml TGF-β1 for 48 h). Data are means ± SD of experiments utilizing OA synoviocytes from different patients. A : representative dynamic change in intracellular cAMP levels in OA synoviocytes after treatment with FsK (0.01, 0.1, 1, and 10 μM). FsK treatment at 0.1, 1, and 10 μM resulted in detectable cAMP levels in OA synoviocytes. cAMP signal was detected with a cAMP-specific sensor. ΔF/F 0 , ratio of fluorescence intensity reduction at each time point to fluorescence intensity at baseline. B : cAMP levels were elevated in FsK (10 μM)-treated OA synoviocytes ( n = 3 patients). C : FsK treatment at 1 and 10 μM reduced TGF-β1-induced ACTA2 expression ( n = 4 patients). D : FsK treatment at 10 μM reduced TGF-β1-induced COL1A1 expression ( n = 4 patients). E : FsK treatment at 10 μM reduced TGF-β1-induced TIMP-1 expression ( n = 4 patients). F : FsK treatment (10 μM) did not alter TGF-β1-induced PLOD2 expression ( n = 4 patients). G : Western blot of α-SMA (predicted mol mass: 42 kDa) in control and TGF-β1-, TGF-β1 + FsK-, and FsK-treated OA synoviocytes. GAPDH (predicted mol mass: 40 kDa) was used as loading control. H : semiquantitative densitometry analysis of α-SMA normalized to GAPDH and expressed as ratio to control in cell extracts of control and TGF-β1-, TGF-β1 + FsK-, and FsK-treated OA synoviocytes. FsK (10 μM) treatment reduced TGF-β1-linked increase in α-SMA in OA synoviocytes ( n = 6 patients). I : procollagen type I content in cell extracts of control and TGF-β1-, TGF-β1 + FsK-, and FsK-treated OA synoviocytes. Data were normalized to total protein content. FsK (10 μM) treatment reduced TGF-β1-linked increase in procollagen type I content in OA synoviocytes ( n = 3 patients). J : FsK (10 μM) treatment reduced α-SMA staining and myofibroblast-like phenotype in TGF-β1-stimulated OA synoviocytes. * P

Techniques Used: Expressing, Fluorescence, Western Blot, Staining

17) Product Images from "Posttranscriptional control of the chemokine receptor CXCR4 expression in cancer cells"

Article Title: Posttranscriptional control of the chemokine receptor CXCR4 expression in cancer cells

Journal: Carcinogenesis

doi: 10.1093/carcin/bgu080

CXCR4 expression in breast cancer cells. ( A ) CXCR4 mRNA levels in breast cell lines by quantitative PCR (qPCR) using a human CXCR4 probe normalized to GAPDH. Data are from one representative experiment out of three independent experiments, ** P
Figure Legend Snippet: CXCR4 expression in breast cancer cells. ( A ) CXCR4 mRNA levels in breast cell lines by quantitative PCR (qPCR) using a human CXCR4 probe normalized to GAPDH. Data are from one representative experiment out of three independent experiments, ** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

TTP regulation of CXCR4 mRNA and protein. ( A ) Quantitative PCR (qPCR) quantification of CXCR4 mRNA associated with TTP protein. MDA-MB-231 cells were transfected with TTP or C124R expression plasmids for 24h. Cells were lysed, and TTP and C124R proteins were immunoprecipitated using anti-TTP or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed by qPCR using a FAM-labeled human CXCR4 Taqman expression probe and normalized to a VIC-labeled GAPDH probe. Data are from one experiment representative of two independent experiments * P
Figure Legend Snippet: TTP regulation of CXCR4 mRNA and protein. ( A ) Quantitative PCR (qPCR) quantification of CXCR4 mRNA associated with TTP protein. MDA-MB-231 cells were transfected with TTP or C124R expression plasmids for 24h. Cells were lysed, and TTP and C124R proteins were immunoprecipitated using anti-TTP or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed by qPCR using a FAM-labeled human CXCR4 Taqman expression probe and normalized to a VIC-labeled GAPDH probe. Data are from one experiment representative of two independent experiments * P

Techniques Used: Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Immunoprecipitation, Labeling

Investigation of ARE functionality in CXCR4 3′-UTR. ( A ) Schematic representation of the RPS30-EGFP-control 3′-UTR, RPS30-EGFP-CXCR4 3′-UTR and ARE, and RPS30-EGFP-TNF ARE reporter constructs. ( B ) Regulation of reporter activity by CXCR4 3′-UTR. HEK293 cells were transfected with the RPS30 promoter-linked EGFP reporter plasmid construct containing CXCR4 3′-UTR or a control 3′-UTR (BGH 3′-UTR). GFP fluorescence was measured 24h posttransfection. ( C ) CXCR4-ARE-mediated regulation of reporter expression. HEK293 cells were transfected with RPS30-EGFP reporter plasmid constructs containing CXCR4 ARE, TNF-ARE or control BGH 3′-UTR. GFP fluorescence was measured 24h posttransfection. ( D ) mRNA levels of the EGFP reporters; HEK293 cells were transfected with the reporter constructs in B and C for 24h, then RNA was extracted for real-time PCR (quantitative PCR) using a FAM-labeled probe against EGFP and normalized to a VIC-labeled GAPDH then normalized again to a HEX-labeled RFP probe. Data represent mean ± standard error of the mean from three independent experiments * P = 0.01, ** P
Figure Legend Snippet: Investigation of ARE functionality in CXCR4 3′-UTR. ( A ) Schematic representation of the RPS30-EGFP-control 3′-UTR, RPS30-EGFP-CXCR4 3′-UTR and ARE, and RPS30-EGFP-TNF ARE reporter constructs. ( B ) Regulation of reporter activity by CXCR4 3′-UTR. HEK293 cells were transfected with the RPS30 promoter-linked EGFP reporter plasmid construct containing CXCR4 3′-UTR or a control 3′-UTR (BGH 3′-UTR). GFP fluorescence was measured 24h posttransfection. ( C ) CXCR4-ARE-mediated regulation of reporter expression. HEK293 cells were transfected with RPS30-EGFP reporter plasmid constructs containing CXCR4 ARE, TNF-ARE or control BGH 3′-UTR. GFP fluorescence was measured 24h posttransfection. ( D ) mRNA levels of the EGFP reporters; HEK293 cells were transfected with the reporter constructs in B and C for 24h, then RNA was extracted for real-time PCR (quantitative PCR) using a FAM-labeled probe against EGFP and normalized to a VIC-labeled GAPDH then normalized again to a HEX-labeled RFP probe. Data represent mean ± standard error of the mean from three independent experiments * P = 0.01, ** P

Techniques Used: Construct, Activity Assay, Transfection, Plasmid Preparation, Fluorescence, Expressing, Real-time Polymerase Chain Reaction, Labeling

HuR regulation of CXCR4 mRNA and protein. ( A ) qPCR quantification of CXCR4 mRNA associated with HuR protein. MDA-MB-231 cells were lysed and HuR protein was immunoprecipitated using anti-HuR or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed as described above using a probe specific for human CXCR4 or human uPA as a positive control and both were normalized to human GAPDH. Data are from one experiment representative of two independent experiments, * P
Figure Legend Snippet: HuR regulation of CXCR4 mRNA and protein. ( A ) qPCR quantification of CXCR4 mRNA associated with HuR protein. MDA-MB-231 cells were lysed and HuR protein was immunoprecipitated using anti-HuR or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed as described above using a probe specific for human CXCR4 or human uPA as a positive control and both were normalized to human GAPDH. Data are from one experiment representative of two independent experiments, * P

Techniques Used: Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Immunoprecipitation, Positive Control

18) Product Images from "FIZZ2/RELM-? Induction and Role in Pulmonary Fibrosis"

Article Title: FIZZ2/RELM-? Induction and Role in Pulmonary Fibrosis

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1000964

FIZZ2, FIZZ3, and RELM-γ expression in pulmonary fibrosis. A and B , Murine lungs from saline (SAL)- or BLM-treated mice were analyzed for FIZZ2 mRNA ( A ) and protein ( B ) by RT-PCR and Western blotting, respectively. GAPDH protein served as a loading
Figure Legend Snippet: FIZZ2, FIZZ3, and RELM-γ expression in pulmonary fibrosis. A and B , Murine lungs from saline (SAL)- or BLM-treated mice were analyzed for FIZZ2 mRNA ( A ) and protein ( B ) by RT-PCR and Western blotting, respectively. GAPDH protein served as a loading

Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

19) Product Images from "Single-cell protein profiling in microchambers with barcoded beads"

Article Title: Single-cell protein profiling in microchambers with barcoded beads

Journal: Microsystems & Nanoengineering

doi: 10.1038/s41378-019-0099-5

Immunoassays with barcoded Luminex beads. a Schematic illustration of the immunoassay on the bead surface. Labelling was conducted in two steps with biotinylated antibodies (step 1) and streptavidin-conjugated Phycoerythrin (step 2). ( b ) Calibration of GAPDH, Gal-3 and Gal-3bp in a 96-well plate format. c Multiplexing with all three targets revealed that there was no cross-reaction between the assays. The digital combinations of the three targets are noted underneath the table, with (“1”) indicating the presence and (“0”) indication the absence of the analyte ( d – f ). On-chip calibration curves in microchambers with the respective limits of detection (LODs). The blue dotted lines represent the corresponding background signals plus three times the standard deviation. The red lines represent the linear interpolation fit (please note the semi-logarithmic x -axis scaling that results in the nonlinear appearance of the linear interpolation curves)
Figure Legend Snippet: Immunoassays with barcoded Luminex beads. a Schematic illustration of the immunoassay on the bead surface. Labelling was conducted in two steps with biotinylated antibodies (step 1) and streptavidin-conjugated Phycoerythrin (step 2). ( b ) Calibration of GAPDH, Gal-3 and Gal-3bp in a 96-well plate format. c Multiplexing with all three targets revealed that there was no cross-reaction between the assays. The digital combinations of the three targets are noted underneath the table, with (“1”) indicating the presence and (“0”) indication the absence of the analyte ( d – f ). On-chip calibration curves in microchambers with the respective limits of detection (LODs). The blue dotted lines represent the corresponding background signals plus three times the standard deviation. The red lines represent the linear interpolation fit (please note the semi-logarithmic x -axis scaling that results in the nonlinear appearance of the linear interpolation curves)

Techniques Used: Luminex, Multiplexing, Chromatin Immunoprecipitation, Standard Deviation

Multiplexed single-cell analysis. a Fluorescent images of selected microchambers that show the acquired images prior to analysis. Cells were visualized by staining of the nuclei, while the barcoded beads were identified by the ratio of emission at 658 and 712 nm. b Based on the fluorescent signals of the two barcoded labels, the bead identity could be determined. c Profiles of Gal-3 and Gal-3bp in the three investigated cell types (depicted in different colours). The data were derived from microchambers in which both types of functionalized beads (Gal-3 and Gal-3bp) were co-immobilized with one individual cell. With few exceptions, each cell type exhibited a clear expression pattern for the two proteins. d – f Fluorescence signals derived from the functionalized beads for all tested cells. Here, we used microchambers in which at least one type of barcoded bead was co-captured. While SK-BR-3 cells expressed higher Gal-3bp levels than HEK-293T and MCF-7 cells, the latter showed stronger Gal-3 expression when compared with HEK-293T and SK-BR-3 cells. For GAPDH ( d ), we observed an effect similar to the high-dose Hook effect, resulting in a signal decrease at increasing target concentrations (Fig. 4d ). Hence, HEK-293T cells, which showed the highest fluorescence in the GAPDH assay, actually had lower GAPDH concentrations than the other two cell lines; significance levels are indicated with n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001
Figure Legend Snippet: Multiplexed single-cell analysis. a Fluorescent images of selected microchambers that show the acquired images prior to analysis. Cells were visualized by staining of the nuclei, while the barcoded beads were identified by the ratio of emission at 658 and 712 nm. b Based on the fluorescent signals of the two barcoded labels, the bead identity could be determined. c Profiles of Gal-3 and Gal-3bp in the three investigated cell types (depicted in different colours). The data were derived from microchambers in which both types of functionalized beads (Gal-3 and Gal-3bp) were co-immobilized with one individual cell. With few exceptions, each cell type exhibited a clear expression pattern for the two proteins. d – f Fluorescence signals derived from the functionalized beads for all tested cells. Here, we used microchambers in which at least one type of barcoded bead was co-captured. While SK-BR-3 cells expressed higher Gal-3bp levels than HEK-293T and MCF-7 cells, the latter showed stronger Gal-3 expression when compared with HEK-293T and SK-BR-3 cells. For GAPDH ( d ), we observed an effect similar to the high-dose Hook effect, resulting in a signal decrease at increasing target concentrations (Fig. 4d ). Hence, HEK-293T cells, which showed the highest fluorescence in the GAPDH assay, actually had lower GAPDH concentrations than the other two cell lines; significance levels are indicated with n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001

Techniques Used: Single-cell Analysis, Staining, Derivative Assay, Expressing, Fluorescence

20) Product Images from "Doxycycline attenuates cisplatin-induced acute kidney injury through pleiotropic effects"

Article Title: Doxycycline attenuates cisplatin-induced acute kidney injury through pleiotropic effects

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00648.2017

Effects of doxycycline (Dox) on inflammation and inflammasome induced by cisplatin (CDDP) in the kidneys. A : mRNA expression of TNF-α, IL-1β, MCP-1, TGF-β1, IL-6, and GAPDH was determined by real-time PCR. The abundance of each mRNA was normalized to GAPDH. B : protein levels of p-NF-κB p65 and TNF-α were evaluated by immunoblotting analysis. C : ratios of p-NF-κB p65 and TNF-α to β-actin were calculated. D : representative photomicrographs of immunofluorescence staining for F4/80. White arrows indicate F4/80-positive cells. Scale bars = 50 µm. E : quantitative analysis of F4/80-positive cells. HPF, high power field. F : protein levels of NLRP3, cleaved caspase-1, and cleaved IL-1β were evaluated by immunoblotting analysis. G : ratios of NLRP3, cleaved caspase-3, and cleaved IL-1β to β-actin were calculated. Control (Cont., n = 4); Dox-treated ( n = 4); CDDP-injected ( n = 6); and CDDP+Dox treatment ( n = 6). Values are means ± SE, * P
Figure Legend Snippet: Effects of doxycycline (Dox) on inflammation and inflammasome induced by cisplatin (CDDP) in the kidneys. A : mRNA expression of TNF-α, IL-1β, MCP-1, TGF-β1, IL-6, and GAPDH was determined by real-time PCR. The abundance of each mRNA was normalized to GAPDH. B : protein levels of p-NF-κB p65 and TNF-α were evaluated by immunoblotting analysis. C : ratios of p-NF-κB p65 and TNF-α to β-actin were calculated. D : representative photomicrographs of immunofluorescence staining for F4/80. White arrows indicate F4/80-positive cells. Scale bars = 50 µm. E : quantitative analysis of F4/80-positive cells. HPF, high power field. F : protein levels of NLRP3, cleaved caspase-1, and cleaved IL-1β were evaluated by immunoblotting analysis. G : ratios of NLRP3, cleaved caspase-3, and cleaved IL-1β to β-actin were calculated. Control (Cont., n = 4); Dox-treated ( n = 4); CDDP-injected ( n = 6); and CDDP+Dox treatment ( n = 6). Values are means ± SE, * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Injection

Effects of doxycycline (Dox) on apoptosis induced by cisplatin (CDDP) in the kidneys. A : representative image of TUNEL staining in the cortex. Apoptotic cells are visualized as green and nuclei are stained with DAPI (blue). Scale bars = 50 µm. B : quantitative analysis for the number of TUNEL-staining positive cells. C : mRNA expression of TNFR1, TNFR2, Fas, FasL, and GAPDH was determined by real-time PCR. The abundance of each mRNA was normalized to GAPDH. D : protein levels of Bax, Bcl-2, caspase-3, and cleaved caspase-3 were evaluated by immunoblotting analysis. E : ratios of Bax, Bcl-2, Bax, caspase-3, and cleaved caspase-3 to β-actin were calculated. Control (Cont., n = 4); Dox-treated ( n = 4); CDDP-injected ( n = 6); CDDP+Dox treatment ( n = 6). Values are means ± SE, * P
Figure Legend Snippet: Effects of doxycycline (Dox) on apoptosis induced by cisplatin (CDDP) in the kidneys. A : representative image of TUNEL staining in the cortex. Apoptotic cells are visualized as green and nuclei are stained with DAPI (blue). Scale bars = 50 µm. B : quantitative analysis for the number of TUNEL-staining positive cells. C : mRNA expression of TNFR1, TNFR2, Fas, FasL, and GAPDH was determined by real-time PCR. The abundance of each mRNA was normalized to GAPDH. D : protein levels of Bax, Bcl-2, caspase-3, and cleaved caspase-3 were evaluated by immunoblotting analysis. E : ratios of Bax, Bcl-2, Bax, caspase-3, and cleaved caspase-3 to β-actin were calculated. Control (Cont., n = 4); Dox-treated ( n = 4); CDDP-injected ( n = 6); CDDP+Dox treatment ( n = 6). Values are means ± SE, * P

Techniques Used: TUNEL Assay, Staining, Expressing, Real-time Polymerase Chain Reaction, Injection

21) Product Images from "Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts"

Article Title: Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0190217

Agonist activated MAS receptor induces CTGF through ERK1/2 and regulates collagen expression in HEK293 cells stably expressing MAS. (A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS receptor agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist suppresses the expression of CTGF below the basal level. (B) Western-blot showing significant upregulation of CTGF in MAS agonist (AR234960) activated samples whereas CTGF expression decreases in presence of inverse-agonist (AR244555). MAS activated by AR234960 induces phosphorylation of ERK1/2, MAS inhibition by the inverse-agonist (AR244555) reduces ERK1/2 activation. MAS expressing HEK293 cells also show significant down-regulation of CTGF in presence of MEK1 inhibitor (PD98059). CTGF and p-ERK1/2 expression were normalized by GAPDH and ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graph, (* p
Figure Legend Snippet: Agonist activated MAS receptor induces CTGF through ERK1/2 and regulates collagen expression in HEK293 cells stably expressing MAS. (A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS receptor agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist suppresses the expression of CTGF below the basal level. (B) Western-blot showing significant upregulation of CTGF in MAS agonist (AR234960) activated samples whereas CTGF expression decreases in presence of inverse-agonist (AR244555). MAS activated by AR234960 induces phosphorylation of ERK1/2, MAS inhibition by the inverse-agonist (AR244555) reduces ERK1/2 activation. MAS expressing HEK293 cells also show significant down-regulation of CTGF in presence of MEK1 inhibitor (PD98059). CTGF and p-ERK1/2 expression were normalized by GAPDH and ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graph, (* p

Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot, Inhibition, Activation Assay

CTGF regulates collagen in HEK293-MAS cells treated with MAS agonist (AR234960). MAS expressing HEK293 cells were transiently transfected with control siRNA and CTGF siRNA followed by treatment with MAS agonist (AR234960). (A) Real-time PCR analysis confirms CTGF down-regulation by CTGF siRNA as compared to control siRNA transfected and followed by MAS agonist treatment. GAPDH was used as loading control. CTGF siRNA is specific and has no off-target effect on MAS expression. (B). Western-blot confirmation of CTGF protein levels. The western blot image shown is a representative of all the experiment done under similar experimental condition and data from multiple experiments quantitated and cumulative data were presented as bar graphs (** p
Figure Legend Snippet: CTGF regulates collagen in HEK293-MAS cells treated with MAS agonist (AR234960). MAS expressing HEK293 cells were transiently transfected with control siRNA and CTGF siRNA followed by treatment with MAS agonist (AR234960). (A) Real-time PCR analysis confirms CTGF down-regulation by CTGF siRNA as compared to control siRNA transfected and followed by MAS agonist treatment. GAPDH was used as loading control. CTGF siRNA is specific and has no off-target effect on MAS expression. (B). Western-blot confirmation of CTGF protein levels. The western blot image shown is a representative of all the experiment done under similar experimental condition and data from multiple experiments quantitated and cumulative data were presented as bar graphs (** p

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

MAS receptor present on adult human cardiac fibroblast primary cells induces CTGF and collagen expression in response to its agonist. (A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist (AR234960; 10μM) reduced CTGF expression significantly. CTGF expression decreases when MAS signaling is blocked by MEK1 inhibitor treatment. (B) Western-blot showing significant upregulation of CTGF in HCF cells treated with MAS agonist (AR234960; 10μM); the CTGF expression decreases when treated with inverse-agonist (AR244555; 10μM). MAS agonist (AR234960) activation also induces phosphorylation of ERK1/2 in HCF cells. In MAS agonist (AR234960) treated HCF cells, CTGF expression as well as ERK1/2 activation were significantly down-regulated in presence of MEK1 inhibitor (PD98059). CTGF expression and p-ERK1/2 levels were normalized by GAPDH and total ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graphs. (C) Bar graphs showing real-time PCR analysis of different collagen sub-types (Col1A2 and Col3A1) [represented as fold increase (2 ‒ΔΔCt )] in HCF cells. Activated MAS induces collagen synthesis while inhibition of MAS receptor shows significant down-regulation of the same collagen sub-types. Inhibiting MEK1 also reduces expression of the same collagen sub-types. RT-qPCR was normalized by GAPDH. (* p
Figure Legend Snippet: MAS receptor present on adult human cardiac fibroblast primary cells induces CTGF and collagen expression in response to its agonist. (A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist (AR234960; 10μM) reduced CTGF expression significantly. CTGF expression decreases when MAS signaling is blocked by MEK1 inhibitor treatment. (B) Western-blot showing significant upregulation of CTGF in HCF cells treated with MAS agonist (AR234960; 10μM); the CTGF expression decreases when treated with inverse-agonist (AR244555; 10μM). MAS agonist (AR234960) activation also induces phosphorylation of ERK1/2 in HCF cells. In MAS agonist (AR234960) treated HCF cells, CTGF expression as well as ERK1/2 activation were significantly down-regulated in presence of MEK1 inhibitor (PD98059). CTGF expression and p-ERK1/2 levels were normalized by GAPDH and total ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graphs. (C) Bar graphs showing real-time PCR analysis of different collagen sub-types (Col1A2 and Col3A1) [represented as fold increase (2 ‒ΔΔCt )] in HCF cells. Activated MAS induces collagen synthesis while inhibition of MAS receptor shows significant down-regulation of the same collagen sub-types. Inhibiting MEK1 also reduces expression of the same collagen sub-types. RT-qPCR was normalized by GAPDH. (* p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activation Assay, Inhibition, Quantitative RT-PCR

CTGF regulates agonist (AR234960) induced collagen expression in HCF cells. HCF cells were transiently transfected with control siRNA and CTGF siRNA followed by treatment with MAS agonist (AR234960). (A) Real-time PCR analysis confirms CTGF down-regulation by CTGF siRNA as compared to control siRNA transfected and followed by MAS agonist treatment. GAPDH was used as loading control. CTGF siRNA did not affect MAS expression in HCF ( S3 Fig ). (B) . Western-blot confirmation of CTGF protein levels. The western blot image shown is a representative of all the experiments done under similar experimental condition and data from multiple experiments quantitated and cumulative data were presented as bar graphs (left) (** p
Figure Legend Snippet: CTGF regulates agonist (AR234960) induced collagen expression in HCF cells. HCF cells were transiently transfected with control siRNA and CTGF siRNA followed by treatment with MAS agonist (AR234960). (A) Real-time PCR analysis confirms CTGF down-regulation by CTGF siRNA as compared to control siRNA transfected and followed by MAS agonist treatment. GAPDH was used as loading control. CTGF siRNA did not affect MAS expression in HCF ( S3 Fig ). (B) . Western-blot confirmation of CTGF protein levels. The western blot image shown is a representative of all the experiments done under similar experimental condition and data from multiple experiments quantitated and cumulative data were presented as bar graphs (left) (** p

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

22) Product Images from "Rotavirus NSP1 Inhibits NF?B Activation by Inducing Proteasome-Dependent Degradation of ?-TrCP: A Novel Mechanism of IFN Antagonism"

Article Title: Rotavirus NSP1 Inhibits NF?B Activation by Inducing Proteasome-Dependent Degradation of ?-TrCP: A Novel Mechanism of IFN Antagonism

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000280

NFκB subunit p65 is stable in rotavirus infected cells. (A) MA104 cells were infected with three pfu/cell of the indicated virus strain. Lysates were prepared at 2, 4, 6, 8, and 10 hpi and the abundance of p65 was determined by immunoblot using anti-p65 antibody. Blots were probed with anti-GAPDH as a loading control. (B) The subcellular localization of p65 in MA104 cells infected with three pfu/cell of the indicated virus strain was determined at six hpi by confocal microscopy (63×, NA 1.40). Cells were stained with anti-p65 antibody, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG.
Figure Legend Snippet: NFκB subunit p65 is stable in rotavirus infected cells. (A) MA104 cells were infected with three pfu/cell of the indicated virus strain. Lysates were prepared at 2, 4, 6, 8, and 10 hpi and the abundance of p65 was determined by immunoblot using anti-p65 antibody. Blots were probed with anti-GAPDH as a loading control. (B) The subcellular localization of p65 in MA104 cells infected with three pfu/cell of the indicated virus strain was determined at six hpi by confocal microscopy (63×, NA 1.40). Cells were stained with anti-p65 antibody, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG.

Techniques Used: Infection, Confocal Microscopy, Staining

IκBα is stable in cells infected with strains that encode NSP1. (A) MA104 cells were infected with three pfu/cell of the indicated virus strain and lysates were prepared every two hours for ten hours. Immunoblots were probed with anti-IκBα antibody. All blots were probed with anti-GAPDH antibody as a loading control. (B) IκBα levels were quantified by densitometry and are plotted as the ratio of IκBα to GAPDH, normalized to mock infected control. Error bars are the standard error of the mean. (C) Immunblot of lysates in (A) probed with anti-p-IκBα antibody (D) MA104 cells were infected with three pfu/cell of the indicated virus strain. Eight hours post-infection, cells were treated with 50 ng/mL TNFα for 15 minutes. Immunoblots were probed with anti-IκBα or anti-p-IκBα, and anti-GAPDH antibodies. IκBα is not detectable in A5-16 infected cells because it is degraded by six hours post-infection (see panel A).
Figure Legend Snippet: IκBα is stable in cells infected with strains that encode NSP1. (A) MA104 cells were infected with three pfu/cell of the indicated virus strain and lysates were prepared every two hours for ten hours. Immunoblots were probed with anti-IκBα antibody. All blots were probed with anti-GAPDH antibody as a loading control. (B) IκBα levels were quantified by densitometry and are plotted as the ratio of IκBα to GAPDH, normalized to mock infected control. Error bars are the standard error of the mean. (C) Immunblot of lysates in (A) probed with anti-p-IκBα antibody (D) MA104 cells were infected with three pfu/cell of the indicated virus strain. Eight hours post-infection, cells were treated with 50 ng/mL TNFα for 15 minutes. Immunoblots were probed with anti-IκBα or anti-p-IκBα, and anti-GAPDH antibodies. IκBα is not detectable in A5-16 infected cells because it is degraded by six hours post-infection (see panel A).

Techniques Used: Infection, Western Blot

p65 activation and nuclear translocation. MA104 cells were infected with A5-16, OSU or NCDV at an moi of three pfu/cell. Six hours post-infection, nuclear and cytoplasmic fractions were separated with the nuclear extract kit following the manufacturer's instructions (Active Motif). (A) p65 activation measured by p65 TransAm ELISA. Error bars are the standard error of the mean. (B) Nuclear fractions were probed with anti-p65 antibody, anti-laminA/C (nuclear, BD Biosciences), and anti- GAPDH (cytoplasmic) antibodies.
Figure Legend Snippet: p65 activation and nuclear translocation. MA104 cells were infected with A5-16, OSU or NCDV at an moi of three pfu/cell. Six hours post-infection, nuclear and cytoplasmic fractions were separated with the nuclear extract kit following the manufacturer's instructions (Active Motif). (A) p65 activation measured by p65 TransAm ELISA. Error bars are the standard error of the mean. (B) Nuclear fractions were probed with anti-p65 antibody, anti-laminA/C (nuclear, BD Biosciences), and anti- GAPDH (cytoplasmic) antibodies.

Techniques Used: Activation Assay, Translocation Assay, Infection, Enzyme-linked Immunosorbent Assay

23) Product Images from "The instability of the BTB-KELCH protein Gigaxonin causes Giant Axonal Neuropathy and constitutes a new penetrant and specific diagnostic test"

Article Title: The instability of the BTB-KELCH protein Gigaxonin causes Giant Axonal Neuropathy and constitutes a new penetrant and specific diagnostic test

Journal: Acta Neuropathologica Communications

doi: 10.1186/2051-5960-2-47

Diminished levels of Gigaxonin corroborate with identification of mutations in the GAN locus. A Immunodetection of Gigaxonin in new patient’s lymphoblast cell lines. (S1) and (S2) are unaffected sisters of patient F24. B Quantification of Gigaxonin in patients and their relatives, using Tubulin or GAPDH as internal controls. Individual level of Gigaxonin is compared with the range of wild type Gigaxonin (left panel) and mutated Gigaxonin in known GAN patients (as presented in Figure 1 , right panel). The red lines correspond to the maximum individual mean value from patients. Please note that Gigaxonin abundance was so low (undetectable) for F24 and F30 that it was detected as significantly different from mutated Gigaxonin. N = 3-5 experiments. (T-test, *, p
Figure Legend Snippet: Diminished levels of Gigaxonin corroborate with identification of mutations in the GAN locus. A Immunodetection of Gigaxonin in new patient’s lymphoblast cell lines. (S1) and (S2) are unaffected sisters of patient F24. B Quantification of Gigaxonin in patients and their relatives, using Tubulin or GAPDH as internal controls. Individual level of Gigaxonin is compared with the range of wild type Gigaxonin (left panel) and mutated Gigaxonin in known GAN patients (as presented in Figure 1 , right panel). The red lines correspond to the maximum individual mean value from patients. Please note that Gigaxonin abundance was so low (undetectable) for F24 and F30 that it was detected as significantly different from mutated Gigaxonin. N = 3-5 experiments. (T-test, *, p

Techniques Used: Immunodetection, T-Test

Decreased abundance of disease-associated Gigaxonin. A Schematic representation of Gigaxonin and the corresponding known mutations in GAN patients. The N-terminal BTB and C-terminal KELCH domains are represented in blue. Lymphoblast cell lines derived from GAN patients are numbered F1-F25 and their respective mutations are mapped on Gigaxonin. All patients are severely affected by the disease with the exception of patients F2 and F13, who are mild cases reported previously. B Abundance of Gigaxonin, as revealed by immunoblotting using the GigA antibody [ 8 ]. Cost and c1-c3 correspond to ectopic Flag-tagged Gigaxonin expressed in COS cells and to unrelated control individuals, respectively. (A), (B), (F) and (M) stand for Affected, non-affected Brother, Father and Mother, respectively. A1 and A2 are two affected children from the same family. Please note that immunoblottings of patients F18 and F25 are shown in Figure 2 A. C Quantification of Gigaxonin in GAN patients and their relatives. Left: Percentage of Gigaxonin for each individual in comparison to wild type Gigaxonin, as the average of 3–5 independent experiments, after normalization with tubulin and GAPDH. Right: Mean abundance of Gigaxonin in patients and heterozygous individuals, as measured by the percentage in comparison to wild type Gigaxonin. (T-test, *, p
Figure Legend Snippet: Decreased abundance of disease-associated Gigaxonin. A Schematic representation of Gigaxonin and the corresponding known mutations in GAN patients. The N-terminal BTB and C-terminal KELCH domains are represented in blue. Lymphoblast cell lines derived from GAN patients are numbered F1-F25 and their respective mutations are mapped on Gigaxonin. All patients are severely affected by the disease with the exception of patients F2 and F13, who are mild cases reported previously. B Abundance of Gigaxonin, as revealed by immunoblotting using the GigA antibody [ 8 ]. Cost and c1-c3 correspond to ectopic Flag-tagged Gigaxonin expressed in COS cells and to unrelated control individuals, respectively. (A), (B), (F) and (M) stand for Affected, non-affected Brother, Father and Mother, respectively. A1 and A2 are two affected children from the same family. Please note that immunoblottings of patients F18 and F25 are shown in Figure 2 A. C Quantification of Gigaxonin in GAN patients and their relatives. Left: Percentage of Gigaxonin for each individual in comparison to wild type Gigaxonin, as the average of 3–5 independent experiments, after normalization with tubulin and GAPDH. Right: Mean abundance of Gigaxonin in patients and heterozygous individuals, as measured by the percentage in comparison to wild type Gigaxonin. (T-test, *, p

Techniques Used: Derivative Assay, T-Test

24) Product Images from "Role of myeloperoxidase in abdominal aortic aneurysm formation: mitigation by taurine"

Article Title: Role of myeloperoxidase in abdominal aortic aneurysm formation: mitigation by taurine

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00296.2017

Serum amyloid A (SAA) expression levels in apolipoprotein E-deficient (ApoE −/− ) mice versus ApoE/myeloperoxidase (MPO) double-knockout mice infused with ANG II. A : representative Western blot showing SAA protein expression in the plasma, liver, and aorta in ApoE −/− , ApoE −/− /MPO +/− , and ApoE −/− /MPO −/− mice infused with ANG II. Transferrin and GAPDH are shown as loading controls. B : densitometric analysis of SAA protein expression. * P
Figure Legend Snippet: Serum amyloid A (SAA) expression levels in apolipoprotein E-deficient (ApoE −/− ) mice versus ApoE/myeloperoxidase (MPO) double-knockout mice infused with ANG II. A : representative Western blot showing SAA protein expression in the plasma, liver, and aorta in ApoE −/− , ApoE −/− /MPO +/− , and ApoE −/− /MPO −/− mice infused with ANG II. Transferrin and GAPDH are shown as loading controls. B : densitometric analysis of SAA protein expression. * P

Techniques Used: Expressing, Mouse Assay, Double Knockout, Western Blot

Serum amyloid A (SAA) expression levels in apolipoprotein E-deficient (ApoE −/− ) mice treated with or without taurine and infused with ANG II. A : representative Western blot demonstrating effects of ANG II with and without taurine supplementation on SAA protein expression in the plasma, liver, and aorta. Transferrin and GAPDH are shown as loading controls. B : densitometric analysis of SAA protein expression. * P
Figure Legend Snippet: Serum amyloid A (SAA) expression levels in apolipoprotein E-deficient (ApoE −/− ) mice treated with or without taurine and infused with ANG II. A : representative Western blot demonstrating effects of ANG II with and without taurine supplementation on SAA protein expression in the plasma, liver, and aorta. Transferrin and GAPDH are shown as loading controls. B : densitometric analysis of SAA protein expression. * P

Techniques Used: Expressing, Mouse Assay, Western Blot

25) Product Images from "miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis"

Article Title: miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02643

Expression of miR-636, IL1R1, RANK, IKBKB, FAM13A, IL-8 , and IL-6 in CF and non-CF bronchial explants. Quantification of the expression of miR-636 ( A , p = 0.1781), IL1R1 ( B , p = 0.0024), RANK ( C , p = 0.0925), IKBKB ( D , p = 0.0053), FAM13A ( E , p = 0.0141), IL-8 ( F , p = 0.0332), and IL-6 ( G , p = 0.1063) relative to RNU6B and GAPDH , respectively, in bronchial explants CF ( n = 14) and non-CF ( n = 16). A Mann–Whitney test was used to determine significance, * p ≤ 0.05 and ** p ≤ 0.01.
Figure Legend Snippet: Expression of miR-636, IL1R1, RANK, IKBKB, FAM13A, IL-8 , and IL-6 in CF and non-CF bronchial explants. Quantification of the expression of miR-636 ( A , p = 0.1781), IL1R1 ( B , p = 0.0024), RANK ( C , p = 0.0925), IKBKB ( D , p = 0.0053), FAM13A ( E , p = 0.0141), IL-8 ( F , p = 0.0332), and IL-6 ( G , p = 0.1063) relative to RNU6B and GAPDH , respectively, in bronchial explants CF ( n = 14) and non-CF ( n = 16). A Mann–Whitney test was used to determine significance, * p ≤ 0.05 and ** p ≤ 0.01.

Techniques Used: Expressing, MANN-WHITNEY

26) Product Images from "Nuclear retention of importin α coordinates cell fate through changes in gene expression"

Article Title: Nuclear retention of importin α coordinates cell fate through changes in gene expression

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.360

Nuclear accumulation of importin α is a feature in caspase-independent cell death mode. ( A ) HeLa cells were exposed either to 5 mM H 2 O 2 or to 0.5 μM staurosporine (STS) at the indicated times. After incubation, the intracellular ATP levels were measured using CellTiter-Glo assay kit. The results were risen from three independent experiments and presented in comparison with the values in 0 h cells as the mean±s.e.m. ( n =3 each). ( B ) HeLa cells were exposed either to 5 mM H 2 O 2 or to 0.5 μM STS for 4 h. After incubation, the cells were fixed with 3.7% formalin and indirect immunofluorescence was performed to detect endogenous importin α2 using a specific antibody. DNA was visualized by Hoechst 33342. ( C ) HeLa cells were pretreated in the presence or absence of 50 μM zVAD-fmk for 30 min and treated with 5 mM H 2 O 2 or 0.5 μM STS for 4 h. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS–PAGE and analysed by western blotting for PARP and GAPDH. ( D ) HeLa cells were exposed to 5 mM H 2 O 2 for 4 h and were stained with PI and then sorted by FACS. Untreated cells were showed as a control (Cont.). Values are means±s.e.m. ( n =3 each) of PI-positive cells. ** P
Figure Legend Snippet: Nuclear accumulation of importin α is a feature in caspase-independent cell death mode. ( A ) HeLa cells were exposed either to 5 mM H 2 O 2 or to 0.5 μM staurosporine (STS) at the indicated times. After incubation, the intracellular ATP levels were measured using CellTiter-Glo assay kit. The results were risen from three independent experiments and presented in comparison with the values in 0 h cells as the mean±s.e.m. ( n =3 each). ( B ) HeLa cells were exposed either to 5 mM H 2 O 2 or to 0.5 μM STS for 4 h. After incubation, the cells were fixed with 3.7% formalin and indirect immunofluorescence was performed to detect endogenous importin α2 using a specific antibody. DNA was visualized by Hoechst 33342. ( C ) HeLa cells were pretreated in the presence or absence of 50 μM zVAD-fmk for 30 min and treated with 5 mM H 2 O 2 or 0.5 μM STS for 4 h. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS–PAGE and analysed by western blotting for PARP and GAPDH. ( D ) HeLa cells were exposed to 5 mM H 2 O 2 for 4 h and were stained with PI and then sorted by FACS. Untreated cells were showed as a control (Cont.). Values are means±s.e.m. ( n =3 each) of PI-positive cells. ** P

Techniques Used: Incubation, Glo Assay, Immunofluorescence, SDS Page, Western Blot, Staining, FACS

27) Product Images from "MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma"

Article Title: MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.25117

C-terminal region of MTBP is required for binding to IPO7 and inhibiting p-Erk nuclear translocation (A) Deletion constructs of MTBP tagged with the FLAG peptide at the N-terminus. MTBP: full-length MTBP; F4-MTBP (F4): N-terminal region deleted MTBP; ΔC-MTBP (ΔC): C-terminal region deleted MTBP. Numbers indicate amino acid of MTBP. Black bars indicate a nuclear localization signal (NLS) spanning between codons 730 and 753. (B) Co-immunoprecipitation (IP) studies between MTBP (FLAG tag) and IPO7 using protein extracts (~200 μg) from Huh7 cells expressing full-length MTBP (MTBP), F4-MTBP (F4), and ΔC-MTBP (ΔC). Isotypes were used as negative controls (m: mouse). 10% of the total amount of protein lysate (~20 μg) was used for input. (C) Immunofluorescence studies for p-Erk following treatment with solvent (EGF-) or 50 ng/ml of EGF for 30 min using PLC/PRF/5 cells with (ΔC) or without (control) overexpression of ΔC-MTBP. Scale bar, 50 μm. (D) Results of qRT-PCR to measure mRNA expression of EGR1 , c-fos , and PKCα using PLC/PRF/5 (top) and Huh7 (bottom) cells infected with lentiviral vectors encoding empty (control, grey) or ΔC-MTBP cDNA (ΔC, black), with (+) or without (−) 50 ng/ml of EGF stimulation for 15 min. Data are normalized by values of GAPDH mRNA. Error bars: means ± S.D. from three independent experiments. Student's t test. NS, not significant. (E) Transwell migration assays for 12 hours using PLC/PRF/5 (left) and Huh7 (right) cells infected with lentiviral vectors encoding empty (control) and ΔC-MTBP cDNA (ΔC). Error bars: means ± S.D. from three independent experiments. Student's t test. NS, not significant.
Figure Legend Snippet: C-terminal region of MTBP is required for binding to IPO7 and inhibiting p-Erk nuclear translocation (A) Deletion constructs of MTBP tagged with the FLAG peptide at the N-terminus. MTBP: full-length MTBP; F4-MTBP (F4): N-terminal region deleted MTBP; ΔC-MTBP (ΔC): C-terminal region deleted MTBP. Numbers indicate amino acid of MTBP. Black bars indicate a nuclear localization signal (NLS) spanning between codons 730 and 753. (B) Co-immunoprecipitation (IP) studies between MTBP (FLAG tag) and IPO7 using protein extracts (~200 μg) from Huh7 cells expressing full-length MTBP (MTBP), F4-MTBP (F4), and ΔC-MTBP (ΔC). Isotypes were used as negative controls (m: mouse). 10% of the total amount of protein lysate (~20 μg) was used for input. (C) Immunofluorescence studies for p-Erk following treatment with solvent (EGF-) or 50 ng/ml of EGF for 30 min using PLC/PRF/5 cells with (ΔC) or without (control) overexpression of ΔC-MTBP. Scale bar, 50 μm. (D) Results of qRT-PCR to measure mRNA expression of EGR1 , c-fos , and PKCα using PLC/PRF/5 (top) and Huh7 (bottom) cells infected with lentiviral vectors encoding empty (control, grey) or ΔC-MTBP cDNA (ΔC, black), with (+) or without (−) 50 ng/ml of EGF stimulation for 15 min. Data are normalized by values of GAPDH mRNA. Error bars: means ± S.D. from three independent experiments. Student's t test. NS, not significant. (E) Transwell migration assays for 12 hours using PLC/PRF/5 (left) and Huh7 (right) cells infected with lentiviral vectors encoding empty (control) and ΔC-MTBP cDNA (ΔC). Error bars: means ± S.D. from three independent experiments. Student's t test. NS, not significant.

Techniques Used: Binding Assay, Translocation Assay, Construct, Immunoprecipitation, FLAG-tag, Expressing, Immunofluorescence, Planar Chromatography, Over Expression, Quantitative RT-PCR, Infection, Migration

Cytoplasmic MTBP plays a role in inhibiting the Erk1/2-Elk-1 signaling (A) Co-immunoprecipitation studies between a NLS mutant MTBP (MTBP NLS ) and IPO7 using protein lysates (~200 μg) from PLC/PRF/5 cells expressing FLAG-tagged MTBP NLS . MTBP NLS was precipitated using anti-FLAG M2 affinity gel. Isotypes and protein A/G agarose beads were used as negative controls. 10% of the total amount of protein lysate (~20 μg) was used for input. (B) Immunofluorescence studies for p-Erk following treatment with solvent (EGF-) or 50 ng/ml of EGF for 30 min using PLC/PRF/5 (left) and Huh7 (right) cells with (MTBP NLS ) or without (control) overexpression of MTBP NLS . Scale bar, 25 μm. (C) Results of qRT-PCR to measure EGR1 , c-fos , and PKCα mRNA expression using PLC/PRF/5 (top) and Huh7 (bottom) cells infected with lentiviral vectors encoding empty (control, grey) or MTBP NLS cDNA (NLS, black), with (+) or without (−) 50 ng/ml of EGF stimulation for 15 min. Data are normalized by values of GAPDH mRNA. Error bars: means ± S.D. from three independent experiments. * , P
Figure Legend Snippet: Cytoplasmic MTBP plays a role in inhibiting the Erk1/2-Elk-1 signaling (A) Co-immunoprecipitation studies between a NLS mutant MTBP (MTBP NLS ) and IPO7 using protein lysates (~200 μg) from PLC/PRF/5 cells expressing FLAG-tagged MTBP NLS . MTBP NLS was precipitated using anti-FLAG M2 affinity gel. Isotypes and protein A/G agarose beads were used as negative controls. 10% of the total amount of protein lysate (~20 μg) was used for input. (B) Immunofluorescence studies for p-Erk following treatment with solvent (EGF-) or 50 ng/ml of EGF for 30 min using PLC/PRF/5 (left) and Huh7 (right) cells with (MTBP NLS ) or without (control) overexpression of MTBP NLS . Scale bar, 25 μm. (C) Results of qRT-PCR to measure EGR1 , c-fos , and PKCα mRNA expression using PLC/PRF/5 (top) and Huh7 (bottom) cells infected with lentiviral vectors encoding empty (control, grey) or MTBP NLS cDNA (NLS, black), with (+) or without (−) 50 ng/ml of EGF stimulation for 15 min. Data are normalized by values of GAPDH mRNA. Error bars: means ± S.D. from three independent experiments. * , P

Techniques Used: Immunoprecipitation, Mutagenesis, Planar Chromatography, Expressing, Immunofluorescence, Over Expression, Quantitative RT-PCR, Infection

28) Product Images from "Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells"

Article Title: Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

Journal: Oncotarget

doi:

Obscurin knockdown in MCF10A cells causes increased microtentacle formation and attachment A) MCF10A obscurin knockdown cells express more detyrosinated tubulin (glu-tubulin) and vimentin than shCtrl cells, as shown by immunoblot analysis. GAPDH serves as loading control. B) Approximately 40% of MCF10A cells expressing shObsc-1 or -2 form microtentacles compared to 10% shCtrl cells. N=3. Error Bars: +/- S.D. Asterisks: p
Figure Legend Snippet: Obscurin knockdown in MCF10A cells causes increased microtentacle formation and attachment A) MCF10A obscurin knockdown cells express more detyrosinated tubulin (glu-tubulin) and vimentin than shCtrl cells, as shown by immunoblot analysis. GAPDH serves as loading control. B) Approximately 40% of MCF10A cells expressing shObsc-1 or -2 form microtentacles compared to 10% shCtrl cells. N=3. Error Bars: +/- S.D. Asterisks: p

Techniques Used: Expressing

29) Product Images from "Role of forward and reverse signaling in Eph receptor and ephrin mediated cell segregation"

Article Title: Role of forward and reverse signaling in Eph receptor and ephrin mediated cell segregation

Journal: Experimental Cell Research

doi: 10.1016/j.yexcr.2019.04.040

Activation of EphB2 by ephrinB1 and ephrinB1-6F. (A): Cells expressing EphB2, ephrinB1 or ephrinB1-6F, or control HEK293 cells, were mixed in different combinations in a tube and centrifuged to force them into contact. The cells were lysed at the indicated time points, then subjected to Western blot analysis to detect EphB2, phospho-EphB2 and ephrinB1, and Gapdh as loading control. (B): Quantitation of phospho-EphB2 detected, normalised to the amount of EphB2 protein. The low amount of endogenous ephrinBs in HEK293 cells elicit low level EphB2 phosphorylation. There is a similar time course of EphB2 phosphorylation after interaction with ephrinB1 or ephrinB1-6F cells, with a > 30-fold increase by 5 min that progressively declines at 15 min and 30 min. (C-H): immunostaining of co-cultures of EphB2/ephrinB1 (C–E) and EphB2/ephrinB1-6F cells (F-H) to detect p-EphB2 and a marker of intracellular vesicles, Rab11. EphB2 cells co-express GFP (green signal in C, F). Activation of EphB2 with ephrinB1 or ephrinB1-6F leads to translocation from the cell surface to intracellular vesicles. Scale bar, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Figure Legend Snippet: Activation of EphB2 by ephrinB1 and ephrinB1-6F. (A): Cells expressing EphB2, ephrinB1 or ephrinB1-6F, or control HEK293 cells, were mixed in different combinations in a tube and centrifuged to force them into contact. The cells were lysed at the indicated time points, then subjected to Western blot analysis to detect EphB2, phospho-EphB2 and ephrinB1, and Gapdh as loading control. (B): Quantitation of phospho-EphB2 detected, normalised to the amount of EphB2 protein. The low amount of endogenous ephrinBs in HEK293 cells elicit low level EphB2 phosphorylation. There is a similar time course of EphB2 phosphorylation after interaction with ephrinB1 or ephrinB1-6F cells, with a > 30-fold increase by 5 min that progressively declines at 15 min and 30 min. (C-H): immunostaining of co-cultures of EphB2/ephrinB1 (C–E) and EphB2/ephrinB1-6F cells (F-H) to detect p-EphB2 and a marker of intracellular vesicles, Rab11. EphB2 cells co-express GFP (green signal in C, F). Activation of EphB2 with ephrinB1 or ephrinB1-6F leads to translocation from the cell surface to intracellular vesicles. Scale bar, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Techniques Used: Activation Assay, Expressing, Western Blot, Quantitation Assay, Immunostaining, Marker, Translocation Assay

30) Product Images from "The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence from an In Vitro Tumor Microenvironment Crosstalk Model"

Article Title: The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence from an In Vitro Tumor Microenvironment Crosstalk Model

Journal: Cancers

doi: 10.3390/cancers11121931

H295R cells stimulate ASC proliferation and drive ASC differentiation toward a myofibroblast-like phenotype. ( A ) ASCs alone (ASC) or co-cultured with H295R (ASC+H295R) were assessed for cell proliferation at the indicated time points (2, 3, 7 and 9 days) by direct cell count. The proliferative rate was calculated as fold increase (FI) versus the co-culture starting time (Time point = 0), n = 5. ( B ) Glucose uptake measurement and western blot analysis of GLUT-1 and GLUT-4 expression (inset, fold increase intensity vs. ASC after normalization on actin band is indicated to the right of the bands) assessed in ASCs after 7-day mono- or co-culture, n = 3. ( C ) Gene expression of specific mesenchymal stem-related markers revealed by RT-qPCR Taqman assay in 7-day co-cultured ASCs compared with the ASC mono-culture, n = 3. ( D ) Western blot analysis of α-SMA expression and optical microscopy of ASCs cultured alone or in the presence of H295R cells for 7 days. Original magnification: 10×; zoom in: 2×. For western blot analysis, GAPDH or actin were used as internal loading control. Gene expression and glucose uptake are indicated as fold increase (FI) versus ASCs alone. Data are expressed as the mean ± SE in at least three independent experiments; * p
Figure Legend Snippet: H295R cells stimulate ASC proliferation and drive ASC differentiation toward a myofibroblast-like phenotype. ( A ) ASCs alone (ASC) or co-cultured with H295R (ASC+H295R) were assessed for cell proliferation at the indicated time points (2, 3, 7 and 9 days) by direct cell count. The proliferative rate was calculated as fold increase (FI) versus the co-culture starting time (Time point = 0), n = 5. ( B ) Glucose uptake measurement and western blot analysis of GLUT-1 and GLUT-4 expression (inset, fold increase intensity vs. ASC after normalization on actin band is indicated to the right of the bands) assessed in ASCs after 7-day mono- or co-culture, n = 3. ( C ) Gene expression of specific mesenchymal stem-related markers revealed by RT-qPCR Taqman assay in 7-day co-cultured ASCs compared with the ASC mono-culture, n = 3. ( D ) Western blot analysis of α-SMA expression and optical microscopy of ASCs cultured alone or in the presence of H295R cells for 7 days. Original magnification: 10×; zoom in: 2×. For western blot analysis, GAPDH or actin were used as internal loading control. Gene expression and glucose uptake are indicated as fold increase (FI) versus ASCs alone. Data are expressed as the mean ± SE in at least three independent experiments; * p

Techniques Used: Cell Culture, Cell Counting, Co-Culture Assay, Western Blot, Expressing, Quantitative RT-PCR, TaqMan Assay, Microscopy

The potential molecular crosstalk underlying H295R/ASCs interaction . RT-qPCR Taqman analysis of gene expression in both ASCs and H295R cells after 7-day mono- or co-culture (ASC or H295R cells and ASC + H295R or H295R + ASC, respectively). Leptin and IL-8 ( A ), and CXCL12 (SDF-1) and DDP4 ( C ) gene expression was evaluated in ASCs, and the intracellular SDF-1 protein expression was assessed by western blot analysis ( C ), inset; fold increase intensity vs. ASCs after normalization on GAPDH band is indicated to the right of the band). IGF2, IGF-1R and Ob-R gene expression was evaluated in H295R cells ( B ). For all genes, the relative expression level was calculated as fold increase (FI) versus the mono-culture. For western blot analysis, GAPDH was used as internal loading control. ( D ) Extracellular levels of SDF-1 measured by ELISA assay in the conditioned medium of ASCs after 7-day mono- and co-culture. The absolute SDF-1 concentrations were normalized on the relative cell number and expressed as fold increase (FI) versus ASCs. ( E ) CXCR4 and CXCR7 expression was evaluated in H295R cells alone or in co-culture with ASCs. Data are expressed as the mean ± SE vs. the respective monoculture in at least three independent experiments; * p
Figure Legend Snippet: The potential molecular crosstalk underlying H295R/ASCs interaction . RT-qPCR Taqman analysis of gene expression in both ASCs and H295R cells after 7-day mono- or co-culture (ASC or H295R cells and ASC + H295R or H295R + ASC, respectively). Leptin and IL-8 ( A ), and CXCL12 (SDF-1) and DDP4 ( C ) gene expression was evaluated in ASCs, and the intracellular SDF-1 protein expression was assessed by western blot analysis ( C ), inset; fold increase intensity vs. ASCs after normalization on GAPDH band is indicated to the right of the band). IGF2, IGF-1R and Ob-R gene expression was evaluated in H295R cells ( B ). For all genes, the relative expression level was calculated as fold increase (FI) versus the mono-culture. For western blot analysis, GAPDH was used as internal loading control. ( D ) Extracellular levels of SDF-1 measured by ELISA assay in the conditioned medium of ASCs after 7-day mono- and co-culture. The absolute SDF-1 concentrations were normalized on the relative cell number and expressed as fold increase (FI) versus ASCs. ( E ) CXCR4 and CXCR7 expression was evaluated in H295R cells alone or in co-culture with ASCs. Data are expressed as the mean ± SE vs. the respective monoculture in at least three independent experiments; * p

Techniques Used: Quantitative RT-PCR, Expressing, Co-Culture Assay, Western Blot, Enzyme-linked Immunosorbent Assay

31) Product Images from "Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma"

Article Title: Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma

Journal: Scientific Reports

doi: 10.1038/s41598-020-62690-9

Narciclasine downregulates MYC. ( A) BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 48 hours) or DMSO control, followed by western blotting of whole cell lysates for MYC and GAPDH (loading control). Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S5 . Blots are representative of at least 3 independent experiments. ( B) BC-1 and BC-3 cell lines were treated with narciclasine (50 nM for 24 hours) or DMSO control followed by qRT-PCR analysis of TERT, SLC19A1, MYB and PMM2 mRNA (direct target genes of MYC protein). Real-time PCR reactions were carried out in triplicate and the data were presented as fold change in target gene expression (mean ± SE) from a representative of 2 independent experiments. Statistically significant differences were shown by asterisks (*) at a level of p ≤ 0.05, (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001.
Figure Legend Snippet: Narciclasine downregulates MYC. ( A) BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 48 hours) or DMSO control, followed by western blotting of whole cell lysates for MYC and GAPDH (loading control). Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S5 . Blots are representative of at least 3 independent experiments. ( B) BC-1 and BC-3 cell lines were treated with narciclasine (50 nM for 24 hours) or DMSO control followed by qRT-PCR analysis of TERT, SLC19A1, MYB and PMM2 mRNA (direct target genes of MYC protein). Real-time PCR reactions were carried out in triplicate and the data were presented as fold change in target gene expression (mean ± SE) from a representative of 2 independent experiments. Statistically significant differences were shown by asterisks (*) at a level of p ≤ 0.05, (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001.

Techniques Used: Western Blot, Derivative Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing

Narciclasine induces apoptosis in PEL cells. ( A) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, stained with Annexin V-FITC/propidium iodide, and analyzed for apoptosis by flow cytometry. Data are representative of 3 independent experiments. The gating of cells for analysis were presented in Supplementary Fig. S3 . ( B) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, followed by measurement of active caspase-3/7 using Apo-ONE homogeneous assay kit. Data are representative of 2 independent experiments. Statistically significant differences were shown by asterisks (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001. ns – not significant. ( C) BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 48 hours) or DMSO control, followed by western blotting of whole cell lysates for cleavage of PARP and GAPDH (loading control). Cl – Cleaved; FL – Full Length. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S4 . Blots are representative of 3 independent experiments.
Figure Legend Snippet: Narciclasine induces apoptosis in PEL cells. ( A) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, stained with Annexin V-FITC/propidium iodide, and analyzed for apoptosis by flow cytometry. Data are representative of 3 independent experiments. The gating of cells for analysis were presented in Supplementary Fig. S3 . ( B) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, followed by measurement of active caspase-3/7 using Apo-ONE homogeneous assay kit. Data are representative of 2 independent experiments. Statistically significant differences were shown by asterisks (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001. ns – not significant. ( C) BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 48 hours) or DMSO control, followed by western blotting of whole cell lysates for cleavage of PARP and GAPDH (loading control). Cl – Cleaved; FL – Full Length. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S4 . Blots are representative of 3 independent experiments.

Techniques Used: Staining, Flow Cytometry, Western Blot, Derivative Assay

MYC is not a primary target of narciclasine. BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 12, 24, 36 and 48 hours) or DMSO control, followed by western blotting of whole cell lysates for PARP, MYC, Caspase-3 and GAPDH (loading control). Cl – Cleaved; FL – Full Length. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Figs. S6 – S8 .
Figure Legend Snippet: MYC is not a primary target of narciclasine. BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 12, 24, 36 and 48 hours) or DMSO control, followed by western blotting of whole cell lysates for PARP, MYC, Caspase-3 and GAPDH (loading control). Cl – Cleaved; FL – Full Length. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Figs. S6 – S8 .

Techniques Used: Western Blot, Derivative Assay

32) Product Images from "Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer"

Article Title: Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer

Journal: Molecular Therapy Oncolytics

doi: 10.1016/j.omto.2020.06.021

PTX Enhances the Replication Efficiency of OBP-401 in Human Gastric Cancer Cells (A) Time-lapse imaging of GCIY cells treated with OBP-401, PTX, or both. Green color indicates OBP-401 replication, and red color indicates PTX uptake. Scale bar, 50 μm. (B) Expression of adenoviral E1A proteins in GCIY and KATOIII cells treated with OBP-401 and PTX. β-actin was used as a loading control. The band intensity of E1A was normalized against that of β-actin and the ratios are shown. (C) Assessment of viral replication in GCIY cells. GCIY cells were treated with OBP-401 (1 MOI) or the combination of OBP-401 (1 MOI) and PTX (0.1 μM). Quantitative real-time PCR assay was performed to quantify the amount of viral E1A copy number. The copy number of viral E1A is defined as the E1A/GAPDH ratio relative to that of the sample 2 h after OBP-401 infection (2 h after OBP-401 infection = 1). Data are shown as means ± SD. Statistical significance was defined as p
Figure Legend Snippet: PTX Enhances the Replication Efficiency of OBP-401 in Human Gastric Cancer Cells (A) Time-lapse imaging of GCIY cells treated with OBP-401, PTX, or both. Green color indicates OBP-401 replication, and red color indicates PTX uptake. Scale bar, 50 μm. (B) Expression of adenoviral E1A proteins in GCIY and KATOIII cells treated with OBP-401 and PTX. β-actin was used as a loading control. The band intensity of E1A was normalized against that of β-actin and the ratios are shown. (C) Assessment of viral replication in GCIY cells. GCIY cells were treated with OBP-401 (1 MOI) or the combination of OBP-401 (1 MOI) and PTX (0.1 μM). Quantitative real-time PCR assay was performed to quantify the amount of viral E1A copy number. The copy number of viral E1A is defined as the E1A/GAPDH ratio relative to that of the sample 2 h after OBP-401 infection (2 h after OBP-401 infection = 1). Data are shown as means ± SD. Statistical significance was defined as p

Techniques Used: Imaging, Expressing, Real-time Polymerase Chain Reaction, Infection

33) Product Images from "Inhibitory Effects of Sigma-2 Receptor Agonists on T Lymphocyte Activation"

Article Title: Inhibitory Effects of Sigma-2 Receptor Agonists on T Lymphocyte Activation

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2013.00023

Effect of σ-2 ligands on the expression of IL-2, TNFα, and COX-2 in Jurkat T cells . (A) Total RNA from Jurkat cells, treated with σ-2 agonists BD-737 and CB-184 or the antagonist AC-927 at the doses indicated (1, 5, and 10 μM) and grown in the absence or presence of PMA (15 ng/ml) + Ion (1 μM), was analyzed by quantitative RT-PCR. Relative quantification (RQ) of mRNA levels of IL-2, TNFα, and COX-2 was determined using endogenous expression of GAPDH and is shown as RQ ± SD. (B) PGE 2 production by Jurkat cells treated with σ-2 agonists BD-737 and CB-184 at the doses indicated (1 and 10 μM), grown in the absence or presence of PMA + Ion. PGE 2 was measured by a standard EIA assay as described in Section “ Materials and Methods .” Results are shown as the mean ± SD of PGE 2 in pg/ml of determinations conducted in triplicate. (n.s, not significant; * p
Figure Legend Snippet: Effect of σ-2 ligands on the expression of IL-2, TNFα, and COX-2 in Jurkat T cells . (A) Total RNA from Jurkat cells, treated with σ-2 agonists BD-737 and CB-184 or the antagonist AC-927 at the doses indicated (1, 5, and 10 μM) and grown in the absence or presence of PMA (15 ng/ml) + Ion (1 μM), was analyzed by quantitative RT-PCR. Relative quantification (RQ) of mRNA levels of IL-2, TNFα, and COX-2 was determined using endogenous expression of GAPDH and is shown as RQ ± SD. (B) PGE 2 production by Jurkat cells treated with σ-2 agonists BD-737 and CB-184 at the doses indicated (1 and 10 μM), grown in the absence or presence of PMA + Ion. PGE 2 was measured by a standard EIA assay as described in Section “ Materials and Methods .” Results are shown as the mean ± SD of PGE 2 in pg/ml of determinations conducted in triplicate. (n.s, not significant; * p

Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

34) Product Images from "Apigenin Sensitizes Prostate Cancer Cells to Apo2L/TRAIL by Targeting Adenine Nucleotide Translocase-2"

Article Title: Apigenin Sensitizes Prostate Cancer Cells to Apo2L/TRAIL by Targeting Adenine Nucleotide Translocase-2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055922

Apigenin enhances Apo2L/TRAIL-induced apoptosis by inhibiting ANT2 in LNCaP cells. LNCaP cells were transfected with siANT2 or siCtrl and were incubated for 48 hr. A, ANT2 mRNA was quantified by real-time RT-PCR and normalized by GAPDH. B, Protein levels of DR5 and β-actin were analyzed by Western blotting. C, The cells were incubated with or without 50 ng/ml Apo2L/TRAIL. After 24 hr, the sub-G1 population of the cells was measured with flow cytometry. D, The cells were incubated with 20 µmol/L apigenin for 24 hr, followed by treatment with or without 50 ng/ml Apo2L/TRAIL. The Apo2L/TRAIL activity in samples without apigenin was normalized to 1. E, The cells were incubated with 20 µmol/L apigenin for 24 hr. Protein levels of DR5 and β-actin were analyzed by Western blotting. Columns, mean; bars, SD (n = 3). * P
Figure Legend Snippet: Apigenin enhances Apo2L/TRAIL-induced apoptosis by inhibiting ANT2 in LNCaP cells. LNCaP cells were transfected with siANT2 or siCtrl and were incubated for 48 hr. A, ANT2 mRNA was quantified by real-time RT-PCR and normalized by GAPDH. B, Protein levels of DR5 and β-actin were analyzed by Western blotting. C, The cells were incubated with or without 50 ng/ml Apo2L/TRAIL. After 24 hr, the sub-G1 population of the cells was measured with flow cytometry. D, The cells were incubated with 20 µmol/L apigenin for 24 hr, followed by treatment with or without 50 ng/ml Apo2L/TRAIL. The Apo2L/TRAIL activity in samples without apigenin was normalized to 1. E, The cells were incubated with 20 µmol/L apigenin for 24 hr. Protein levels of DR5 and β-actin were analyzed by Western blotting. Columns, mean; bars, SD (n = 3). * P

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

Apigenin enhances Apo2L/TRAIL-induced apoptosis via upregulation of DR5. A, DU145 cells were treated with various concentrations of each flavonoid for 24 hr, followed by treatment with or without 50 ng/ml Apo2L/TRAIL. After 24 hr, the sub-G1 population of the cells was measured using flow cytometry. B, DU145 cells were treated with the indicated concentrations of each flavonoid for 24 hr and harvested. The lysates were analyzed using Western blotting with an anti-DR5 antibody. β-actin was used as a loading control. C, DR5 mRNA was quantified by real-time RT-PCR and normalized by GAPDH. Columns, mean; bars, SD (n = 3). ** P
Figure Legend Snippet: Apigenin enhances Apo2L/TRAIL-induced apoptosis via upregulation of DR5. A, DU145 cells were treated with various concentrations of each flavonoid for 24 hr, followed by treatment with or without 50 ng/ml Apo2L/TRAIL. After 24 hr, the sub-G1 population of the cells was measured using flow cytometry. B, DU145 cells were treated with the indicated concentrations of each flavonoid for 24 hr and harvested. The lysates were analyzed using Western blotting with an anti-DR5 antibody. β-actin was used as a loading control. C, DR5 mRNA was quantified by real-time RT-PCR and normalized by GAPDH. Columns, mean; bars, SD (n = 3). ** P

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

Knockdown of ANT2 enhances Apo2L/TRAIL-induced apoptosis by upregulating DR5 at the post-transcriptional level. DU145 cells were transfected with two different ANT2 siRNAs (siANT2 #1 and #2) or a non-targeting siRNA (siCtrl) and were incubated for 48 hr. A, ANT2 mRNA was quantified by real-time RT-PCR and normalized by GAPDH. B, The cells were treated with or without 50 ng/ml Apo2L/TRAIL. After 24 hr, the sub-G1 population of the cells was measured with flow cytometry. C, Protein levels of DR5 and β-actin were analyzed by Western blotting. D, mRNA levels of DR5 were measured by real-time RT-PCR and normalized by GAPDH. Columns, mean; bars, SD (n = 3). * P
Figure Legend Snippet: Knockdown of ANT2 enhances Apo2L/TRAIL-induced apoptosis by upregulating DR5 at the post-transcriptional level. DU145 cells were transfected with two different ANT2 siRNAs (siANT2 #1 and #2) or a non-targeting siRNA (siCtrl) and were incubated for 48 hr. A, ANT2 mRNA was quantified by real-time RT-PCR and normalized by GAPDH. B, The cells were treated with or without 50 ng/ml Apo2L/TRAIL. After 24 hr, the sub-G1 population of the cells was measured with flow cytometry. C, Protein levels of DR5 and β-actin were analyzed by Western blotting. D, mRNA levels of DR5 were measured by real-time RT-PCR and normalized by GAPDH. Columns, mean; bars, SD (n = 3). * P

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

35) Product Images from "Intrarenal Renin Angiotensin System Revisited"

Article Title: Intrarenal Renin Angiotensin System Revisited

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.150284

Quantitative RT-PCR analysis of AGT mRNA expression from isolated single-nephron preparations. The percentages of AGT mRNA relative to the respective GAPDH mRNA abundances are shown for proximal S1, S1/S2, and S3 segments. *, p
Figure Legend Snippet: Quantitative RT-PCR analysis of AGT mRNA expression from isolated single-nephron preparations. The percentages of AGT mRNA relative to the respective GAPDH mRNA abundances are shown for proximal S1, S1/S2, and S3 segments. *, p

Techniques Used: Quantitative RT-PCR, Expressing, Isolation

36) Product Images from "Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis"

Article Title: Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis

Journal: Oncotarget

doi: 10.18632/oncotarget.12189

MS-444 inhibits COX-2 expression and promotes ARE-containing mRNA trafficking to P-bodies A. HCA-7 cells were treated with 12 μM MS-444 or a vehicle control for the indicated times. COX-2 mRNA levels were assayed by qPCR using GAPDH as a loading control and normalized to non-treated cells. Each value represents an average of triplicates ± SEM. B. HCA-7 cells were with the indicated concentrations of MS-444 for 8 hr and COX-2 mRNA levels were assayed by qPCR. The data was fitted to a dose-response curve to determine the IC 50 with respect to COX-2 mRNA expression. Each data point represents an average of triplicates ± SEM. C. HCA-7 cells were treated with the indicated concentrations of MS-444 for 8 hr and COX-2 protein expression was assayed by western blot. Actin was used as a loading control. D. HCA-7 cells were treated with DMSO (control) or 10 μM MS-444 for 6 hr and RNP-IP of HuR or control IgG was done to isolate mRNAs bound by HuR. qPCR was used to quantitate COX-2 mRNA levels normalized to IgG control as the average from 3 independent experiments ± SEM. E. HCT116 cells were transfected with luciferase reporter constructs without the COX-2 3′UTR (LucΔ3′UTR) or fused to the full-length COX-2 3′UTR and treated with 10 μM MS-444 for 8 hr. Luciferase activity was normalized to total protein activity and is the average of 3 experiments ± SEM. N.S., not significant. F. Representation of the MS2 system showing the reporter mRNA containing MS2 sites present upstream of the ARE-containing COX-2 3′UTR bound by YFP-tagged MS2 binding protein, allowing for fluorescent visualization of the mRNA. HCT116 cells were transfected with the MS2 dual plasmid system using the MS2-COX-2 3′UTR mRNA expression constructs, along with MS2-YFP and mCherry-tagged Dcp1a to visualize mRNA and P-bodies, respectively. Cells were treated with 10 μM MS-444 for 8 hr and visualized for P-bodies (red) and MS2-YFP-bound mRNA (pseudo-colored green). Arrowheads indicate representative P-body signal with boxed areas enlarged on right showing Dcp1a and YFP-MS2 signal. Scale bar = 10 μm. G. Co-localization between the reporter MS2-COX-2 3′UTR and P-bodies is shown as percentage of P-bodies per cell exhibiting co-localization between the MS-2-bound YFP mRNA reporter and Dcp1a signal ± SEM (n = 10 cells per group).
Figure Legend Snippet: MS-444 inhibits COX-2 expression and promotes ARE-containing mRNA trafficking to P-bodies A. HCA-7 cells were treated with 12 μM MS-444 or a vehicle control for the indicated times. COX-2 mRNA levels were assayed by qPCR using GAPDH as a loading control and normalized to non-treated cells. Each value represents an average of triplicates ± SEM. B. HCA-7 cells were with the indicated concentrations of MS-444 for 8 hr and COX-2 mRNA levels were assayed by qPCR. The data was fitted to a dose-response curve to determine the IC 50 with respect to COX-2 mRNA expression. Each data point represents an average of triplicates ± SEM. C. HCA-7 cells were treated with the indicated concentrations of MS-444 for 8 hr and COX-2 protein expression was assayed by western blot. Actin was used as a loading control. D. HCA-7 cells were treated with DMSO (control) or 10 μM MS-444 for 6 hr and RNP-IP of HuR or control IgG was done to isolate mRNAs bound by HuR. qPCR was used to quantitate COX-2 mRNA levels normalized to IgG control as the average from 3 independent experiments ± SEM. E. HCT116 cells were transfected with luciferase reporter constructs without the COX-2 3′UTR (LucΔ3′UTR) or fused to the full-length COX-2 3′UTR and treated with 10 μM MS-444 for 8 hr. Luciferase activity was normalized to total protein activity and is the average of 3 experiments ± SEM. N.S., not significant. F. Representation of the MS2 system showing the reporter mRNA containing MS2 sites present upstream of the ARE-containing COX-2 3′UTR bound by YFP-tagged MS2 binding protein, allowing for fluorescent visualization of the mRNA. HCT116 cells were transfected with the MS2 dual plasmid system using the MS2-COX-2 3′UTR mRNA expression constructs, along with MS2-YFP and mCherry-tagged Dcp1a to visualize mRNA and P-bodies, respectively. Cells were treated with 10 μM MS-444 for 8 hr and visualized for P-bodies (red) and MS2-YFP-bound mRNA (pseudo-colored green). Arrowheads indicate representative P-body signal with boxed areas enlarged on right showing Dcp1a and YFP-MS2 signal. Scale bar = 10 μm. G. Co-localization between the reporter MS2-COX-2 3′UTR and P-bodies is shown as percentage of P-bodies per cell exhibiting co-localization between the MS-2-bound YFP mRNA reporter and Dcp1a signal ± SEM (n = 10 cells per group).

Techniques Used: Mass Spectrometry, Expressing, High Content Screening, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Binding Assay, Plasmid Preparation

MS-444 inhibits COX-2 expression in vivo A. Tumor growth of HCA-7 cell implants in nude mice treated with 25 mg/kg MS-444 or vehicle control every 48 hr. Representative tumors excised at day 44 and are shown. *, P ≤ 0.05. B. IHC detection of COX-2. Graph indicates the average immunoreactivity scores (IRS) from stained tumor sections (n=10 tumors). C. Tumor growth of established HCA-7 tumors in mice treated with 25 mg/kg bw MS-444 or vehicle control by IP for 3 weeks as indicated. *, P ≤ 0.01. D. After 21 days, mice were injected with 2 mg/kg Fluorocoxib, tumors were excised and imaged to detect COX-2 expression ex vivo . Epi-fluorescence scale is shown. E. COX-2 mRNA expression of excised tumors was measured by qPCR using GAPDH as a loading control.
Figure Legend Snippet: MS-444 inhibits COX-2 expression in vivo A. Tumor growth of HCA-7 cell implants in nude mice treated with 25 mg/kg MS-444 or vehicle control every 48 hr. Representative tumors excised at day 44 and are shown. *, P ≤ 0.05. B. IHC detection of COX-2. Graph indicates the average immunoreactivity scores (IRS) from stained tumor sections (n=10 tumors). C. Tumor growth of established HCA-7 tumors in mice treated with 25 mg/kg bw MS-444 or vehicle control by IP for 3 weeks as indicated. *, P ≤ 0.01. D. After 21 days, mice were injected with 2 mg/kg Fluorocoxib, tumors were excised and imaged to detect COX-2 expression ex vivo . Epi-fluorescence scale is shown. E. COX-2 mRNA expression of excised tumors was measured by qPCR using GAPDH as a loading control.

Techniques Used: Mass Spectrometry, Expressing, In Vivo, High Content Screening, Mouse Assay, Immunohistochemistry, Staining, Injection, Ex Vivo, Fluorescence, Real-time Polymerase Chain Reaction

37) Product Images from "Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-?B activation: independence from endogenous TNF-? and platelet-activating factor"

Article Title: Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-?B activation: independence from endogenous TNF-? and platelet-activating factor

Journal: Immunology

doi: 10.1111/j.1365-2567.2006.02344.x

Anti-TNF antibodies do not block LPS-induced CXCL2 transcription in IEC-6 cells. Cells were treated with anti-TNF antibodies (0·08 µg/ml) or control antibodies (0·08 µg/ml) for 30 min, then incubated with LPS (5 µg/ml) or culture medium for 2 hr. Cellular RNA was extracted and RT–PCR performed using primer specific for CXCL2 and GAPDH. PCR products were run on a 1·5% agarose gel and stained with SYBR green I. Similar results were obtained in two independent experiments with three samples per group.
Figure Legend Snippet: Anti-TNF antibodies do not block LPS-induced CXCL2 transcription in IEC-6 cells. Cells were treated with anti-TNF antibodies (0·08 µg/ml) or control antibodies (0·08 µg/ml) for 30 min, then incubated with LPS (5 µg/ml) or culture medium for 2 hr. Cellular RNA was extracted and RT–PCR performed using primer specific for CXCL2 and GAPDH. PCR products were run on a 1·5% agarose gel and stained with SYBR green I. Similar results were obtained in two independent experiments with three samples per group.

Techniques Used: Blocking Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

WEB2170 does not block LPS-induced CXCL2 transcription in IEC-6 cells. Following pretreatment with either WEB2170 (1, 5 and 10 µg/ml) or culture medium for 30 and 60 min, cells were treated with LPS for 2 hr. Some cells were treated with WEB2170 alone. Cellular RNA was extracted and RT–PCR performed using primer specific for CXCL2 and GAPDH. PCR products were run on a 1·5% agarose gel and stained with SYBR green I. Similar results were obtained in two independent experiments.
Figure Legend Snippet: WEB2170 does not block LPS-induced CXCL2 transcription in IEC-6 cells. Following pretreatment with either WEB2170 (1, 5 and 10 µg/ml) or culture medium for 30 and 60 min, cells were treated with LPS for 2 hr. Some cells were treated with WEB2170 alone. Cellular RNA was extracted and RT–PCR performed using primer specific for CXCL2 and GAPDH. PCR products were run on a 1·5% agarose gel and stained with SYBR green I. Similar results were obtained in two independent experiments.

Techniques Used: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

38) Product Images from "PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions"

Article Title: PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

Journal: Scientific Reports

doi: 10.1038/srep18417

Akt activation induced by serum withdrawal triggers upregulation of P2X7R receptor expression in neuroblastoma cells. ( A ) Changes in Akt phosphorylation in N2a cells cultured in SF medium for the indicated time points. Whole-cell lysates were analyzed by western blotting with antibodies against phospho-Akt (Thr 308 ) or total Akt. Histogram represents relative levels of phospho-Akt (pAkt) during the whole detection period, obtained by densitometry and normalization to total Akt. The values represent mean ± s.e.m. of four independent experiments in duplicate. * P ≤ 0.05, ** P ≤ 0.01 vs time = 0 (ANOVA with the Dunnett’s post hoc test). ( B ) N2a cells were incubated in absence or presence of API-1 (10 μM, Akt inhibitor) in SF medium for 24 h. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Results are mean ± s.e.m. of three independent experiments in triplicate. *** P ≤ 0.001 vs control (t-test). ( C ) Immunoblotting depicting the presence of endogenous P2X7R in whole-cell lysates from N2a cells cultured in SF medium for 24 h in the absence (control) or presence of API-1. Whole-cell lysates were analyzed by western blotting with anti-P2X7R antibody. GAPDH was used as internal loading control. Histogram represents levels of P2X7 protein in control cells compared to treated cells, obtained by densitometry and normalization to GAPDH. Values are mean ± s.e.m. of three independent experiments in duplicate; ** P ≤ 0.01 vs control (t-test). ( D ) Immunofluorescence for P2X7R (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of API-1. Scale bar = 15 μm.
Figure Legend Snippet: Akt activation induced by serum withdrawal triggers upregulation of P2X7R receptor expression in neuroblastoma cells. ( A ) Changes in Akt phosphorylation in N2a cells cultured in SF medium for the indicated time points. Whole-cell lysates were analyzed by western blotting with antibodies against phospho-Akt (Thr 308 ) or total Akt. Histogram represents relative levels of phospho-Akt (pAkt) during the whole detection period, obtained by densitometry and normalization to total Akt. The values represent mean ± s.e.m. of four independent experiments in duplicate. * P ≤ 0.05, ** P ≤ 0.01 vs time = 0 (ANOVA with the Dunnett’s post hoc test). ( B ) N2a cells were incubated in absence or presence of API-1 (10 μM, Akt inhibitor) in SF medium for 24 h. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Results are mean ± s.e.m. of three independent experiments in triplicate. *** P ≤ 0.001 vs control (t-test). ( C ) Immunoblotting depicting the presence of endogenous P2X7R in whole-cell lysates from N2a cells cultured in SF medium for 24 h in the absence (control) or presence of API-1. Whole-cell lysates were analyzed by western blotting with anti-P2X7R antibody. GAPDH was used as internal loading control. Histogram represents levels of P2X7 protein in control cells compared to treated cells, obtained by densitometry and normalization to GAPDH. Values are mean ± s.e.m. of three independent experiments in duplicate; ** P ≤ 0.01 vs control (t-test). ( D ) Immunofluorescence for P2X7R (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of API-1. Scale bar = 15 μm.

Techniques Used: Activation Assay, Expressing, Cell Culture, Western Blot, Incubation, Polymerase Chain Reaction, T-Test, Immunofluorescence

Involvement of PI3K and PKC in the regulation of P2X7R expression in neuroblastoma cells following serum withdrawal. ( A ) N2a cells were incubated in FBS medium for 24 h or in SF medium in absence (control) or presence of LY294002 (50 μM, PI3K inhibitor), U0126 (10 μM, MEK/ERK1/2 inhibitor), H89 (1 μM, PKA inhibitor), GF109203X (5 μM, pan PKC inhibitor) or KN93 (1 μM, CaMKII inhibitor) in SF medium for 24 h. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Normalized P2X7 transcript levels in cells cultured in FBS was set as 100%. Results are mean ± s.e.m. of three independent experiments in triplicate. ** P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test). ( B ) Immunoblotting showing the presence of endogenous P2X7R in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or GF109203X. Whole-cell lysates were analyzed by western blotting with anti-P2X7R antibody. GAPDH was used as internal loading control. Histogram represents levels of P2X7 protein in control cells compared to treated cells and were obtained by densitometry and normalization to GAPDH. Values are mean ± s.e.m. of three independent experiments in duplicate; * P ≤ 0.05, ** P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test). ( C ) Immunofluorescence for P2X7R (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or GF109203X. Scale bar = 15 μm.
Figure Legend Snippet: Involvement of PI3K and PKC in the regulation of P2X7R expression in neuroblastoma cells following serum withdrawal. ( A ) N2a cells were incubated in FBS medium for 24 h or in SF medium in absence (control) or presence of LY294002 (50 μM, PI3K inhibitor), U0126 (10 μM, MEK/ERK1/2 inhibitor), H89 (1 μM, PKA inhibitor), GF109203X (5 μM, pan PKC inhibitor) or KN93 (1 μM, CaMKII inhibitor) in SF medium for 24 h. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Normalized P2X7 transcript levels in cells cultured in FBS was set as 100%. Results are mean ± s.e.m. of three independent experiments in triplicate. ** P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test). ( B ) Immunoblotting showing the presence of endogenous P2X7R in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or GF109203X. Whole-cell lysates were analyzed by western blotting with anti-P2X7R antibody. GAPDH was used as internal loading control. Histogram represents levels of P2X7 protein in control cells compared to treated cells and were obtained by densitometry and normalization to GAPDH. Values are mean ± s.e.m. of three independent experiments in duplicate; * P ≤ 0.05, ** P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test). ( C ) Immunofluorescence for P2X7R (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or GF109203X. Scale bar = 15 μm.

Techniques Used: Expressing, Incubation, Polymerase Chain Reaction, Cell Culture, Western Blot, Immunofluorescence

Sp1 transcription factor is upregulated in serum-deprived neuroblastoma cells. ( A ) Changes in Sp1 transcript levels in N2a cells cultured either in FBS medium for 24 h or in SF medium for the indicated time periods. Total RNA was extracted and Sp1 mRNA was quantified by Q-PCR. GAPDH was used as a control for differences in cDNA input. Results are mean ± s.e.m. of three independent experiments in triplicate. * P ≤ 0.05, *** P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). ( B ) Immunoblotting depicting the presence of endogenous Sp1 in whole-cell lysates from N2a cells cultured in FBS for 24 or in SF at different time points. Cell lysates were analyzed by western blotting with anti-Sp1 antibody. GAPDH was used as an internal loading control. Histogram represents total Sp1 levels during the whole detection period, obtained by densitometry and normalization to GAPDH. The values represent mean ± s.e.m. of three independent experiments in duplicate. * P ≤ 0.05, ** P ≤ 0.01 vs FBS (ANOVA with the Dunnett’s post hoc test). ( C ) Double immunofluorescence for Sp1 (green) and α-tubulin (red) in N2a cells cultured either in FBS for 24 h or in SF medium for 72 h. Nuclei were counterstained with DAPI (blue). Scale bar = 15 μm.
Figure Legend Snippet: Sp1 transcription factor is upregulated in serum-deprived neuroblastoma cells. ( A ) Changes in Sp1 transcript levels in N2a cells cultured either in FBS medium for 24 h or in SF medium for the indicated time periods. Total RNA was extracted and Sp1 mRNA was quantified by Q-PCR. GAPDH was used as a control for differences in cDNA input. Results are mean ± s.e.m. of three independent experiments in triplicate. * P ≤ 0.05, *** P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). ( B ) Immunoblotting depicting the presence of endogenous Sp1 in whole-cell lysates from N2a cells cultured in FBS for 24 or in SF at different time points. Cell lysates were analyzed by western blotting with anti-Sp1 antibody. GAPDH was used as an internal loading control. Histogram represents total Sp1 levels during the whole detection period, obtained by densitometry and normalization to GAPDH. The values represent mean ± s.e.m. of three independent experiments in duplicate. * P ≤ 0.05, ** P ≤ 0.01 vs FBS (ANOVA with the Dunnett’s post hoc test). ( C ) Double immunofluorescence for Sp1 (green) and α-tubulin (red) in N2a cells cultured either in FBS for 24 h or in SF medium for 72 h. Nuclei were counterstained with DAPI (blue). Scale bar = 15 μm.

Techniques Used: Cell Culture, Polymerase Chain Reaction, Western Blot, Immunofluorescence

P2X7R expression is upregulated in serum-deprived neuroblastoma cells. ( A ) Changes in P2X7 transcript levels in N2a cell line cultured either in standard culture medium (FBS) for 24 h or in serum free medium (SF) for the indicated time periods. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR as described in M ethods . GAPDH was used as a control for differences in cDNA input. Results are mean ± s.e.m. of three independent experiments in triplicate; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). ( B ) Immunoblotting depicting the presence of endogenous P2X7R in whole-cell lysates from N2a cells cultured either in FBS for 24 or in SF at the indicated time points. Cell lysates were analyzed by western blotting with anti-P2X7R (intracellular epitope) antibody. GAPDH was used as internal loading control. Histogram represents P2X7 protein levels at the indicated time periods obtained by densitometry and normalization to GAPDH. The values represent mean ± s.e.m. of three independent experiments in duplicate. * P ≤ 0.05, *** P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). ( C ) Double immunofluorescence for P2X7R (green) and α-tubulin (red) in N2a cells cultured either in FBS for 24 h or in SF medium for 72 h. Nuclei were counterstained with DAPI (blue). Insets depict enlarged views (2.5X magnification) of delimited area. Scale bar = 15 μm. ( D ) Proliferation of serum-starved N2a cells treated with LY294002 (50 μM), BBG (5 or 10 μM), A740003 (10 or 50 μM) and/or ARL67156 (100 μM) for 48 h. Values were normalized to those obtained from untreated control cells, set as 100% proliferation rate. Results are mean ± s.e.m. of three independent experiments in triplicate. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test); ### P
Figure Legend Snippet: P2X7R expression is upregulated in serum-deprived neuroblastoma cells. ( A ) Changes in P2X7 transcript levels in N2a cell line cultured either in standard culture medium (FBS) for 24 h or in serum free medium (SF) for the indicated time periods. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR as described in M ethods . GAPDH was used as a control for differences in cDNA input. Results are mean ± s.e.m. of three independent experiments in triplicate; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). ( B ) Immunoblotting depicting the presence of endogenous P2X7R in whole-cell lysates from N2a cells cultured either in FBS for 24 or in SF at the indicated time points. Cell lysates were analyzed by western blotting with anti-P2X7R (intracellular epitope) antibody. GAPDH was used as internal loading control. Histogram represents P2X7 protein levels at the indicated time periods obtained by densitometry and normalization to GAPDH. The values represent mean ± s.e.m. of three independent experiments in duplicate. * P ≤ 0.05, *** P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). ( C ) Double immunofluorescence for P2X7R (green) and α-tubulin (red) in N2a cells cultured either in FBS for 24 h or in SF medium for 72 h. Nuclei were counterstained with DAPI (blue). Insets depict enlarged views (2.5X magnification) of delimited area. Scale bar = 15 μm. ( D ) Proliferation of serum-starved N2a cells treated with LY294002 (50 μM), BBG (5 or 10 μM), A740003 (10 or 50 μM) and/or ARL67156 (100 μM) for 48 h. Values were normalized to those obtained from untreated control cells, set as 100% proliferation rate. Results are mean ± s.e.m. of three independent experiments in triplicate. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test); ### P

Techniques Used: Expressing, Cell Culture, Polymerase Chain Reaction, Western Blot, Immunofluorescence

39) Product Images from "A Powerful Yeast Model to Investigate the Synergistic Interaction of ?-Synuclein and Tau in Neurodegeneration"

Article Title: A Powerful Yeast Model to Investigate the Synergistic Interaction of ?-Synuclein and Tau in Neurodegeneration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055848

ASYN increases Tau insoluble aggregation state. A) Immunofluorescence with an anti-ASYN antibody showed no significant differences between the percentage of cells that contain ASYN (WT and A53T) intracellular inclusions when expressed alone or in combination with tau (WT and P301L). No yeast cells with ASYN big inclusions were observed in the strain expressing ASYN A53T in combination with tau P301L For statistical analysis at least 800 cells were counted. B) Both ASYN (WT and A53T) and tau (WT and P301L) form intracellular sarkosyl insoluble aggregates when expressed either alone or in combination, which are detectable by western blot. GAPDH was used as loading and soluble protein control. *corresponds to an unspecific band. Results are representative of three independent experiments.
Figure Legend Snippet: ASYN increases Tau insoluble aggregation state. A) Immunofluorescence with an anti-ASYN antibody showed no significant differences between the percentage of cells that contain ASYN (WT and A53T) intracellular inclusions when expressed alone or in combination with tau (WT and P301L). No yeast cells with ASYN big inclusions were observed in the strain expressing ASYN A53T in combination with tau P301L For statistical analysis at least 800 cells were counted. B) Both ASYN (WT and A53T) and tau (WT and P301L) form intracellular sarkosyl insoluble aggregates when expressed either alone or in combination, which are detectable by western blot. GAPDH was used as loading and soluble protein control. *corresponds to an unspecific band. Results are representative of three independent experiments.

Techniques Used: Immunofluorescence, Expressing, Western Blot

40) Product Images from "Expression and regulation of proton-coupled oligopeptide transporters in colonic tissue and immune cells of mice"

Article Title: Expression and regulation of proton-coupled oligopeptide transporters in colonic tissue and immune cells of mice

Journal: Biochemical pharmacology

doi: 10.1016/j.bcp.2017.12.025

The expression of IL-6, TNFα and IL-1β in RAW264.7-musPHT2 and RAW264.7-vector cells stimulated with LPS. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with LPS (10 ng/mL) for 4 hr, after which total RNA was isolated for Il-6, Tnf α and Il-1 β detection (A). The CT values of Il-6, Tnf α, Il-1 β and Gapdh ranged from 16~27. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with LPS (10 ng/mL) for 24 hr, after which cell supernatant were collected for IL-6 and TNFα protein detection (B). Data are expressed as mean + SE (n=3). *p
Figure Legend Snippet: The expression of IL-6, TNFα and IL-1β in RAW264.7-musPHT2 and RAW264.7-vector cells stimulated with LPS. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with LPS (10 ng/mL) for 4 hr, after which total RNA was isolated for Il-6, Tnf α and Il-1 β detection (A). The CT values of Il-6, Tnf α, Il-1 β and Gapdh ranged from 16~27. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with LPS (10 ng/mL) for 24 hr, after which cell supernatant were collected for IL-6 and TNFα protein detection (B). Data are expressed as mean + SE (n=3). *p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Isolation

The expression of IL-6, TNFα and IL-1β in RAW264.7-musPHT2 and RAW264.7-vector cells stimulated with MDP or tri-DAP. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with 10 μg/mL MDP (A) or tri-DAP (B) for 0, 4, 6 and 8 hr, after which total RNA was isolated for Il-6, Tnf α and Il-1 β detection. The CT values of Il-6, Tnf α, Il-1 β and Gapdh ranged from 16~27. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with 0, 1, 10 μg/mL MDP (C) or tri-DAP (D) for 24 hr, after which cell supernatant was collected for IL-6 and TNFα detection. Data are expressed as mean ± SE (n=3). *p
Figure Legend Snippet: The expression of IL-6, TNFα and IL-1β in RAW264.7-musPHT2 and RAW264.7-vector cells stimulated with MDP or tri-DAP. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with 10 μg/mL MDP (A) or tri-DAP (B) for 0, 4, 6 and 8 hr, after which total RNA was isolated for Il-6, Tnf α and Il-1 β detection. The CT values of Il-6, Tnf α, Il-1 β and Gapdh ranged from 16~27. RAW264.7 cells were transiently transfected with pcDNA3.1(+)-musPHT2-his tag or vector for 24 hr and then stimulated with 0, 1, 10 μg/mL MDP (C) or tri-DAP (D) for 24 hr, after which cell supernatant was collected for IL-6 and TNFα detection. Data are expressed as mean ± SE (n=3). *p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Isolation

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Amplification:

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Article Snippet: .. Total RNA was extracted from agonist-treated WT and PLCɛ KO astrocytes using an RNeasy Kit (Invitrogen) as previously described ( ). cDNA was amplified using TaqMan Universal Master Mix in the presence of gene-specific primers for COX-2, IL-1β, and IL-6, with GAPDH used as an internal control (Applied Biosystems). ..

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Article Snippet: .. Total RNA was extracted from agonist-treated control and knockdown glioblastoma cells by using an RNeasy kit (Invitrogen) as previously described ( ). cDNA was amplified using TaqMan Universal master mix in the presence of gene-specific primers for CCN1, CTGF, ACTA2, and ANKRD1, with GAPDH as an internal control (Applied Biosystems). ..

Real-time Polymerase Chain Reaction:

Article Title: Functional changes in glutamate transporters and astrocyte biophysical properties in a rodent model of focal cortical dysplasia
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Article Title: Endothelin-1 receptor antagonists regulate cell surface-associated protein disulfide isomerase in sickle cell disease
Article Snippet: .. Real-time PCR was performed using TaqMan Universal PCR mastermix and preformulated primers for PDI and the endogenous controls β-2-microglobulin and GAPDH (Applied Biosystems, Foster City, CA, USA). .. The cycle threshold ( Ct ) values for PDI expression were normalized against two housekeeping genes, β-2-microglobulin and GAPDH.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Activation of the Tie2 Receptor by Angiopoietin-1 Enhances Tumor Vessel Maturation and Impairs Squamous Cell Carcinoma Growth
Article Snippet: .. Each reaction was performed in the presence of a GAPDH internal control detection system using the Taqman EZ RT-PCR Core Reagent (Applied Biosystems). .. All reactions were performed in triplicates using the ABI Sequence Detection System 7000 (Applied Biosystems).

Expressing:

Article Title: Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule morphology
Article Snippet: .. The mRNA expression levels of the Cx43 and ZO-1 were quantified in each sample using TaqMan Gene Expression Assays (Applied Biosystems) as follows: for Cx43 assay ID, Rn01433957_m1; for ZO-1 assay ID, Rn02116071_s1; GAPDH levels were determined as an endogenous control assay (Applied Biosystems, assay ID, Rn01775763_g1). ..

Polymerase Chain Reaction:

Article Title: Endothelin-1 receptor antagonists regulate cell surface-associated protein disulfide isomerase in sickle cell disease
Article Snippet: .. Real-time PCR was performed using TaqMan Universal PCR mastermix and preformulated primers for PDI and the endogenous controls β-2-microglobulin and GAPDH (Applied Biosystems, Foster City, CA, USA). .. The cycle threshold ( Ct ) values for PDI expression were normalized against two housekeeping genes, β-2-microglobulin and GAPDH.

TaqMan Assay:

Article Title: Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening
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Control Assay:

Article Title: Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule morphology
Article Snippet: .. The mRNA expression levels of the Cx43 and ZO-1 were quantified in each sample using TaqMan Gene Expression Assays (Applied Biosystems) as follows: for Cx43 assay ID, Rn01433957_m1; for ZO-1 assay ID, Rn02116071_s1; GAPDH levels were determined as an endogenous control assay (Applied Biosystems, assay ID, Rn01775763_g1). ..

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    Thermo Fisher gapdh reference
    Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. <t>Gapdh</t> ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the <t>SN1</t> transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.
    Gapdh Reference, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh reference/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gapdh reference - by Bioz Stars, 2020-09
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    99
    Thermo Fisher gene exp gapdh mm99999915 g1
    Regulation of the expression of metastasis-related genes by Akt isoforms. GEO cells expressing non-targeting shRNA (sh) or <t>Akt1,</t> Akt2 or Akt3 shRNA were treated with Dox for 5 days and cDNA samples were subjected to real-time <t>PCR</t> using the Human Tumor Metastasis RT 2 Profiler PCR array from Super Array Biosciences (Qiagen). Top panel: Venn diagram showing differentially expressed metastatic genes on knockdown of each of the three Akt isoforms. Genes shown in red have higher expression in Akt isoform knockdown cells relative to non-targeting shRNA (sh) expressing cells, whereas genes in green show reduced expression. Lower panel: list of genes that showed differential expression ( > two-fold) on loss of individual Akt isoforms. MTSS1 is shown in bold.
    Gene Exp Gapdh Mm99999915 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 671 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 671 article reviews
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    Thermo Fisher gene exp gapdh hs99999905 m1
    Quantitative real time polymerase chain reaction <t>(qRT-PCR)</t> of patient <t>survivin</t> levels. A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.
    Gene Exp Gapdh Hs99999905 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.

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

    Article Title: Intact piRNA pathway prevents L1 mobilization in male meiosis

    doi: 10.1073/pnas.1701069114

    Figure Lengend Snippet: Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.

    Article Snippet: The SN1 transgene expression was quantified in duplex with the Gapdh reference using custom TaqMan FAM/VIC-labeled probes and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific).

    Techniques: Derivative Assay, Methylation, RNA Expression, In Situ, Sequencing, Transgenic Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Regulation of the expression of metastasis-related genes by Akt isoforms. GEO cells expressing non-targeting shRNA (sh) or Akt1, Akt2 or Akt3 shRNA were treated with Dox for 5 days and cDNA samples were subjected to real-time PCR using the Human Tumor Metastasis RT 2 Profiler PCR array from Super Array Biosciences (Qiagen). Top panel: Venn diagram showing differentially expressed metastatic genes on knockdown of each of the three Akt isoforms. Genes shown in red have higher expression in Akt isoform knockdown cells relative to non-targeting shRNA (sh) expressing cells, whereas genes in green show reduced expression. Lower panel: list of genes that showed differential expression ( > two-fold) on loss of individual Akt isoforms. MTSS1 is shown in bold.

    Journal: Oncogene

    Article Title: Role of Akt2 in regulation of metastasis suppressor 1 expression and colorectal cancer metastasis

    doi: 10.1038/onc.2016.460

    Figure Lengend Snippet: Regulation of the expression of metastasis-related genes by Akt isoforms. GEO cells expressing non-targeting shRNA (sh) or Akt1, Akt2 or Akt3 shRNA were treated with Dox for 5 days and cDNA samples were subjected to real-time PCR using the Human Tumor Metastasis RT 2 Profiler PCR array from Super Array Biosciences (Qiagen). Top panel: Venn diagram showing differentially expressed metastatic genes on knockdown of each of the three Akt isoforms. Genes shown in red have higher expression in Akt isoform knockdown cells relative to non-targeting shRNA (sh) expressing cells, whereas genes in green show reduced expression. Lower panel: list of genes that showed differential expression ( > two-fold) on loss of individual Akt isoforms. MTSS1 is shown in bold.

    Article Snippet: Two-step quantitative real-time PCR using TaqMan reagents and primers (Akt1: HS00178289_m1; Akt2: HS01086102_m1; Akt3: HS00987350_m1; Human GAPDH: 4352665; Mouse GAPDH: Mm99999915_g1) was performed according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). mRNA expression was normalized to levels of GAPDH.

    Techniques: Expressing, shRNA, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    HSI activates noncoding transcription independent of promoter interactions. ( A ) Map of the hGH locus and the hGH BAC transgene. Each structural gene in the hGH locus is indicated along with its transcriptional orientation. The 123 kb hGH BAC transgene released from the originating BAC clone (BAC535D15) by NotI digestion was used to generate the hGH BAC transgenic mouse lines ( 26 ). The vertical arrows labeled with roman numerals indicate the positions of DNase I hypersensitive sites (HS) that form in pituitary chromatin and constitute the hGH LCR. The distance (14.5 kb) between the 3′ end of the HSI core and the hGH-N gene promoter is indicated with double-headed arrow. Note that this region encompasses the hCD79b gene that encodes the B-cell specific Ig receptor component, Igβ. Pit-1 binding sites that constitute core determinants of HSI are indicated by the shaded ovals (expanded inset). The 99 bp deletion that inactivates HSI functions ( 18 ) is also shown in expanded view (inset above). ( B ) Map of the wild-type hGH BAC and two derivative transgenes. The − 8.0CD79b transgene (12.5 kb) encompasses HSI and the contiguous hCD79b (expanded view below the hGH locus map) and isolates this region from the hGH gene cluster. The −8.0CD79bΔ1.6 transgene was derived from the −8.0CD79b by deletion of a 1.6-kb internal fragment extending from −0.5 kb of the promoter through intron 2, removing all defined hCD79b promoter elements. ( C ) Noncoding transcription across hCD79b in the transgenic mouse pituitary is preserved in the absence of defined promoter elements. Transcription across the hCD79b region generated by the promoterless − 8.0CD79bΔ1.6 was compared to that of the − 8.0CD79b transgene (containing the intact hCD79b promoter but not the hGH-N promoter) and the hGH BAC transgene (containing both the hCD79b and the hGH-N promoters). Pituitary RNA from mice carrying each of these indicated transgenes was assayed for transcription across hCD79b by RT-qPCR. The data shown in the histogram (Y-axis) have been normalized to the corresponding transgene copy numbers and to somatotrope-specific levels of the endogenous mouse growth hormone ( mGH1 ) mRNA. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each line was tested in triplicate using three independent mice. Values represent the average ± SD. The level of hGH BAC was defined as 1.0. ( D ) Map of the 123 kb λΔCD/hGH transgene and the derivative the −8.0λΔCD transgene. This transgene was generated by replacing the hCD79b gene and promoter (to −500 bp) with a size-matched fragment of bacteriophage λ DNA within the context of an otherwise intact hGH BAC (see ‘Materials and Methods' section). The −8.0λΔCD transgene was released from the λΔCD/hGH transgene with boundaries as indicated. ( E ) Noncoding transcription is activated 3′ of HSI within an inserted (λ) DNA segment. Transcription across the λ-DNA region was compared between the λΔCD/hGH and −8.0λΔCD transgenes. λ noncoding transcription was measured by RT-qPCR, normalized to transgene copy-number and to mGH1 mRNA levels. λ noncoding transcription was actively transcribed without hGH-N promoter. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each experiment was carried out in triplicate. The study of the single −8.0λΔCD line was evaluated in each of three independent mice, each studied in triplicate. Values represent the average ± SD.

    Journal: Nucleic Acids Research

    Article Title: Autonomous actions of the human growth hormone long-range enhancer

    doi: 10.1093/nar/gkv093

    Figure Lengend Snippet: HSI activates noncoding transcription independent of promoter interactions. ( A ) Map of the hGH locus and the hGH BAC transgene. Each structural gene in the hGH locus is indicated along with its transcriptional orientation. The 123 kb hGH BAC transgene released from the originating BAC clone (BAC535D15) by NotI digestion was used to generate the hGH BAC transgenic mouse lines ( 26 ). The vertical arrows labeled with roman numerals indicate the positions of DNase I hypersensitive sites (HS) that form in pituitary chromatin and constitute the hGH LCR. The distance (14.5 kb) between the 3′ end of the HSI core and the hGH-N gene promoter is indicated with double-headed arrow. Note that this region encompasses the hCD79b gene that encodes the B-cell specific Ig receptor component, Igβ. Pit-1 binding sites that constitute core determinants of HSI are indicated by the shaded ovals (expanded inset). The 99 bp deletion that inactivates HSI functions ( 18 ) is also shown in expanded view (inset above). ( B ) Map of the wild-type hGH BAC and two derivative transgenes. The − 8.0CD79b transgene (12.5 kb) encompasses HSI and the contiguous hCD79b (expanded view below the hGH locus map) and isolates this region from the hGH gene cluster. The −8.0CD79bΔ1.6 transgene was derived from the −8.0CD79b by deletion of a 1.6-kb internal fragment extending from −0.5 kb of the promoter through intron 2, removing all defined hCD79b promoter elements. ( C ) Noncoding transcription across hCD79b in the transgenic mouse pituitary is preserved in the absence of defined promoter elements. Transcription across the hCD79b region generated by the promoterless − 8.0CD79bΔ1.6 was compared to that of the − 8.0CD79b transgene (containing the intact hCD79b promoter but not the hGH-N promoter) and the hGH BAC transgene (containing both the hCD79b and the hGH-N promoters). Pituitary RNA from mice carrying each of these indicated transgenes was assayed for transcription across hCD79b by RT-qPCR. The data shown in the histogram (Y-axis) have been normalized to the corresponding transgene copy numbers and to somatotrope-specific levels of the endogenous mouse growth hormone ( mGH1 ) mRNA. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each line was tested in triplicate using three independent mice. Values represent the average ± SD. The level of hGH BAC was defined as 1.0. ( D ) Map of the 123 kb λΔCD/hGH transgene and the derivative the −8.0λΔCD transgene. This transgene was generated by replacing the hCD79b gene and promoter (to −500 bp) with a size-matched fragment of bacteriophage λ DNA within the context of an otherwise intact hGH BAC (see ‘Materials and Methods' section). The −8.0λΔCD transgene was released from the λΔCD/hGH transgene with boundaries as indicated. ( E ) Noncoding transcription is activated 3′ of HSI within an inserted (λ) DNA segment. Transcription across the λ-DNA region was compared between the λΔCD/hGH and −8.0λΔCD transgenes. λ noncoding transcription was measured by RT-qPCR, normalized to transgene copy-number and to mGH1 mRNA levels. λ noncoding transcription was actively transcribed without hGH-N promoter. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each experiment was carried out in triplicate. The study of the single −8.0λΔCD line was evaluated in each of three independent mice, each studied in triplicate. Values represent the average ± SD.

    Article Snippet: In all cases, 1 μg of purified RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and RNA levels were assessed by real-time PCR using TaqMan Universal PCR Master mix (Applied Biosystems) and a 7900HT platform with corresponding probes (hCD79b , Hs00236881_m1; λ; mGAPDH , Mm99999915_g1).

    Techniques: BAC Assay, Transgenic Assay, Labeling, Binding Assay, Derivative Assay, Generated, Mouse Assay, Quantitative RT-PCR

    5′ RACE analyses of transcription start sites 3′ to HSI in the hGH BAC and derivative transgenes reveals consistent spacing between the HSI core determinants and the TSS cluster. RNA was isolated from the pituitaries of mice carrying each of the 5 indicated transgenes (Figure 1 ). In each case, cDNA synthesis was primed at the site labeled 1 (left facing arrow), poly G tails were added to the 3′ end of the cDNA, and the product was then amplified between an adapter-polyC 17 primer and a nested primer (arrow 2). The amplified PCR products were cloned and individually sequenced. Each triangle indicates the 5′ terminus of an individual clone and the results were grouped within 50 bp windows. The total numbers of cDNAs containing an additional non-templated terminal G (corresponding to the 5′-capped structure; filled triangles) are shown, and the total numbers of cDNAs with and without the nontemplated G are included in parentheses. The arrows indicate the distance between the HSI core and the center of the most proximal TSS cluster. hGH BAC , 123 kb unmodified human transgenic mouse line (two copies) ( 26 ); CDΔ0.7/hGH BAC , 0.7 kb deletion of 0.5 kb promoter region and exon 1 from hGH BAC (14 copies) ( 25 ); CDΔ1.6/hGH BAC , 1.6 kb deletion of 0.5 kb promoter through exon 2 from the hGH BAC (three copies) ( 23 ); −8.0CD79bΔ1.6 , 1.6 kb deletion of hCD79b promoter through exon 2 from the −8.0CD79b (6 copies); λΔCD/hGH BAC , 3.8 kb λ gene segment replacing hCD79b from hGH BAC (five copies) ( 23 ).

    Journal: Nucleic Acids Research

    Article Title: Autonomous actions of the human growth hormone long-range enhancer

    doi: 10.1093/nar/gkv093

    Figure Lengend Snippet: 5′ RACE analyses of transcription start sites 3′ to HSI in the hGH BAC and derivative transgenes reveals consistent spacing between the HSI core determinants and the TSS cluster. RNA was isolated from the pituitaries of mice carrying each of the 5 indicated transgenes (Figure 1 ). In each case, cDNA synthesis was primed at the site labeled 1 (left facing arrow), poly G tails were added to the 3′ end of the cDNA, and the product was then amplified between an adapter-polyC 17 primer and a nested primer (arrow 2). The amplified PCR products were cloned and individually sequenced. Each triangle indicates the 5′ terminus of an individual clone and the results were grouped within 50 bp windows. The total numbers of cDNAs containing an additional non-templated terminal G (corresponding to the 5′-capped structure; filled triangles) are shown, and the total numbers of cDNAs with and without the nontemplated G are included in parentheses. The arrows indicate the distance between the HSI core and the center of the most proximal TSS cluster. hGH BAC , 123 kb unmodified human transgenic mouse line (two copies) ( 26 ); CDΔ0.7/hGH BAC , 0.7 kb deletion of 0.5 kb promoter region and exon 1 from hGH BAC (14 copies) ( 25 ); CDΔ1.6/hGH BAC , 1.6 kb deletion of 0.5 kb promoter through exon 2 from the hGH BAC (three copies) ( 23 ); −8.0CD79bΔ1.6 , 1.6 kb deletion of hCD79b promoter through exon 2 from the −8.0CD79b (6 copies); λΔCD/hGH BAC , 3.8 kb λ gene segment replacing hCD79b from hGH BAC (five copies) ( 23 ).

    Article Snippet: In all cases, 1 μg of purified RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and RNA levels were assessed by real-time PCR using TaqMan Universal PCR Master mix (Applied Biosystems) and a 7900HT platform with corresponding probes (hCD79b , Hs00236881_m1; λ; mGAPDH , Mm99999915_g1).

    Techniques: BAC Assay, Isolation, Mouse Assay, Labeling, Amplification, Polymerase Chain Reaction, Clone Assay, Transgenic Assay

    Quantitative real time polymerase chain reaction (qRT-PCR) of patient survivin levels. A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.

    Journal: PLoS ONE

    Article Title: Prognostic Value and Targeted Inhibition of Survivin Expression in Esophageal Adenocarcinoma and Cancer-Adjacent Squamous Epithelium

    doi: 10.1371/journal.pone.0078343

    Figure Lengend Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) of patient survivin levels. A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.

    Article Snippet: PCR was performed in a total volume of 20 ul using 600 ng cDNA, 1× Taqman PCR universal mastermix (Invitrogen, Grand Island, NY, 4304437), and 1× Survivin or GAPDH primer (Applied Biosystems, Carlsbad, CA hs03043576_m1 and hs99999905_m1) for each reaction.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing