Structured Review

Qiagen rna
Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, <t>ZT12,</t> ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. <t>RNA</t> was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.
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1) Product Images from "Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2"

Article Title: Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

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

doi: 10.1073/pnas.1800431115

Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.
Figure Legend Snippet: Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.

Techniques Used: Activity Assay, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.
Figure Legend Snippet: Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.

Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Confocal Microscopy

2) Product Images from "Toll-Like Receptors 4, 5, 6 and 7 are Constitutively Expressed in Non-Human Primate Retinal Neurons"

Article Title: Toll-Like Receptors 4, 5, 6 and 7 are Constitutively Expressed in Non-Human Primate Retinal Neurons

Journal: Journal of neuroimmunology

doi: 10.1016/j.jneuroim.2018.06.007

TLR6 co-localizes with the amacrine cell marker calretinin A) The arrow and asterisk indicate TLR6 staining of round cells in INL of macaque retina tissue. B) The arrow and asterisk indicate staining of calretinin positive cells. C) Merged image with arrow and asterisk indicating co-localization of TLR6 and calretinin positive cells. Outer nuclear layer (ONL), inner nuclear layer (INL), ganglion cell layer (GCL). Scale bar= 20 μm.
Figure Legend Snippet: TLR6 co-localizes with the amacrine cell marker calretinin A) The arrow and asterisk indicate TLR6 staining of round cells in INL of macaque retina tissue. B) The arrow and asterisk indicate staining of calretinin positive cells. C) Merged image with arrow and asterisk indicating co-localization of TLR6 and calretinin positive cells. Outer nuclear layer (ONL), inner nuclear layer (INL), ganglion cell layer (GCL). Scale bar= 20 μm.

Techniques Used: Marker, Staining

3) Product Images from "Macrophage LRP1 Promotes Diet-Induced Hepatic Inflammation and Metabolic Dysfunction by Modulating Wnt Signaling"

Article Title: Macrophage LRP1 Promotes Diet-Induced Hepatic Inflammation and Metabolic Dysfunction by Modulating Wnt Signaling

Journal: Mediators of Inflammation

doi: 10.1155/2018/7902841

LRP1 expression increased diet-induced liver inflammation in mice. (a) LDLR −/− and LDLR −/− ; macLRP1 −/− mice were maintained on a Western diet for 2 weeks. Livers were processed and analyzed for abundance of inflammatory cytokines Ccl3, Ccl4, Ccl8, Ccr1, Ccr2, Cxcl9, and Tnf by quantitative RT-PCR ( n = 3). (b) Insulin receptor and Glut2 ( Insr and Slc2a2 , respectively) expression levels were analyzed for abundance by quantitative RT-PCR ( n = 3). The data represent mean ± SEM of results. ∗ p
Figure Legend Snippet: LRP1 expression increased diet-induced liver inflammation in mice. (a) LDLR −/− and LDLR −/− ; macLRP1 −/− mice were maintained on a Western diet for 2 weeks. Livers were processed and analyzed for abundance of inflammatory cytokines Ccl3, Ccl4, Ccl8, Ccr1, Ccr2, Cxcl9, and Tnf by quantitative RT-PCR ( n = 3). (b) Insulin receptor and Glut2 ( Insr and Slc2a2 , respectively) expression levels were analyzed for abundance by quantitative RT-PCR ( n = 3). The data represent mean ± SEM of results. ∗ p

Techniques Used: Expressing, Mouse Assay, Western Blot, Quantitative RT-PCR

4) Product Images from "SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved"

Article Title: SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved

Journal: Biology of Sex Differences

doi: 10.1186/s13293-017-0158-2

Effect of O 3 in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O 3 exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold ( p
Figure Legend Snippet: Effect of O 3 in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O 3 exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold ( p

Techniques Used: Mouse Assay, Quantitative RT-PCR

5) Product Images from "SET contributes to the epithelial-mesenchymal transition of pancreatic cancer"

Article Title: SET contributes to the epithelial-mesenchymal transition of pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.19067

SET induced N-cadherin via the Rac1/JNK/c-Jun signaling pathway (A and B) In an RT-PCR profiler array for 84 EMT-related genes, 47 genes were upregulated and 12 genes were down-regulated (with fold changes ≥ 3) in SET overexpression cells (SET-HA) compared with control cells (pLNCX2). A representative scatter plot (A) and genes with respective fold-changes are shown (B). Red arrow indicates CDH2 (N-cadherin gene) as one of the highly upregulated gene (∼100 folds) in SET overexpression cells. (C) SET isoform 2 expression (red) partially co-localized with N-cadherin expression (green) at the cell surface (yellow) in PANC-1. (D) SET isoform 2 expression alters the protein levels of members in the Rac1/JNK/c-Jun pathway. Oppositely, a reduction in SET led to decreases in Rac1, JNK1, phospho-JNK, c-Jun, and phospho-c-Jun levels. Whole cell lysates (50 μg) were subjected to Western blotting analysis. β-actin, the internal loading control, is shown with a representative blot. (E) SET isoform 2 expression alters the cellular organizations and expressions of members in the Rac1/JNK/c-Jun pathway. (F) Treating cells with SP600125, a JNK inhibitor, decreased N-cadherin in the PANC-1 SET-HA. (G) TGFβ1 (10 ng/mL; 48 h) treatment did not alter SET expression levels in PANC-1. Whole cell lysates (50 - 70 μg) were subjected to Western blotting analysis. β-actin, a loading control is shown with a representative blot. (H) Stable knockdown of EMT-regulating transcription factors (ZEB1, SNAI2, and TWIST1) decreases SET protein levels in PANC-1. (I) Overexpression of SET in PANC-1 (SET-HA) led to significant increases in both SPARC and SNAI2 transcript levels while opposite effects were observed with the knockdown of SET in MIA PaCa-2 (SET-shRNA) as confirmed with qRT-PCR assays with respective TaqMan probes and GUSB as an internal control. (J and K) SET-mediated effects on N-cadherin are PP2A dependent in pancreatic cancer. (J) PP2A activity was decreased with SET overexpression (SET-HA) as compared to control (pLNCX2) in PANC-1 while FTY720 (PP2A activator) treatment (1 μM – 5 μM; 24 h – 48 h) in SET-HA rescued SET-mediated decreases in PP2A activity as determined with the PP2A Immunoprecipitation Phosphatase Assay. (K) . FTY720 treatment decreased SET-mediated effects on N-cadherin in PANC-1 SET-HA. Whole cell lysates (50 - 70 μg) were subjected to Western blotting analysis. β-actin as a loading control is shown with a representative blot. Bars, SD or SEM. n = 2-3.
Figure Legend Snippet: SET induced N-cadherin via the Rac1/JNK/c-Jun signaling pathway (A and B) In an RT-PCR profiler array for 84 EMT-related genes, 47 genes were upregulated and 12 genes were down-regulated (with fold changes ≥ 3) in SET overexpression cells (SET-HA) compared with control cells (pLNCX2). A representative scatter plot (A) and genes with respective fold-changes are shown (B). Red arrow indicates CDH2 (N-cadherin gene) as one of the highly upregulated gene (∼100 folds) in SET overexpression cells. (C) SET isoform 2 expression (red) partially co-localized with N-cadherin expression (green) at the cell surface (yellow) in PANC-1. (D) SET isoform 2 expression alters the protein levels of members in the Rac1/JNK/c-Jun pathway. Oppositely, a reduction in SET led to decreases in Rac1, JNK1, phospho-JNK, c-Jun, and phospho-c-Jun levels. Whole cell lysates (50 μg) were subjected to Western blotting analysis. β-actin, the internal loading control, is shown with a representative blot. (E) SET isoform 2 expression alters the cellular organizations and expressions of members in the Rac1/JNK/c-Jun pathway. (F) Treating cells with SP600125, a JNK inhibitor, decreased N-cadherin in the PANC-1 SET-HA. (G) TGFβ1 (10 ng/mL; 48 h) treatment did not alter SET expression levels in PANC-1. Whole cell lysates (50 - 70 μg) were subjected to Western blotting analysis. β-actin, a loading control is shown with a representative blot. (H) Stable knockdown of EMT-regulating transcription factors (ZEB1, SNAI2, and TWIST1) decreases SET protein levels in PANC-1. (I) Overexpression of SET in PANC-1 (SET-HA) led to significant increases in both SPARC and SNAI2 transcript levels while opposite effects were observed with the knockdown of SET in MIA PaCa-2 (SET-shRNA) as confirmed with qRT-PCR assays with respective TaqMan probes and GUSB as an internal control. (J and K) SET-mediated effects on N-cadherin are PP2A dependent in pancreatic cancer. (J) PP2A activity was decreased with SET overexpression (SET-HA) as compared to control (pLNCX2) in PANC-1 while FTY720 (PP2A activator) treatment (1 μM – 5 μM; 24 h – 48 h) in SET-HA rescued SET-mediated decreases in PP2A activity as determined with the PP2A Immunoprecipitation Phosphatase Assay. (K) . FTY720 treatment decreased SET-mediated effects on N-cadherin in PANC-1 SET-HA. Whole cell lysates (50 - 70 μg) were subjected to Western blotting analysis. β-actin as a loading control is shown with a representative blot. Bars, SD or SEM. n = 2-3.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Over Expression, Expressing, Western Blot, shRNA, Quantitative RT-PCR, Activity Assay, IP Phosphatase Assay

6) Product Images from "Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2"

Article Title: Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

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

doi: 10.1073/pnas.1800431115

Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.
Figure Legend Snippet: Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.

Techniques Used: Activity Assay, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.
Figure Legend Snippet: Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.

Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Confocal Microscopy

7) Product Images from "Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2"

Article Title: Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

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

doi: 10.1073/pnas.1800431115

Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.
Figure Legend Snippet: Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.

Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Confocal Microscopy

8) Product Images from "Toll-Like Receptors 4, 5, 6 and 7 are Constitutively Expressed in Non-Human Primate Retinal Neurons"

Article Title: Toll-Like Receptors 4, 5, 6 and 7 are Constitutively Expressed in Non-Human Primate Retinal Neurons

Journal: Journal of neuroimmunology

doi: 10.1016/j.jneuroim.2018.06.007

Expression patterns of TLRs 4-7 in macaque neural retina tissue TLR 4-7 proteins were detected by immunofluorescence of macaque retina tissue. Arrow in panel A identifies autofluorescence in photoreceptor layer. Arrows in panels B–D identify TLR positive cells. Photoreceptor layer (PR), Outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), ganglion cell layer (GCL). Scale bar= 20 μm.
Figure Legend Snippet: Expression patterns of TLRs 4-7 in macaque neural retina tissue TLR 4-7 proteins were detected by immunofluorescence of macaque retina tissue. Arrow in panel A identifies autofluorescence in photoreceptor layer. Arrows in panels B–D identify TLR positive cells. Photoreceptor layer (PR), Outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), ganglion cell layer (GCL). Scale bar= 20 μm.

Techniques Used: Expressing, Immunofluorescence

Quantitative PCR detects TLRs 4-7 mRNAs in macaque neural retina tissue qPCR primer assays confirmed expression of TLRs 4-7 mRNA in cynomolgus and rhesus retina tissue compared to a no primer control. ACTB=Beta actin house-keeping gene control.
Figure Legend Snippet: Quantitative PCR detects TLRs 4-7 mRNAs in macaque neural retina tissue qPCR primer assays confirmed expression of TLRs 4-7 mRNA in cynomolgus and rhesus retina tissue compared to a no primer control. ACTB=Beta actin house-keeping gene control.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

9) Product Images from "ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice"

Article Title: ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice

Journal: Cancer Cell

doi: 10.1016/j.ccr.2012.04.011

Mdm2 Ser394 phosphorylation is necessary for full p53 stabilization and function in MEFs (A) Primary MEFs proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (B) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were then analyzed by western blot. (C) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times. (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation is necessary for full p53 stabilization and function in MEFs (A) Primary MEFs proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (B) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were then analyzed by western blot. (C) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times. (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. .

Techniques Used: Standard Deviation, Irradiation, Western Blot, Expressing, Quantitative RT-PCR

Mdm2 Ser394 phosphorylation regulates the attenuation of the p53 response to DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested either 4 or 8 hours later. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for either 12 or 18 hours. Protein levels were then analyzed by western blot. (C) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation regulates the attenuation of the p53 response to DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested either 4 or 8 hours later. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for either 12 or 18 hours. Protein levels were then analyzed by western blot. (C) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh .

Techniques Used: Mouse Assay, Irradiation, Western Blot, Ex Vivo, Expressing, Quantitative RT-PCR

Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot. (B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. (D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours. (E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type. .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot. (B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. (D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours. (E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type. .

Techniques Used: Activity Assay, Mouse Assay, Irradiation, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation

Phosphorylation of Mdm2 Ser394 is necessary but not sufficient to stabilize and activate p53 (A) DNA sequence of both the WT and S394D allele at Mdm2 codon 394. The mutations in S394D also introduce a BclI restriction digest site. (B) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.10) and Mdm2 (p=0.88) relative to PI3K were normalized to wild-type. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. (D) Ex vivo thymocytes were either untreated or irradiated with 2Gy for 2 or 4 hours (n=3 per genotype). Relative expression of Mdm2 and Puma was determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (E) Ex vivo thymocytes were isolated and were either left untreated or irradiated with 2Gy for 12 hours (n=3 per genotype). Apoptotic cells were quantified by flow cytometry. Standard deviation indicated by error bars. (F) Primary MEF proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (G) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were analyzed by western blot. .
Figure Legend Snippet: Phosphorylation of Mdm2 Ser394 is necessary but not sufficient to stabilize and activate p53 (A) DNA sequence of both the WT and S394D allele at Mdm2 codon 394. The mutations in S394D also introduce a BclI restriction digest site. (B) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.10) and Mdm2 (p=0.88) relative to PI3K were normalized to wild-type. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. (D) Ex vivo thymocytes were either untreated or irradiated with 2Gy for 2 or 4 hours (n=3 per genotype). Relative expression of Mdm2 and Puma was determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (E) Ex vivo thymocytes were isolated and were either left untreated or irradiated with 2Gy for 12 hours (n=3 per genotype). Apoptotic cells were quantified by flow cytometry. Standard deviation indicated by error bars. (F) Primary MEF proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (G) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were analyzed by western blot. .

Techniques Used: Sequencing, Introduce, Mouse Assay, Western Blot, Irradiation, Ex Vivo, Expressing, Quantitative RT-PCR, Standard Deviation, Isolation, Flow Cytometry, Cytometry

Phosphorylation of Mdm2 Ser394 regulates radiosensitivity in mice (A) DNA sequence of the WT and S394A alleles at Mdm2 codon 394. The mutations in S394A also introduce a BcgI restriction digest site. (B) PCR- BcgI analysis of F 2 generation mice. Primers flanking Mdm2 codon 394 were used to amplify DNA samples from WT, A/+ , and A/A F 2 generation mice, followed by BcgI digestion. (C) Mice that are either heterozygous and homozygous for the S394A allele are viable and were observed at Mendelian ratios after heterozygous intercrosses (p=0.322). (D) Cohorts of wild-type (n=14), A/A (n=14), and p53 −/− (n=8) mice were irradiated with 8Gy and survival was observed over 26 days. (E) WT, A/A , and p53 −/− .
Figure Legend Snippet: Phosphorylation of Mdm2 Ser394 regulates radiosensitivity in mice (A) DNA sequence of the WT and S394A alleles at Mdm2 codon 394. The mutations in S394A also introduce a BcgI restriction digest site. (B) PCR- BcgI analysis of F 2 generation mice. Primers flanking Mdm2 codon 394 were used to amplify DNA samples from WT, A/+ , and A/A F 2 generation mice, followed by BcgI digestion. (C) Mice that are either heterozygous and homozygous for the S394A allele are viable and were observed at Mendelian ratios after heterozygous intercrosses (p=0.322). (D) Cohorts of wild-type (n=14), A/A (n=14), and p53 −/− (n=8) mice were irradiated with 8Gy and survival was observed over 26 days. (E) WT, A/A , and p53 −/− .

Techniques Used: Mouse Assay, Sequencing, Introduce, Polymerase Chain Reaction, Irradiation

10) Product Images from "Gene Expression Profiles Resulting from Stable Loss of p53 Mirrors Its Role in Tissue Differentiation"

Article Title: Gene Expression Profiles Resulting from Stable Loss of p53 Mirrors Its Role in Tissue Differentiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082494

Array Confirmation. Confirmation of targets from gene array by Quantitative PCR verifies array expression values. QPCR of selected genes that showed variation in expression in arrays were selected and tested with three independently isolated single cell clones using the same shRNA as the array(S36), Transient transfections were carried out in triplicate with the same shRNA , and control cells received the scrambled control plasmid (mean + S.E.M., n = 3) for verification. For each gene, the change in expression matched that of the array in both types of transfections.
Figure Legend Snippet: Array Confirmation. Confirmation of targets from gene array by Quantitative PCR verifies array expression values. QPCR of selected genes that showed variation in expression in arrays were selected and tested with three independently isolated single cell clones using the same shRNA as the array(S36), Transient transfections were carried out in triplicate with the same shRNA , and control cells received the scrambled control plasmid (mean + S.E.M., n = 3) for verification. For each gene, the change in expression matched that of the array in both types of transfections.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Isolation, Clone Assay, shRNA, Transfection, Plasmid Preparation

Gene expression o f p53 target genes after p53 loss. (A) Heat map generated by centroid linkage of the Euclidian distance of mean centered fold change values of transient transfection of shRNA plasmid 36 (T36) and stable line created with plasmid 36 (S36) using a control line (created with scrambled shRNA) as a common divisor, 2 -((T36-Normalizers) – Control) compared to 2 -((S36-Normalizers) – Control) , (n = 1). The negative inverse used for fold changes between 0 and 1. Clusters were then divided into two categories, those with higher expression in T36 and those with higher expression in S36. ( B ) Fold change values plotted for genes involved in apoptosis or differentiation with a fold change between treated and control cells greater than two ( C ) Comparison of stable clones of varying p53 expression. Plot of the same gens as in (B) but between S36 and a stable line created with clone 34 (S34) show stable p53 knockdown irrespective of the level of knockdown, have a much more similar expression pattern than seen between transient and stable.
Figure Legend Snippet: Gene expression o f p53 target genes after p53 loss. (A) Heat map generated by centroid linkage of the Euclidian distance of mean centered fold change values of transient transfection of shRNA plasmid 36 (T36) and stable line created with plasmid 36 (S36) using a control line (created with scrambled shRNA) as a common divisor, 2 -((T36-Normalizers) – Control) compared to 2 -((S36-Normalizers) – Control) , (n = 1). The negative inverse used for fold changes between 0 and 1. Clusters were then divided into two categories, those with higher expression in T36 and those with higher expression in S36. ( B ) Fold change values plotted for genes involved in apoptosis or differentiation with a fold change between treated and control cells greater than two ( C ) Comparison of stable clones of varying p53 expression. Plot of the same gens as in (B) but between S36 and a stable line created with clone 34 (S34) show stable p53 knockdown irrespective of the level of knockdown, have a much more similar expression pattern than seen between transient and stable.

Techniques Used: Expressing, Generated, Transfection, shRNA, Plasmid Preparation, Clone Assay

Bone and muscle differentiation markers show different patterns based on the type of transfection. ( A ) Osteocalcin gene promoter shows loss of activity between S36 and control cells created with a scrambled shRNA sequence. ( B ) Conversely, in T Mix samples, an increase in the activity on the osteocalcin promoter when compared with control cells receiving scrambled shRNA. ( C ) Muscle creatine kinase (MCK) gene promoter activity, showed no difference in activity when S36 is compared to control cells. ( D ) Promoter activity increased in T Mix when compared to control cells. Three novel clones (n = 3) were measured in triplicate each, using the mean to calculate the presented mean ± S.E.M. for each group.
Figure Legend Snippet: Bone and muscle differentiation markers show different patterns based on the type of transfection. ( A ) Osteocalcin gene promoter shows loss of activity between S36 and control cells created with a scrambled shRNA sequence. ( B ) Conversely, in T Mix samples, an increase in the activity on the osteocalcin promoter when compared with control cells receiving scrambled shRNA. ( C ) Muscle creatine kinase (MCK) gene promoter activity, showed no difference in activity when S36 is compared to control cells. ( D ) Promoter activity increased in T Mix when compared to control cells. Three novel clones (n = 3) were measured in triplicate each, using the mean to calculate the presented mean ± S.E.M. for each group.

Techniques Used: Transfection, Activity Assay, shRNA, Sequencing, Clone Assay

11) Product Images from "Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2"

Article Title: Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

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

doi: 10.1073/pnas.1800431115

Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.
Figure Legend Snippet: Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.

Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Confocal Microscopy

12) Product Images from "ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice"

Article Title: ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice

Journal: Cancer Cell

doi: 10.1016/j.ccr.2012.04.011

Mdm2 Ser394 phosphorylation is necessary for full p53 stabilization and function in MEFs (A) Primary MEFs proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (B) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were then analyzed by western blot. (C) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times. (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation is necessary for full p53 stabilization and function in MEFs (A) Primary MEFs proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (B) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were then analyzed by western blot. (C) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times. (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. .

Techniques Used: Standard Deviation, Irradiation, Western Blot, Expressing, Quantitative RT-PCR

Mdm2 Ser394 phosphorylation regulates the attenuation of the p53 response to DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested either 4 or 8 hours later. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for either 12 or 18 hours. Protein levels were then analyzed by western blot. (C) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation regulates the attenuation of the p53 response to DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested either 4 or 8 hours later. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for either 12 or 18 hours. Protein levels were then analyzed by western blot. (C) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh .

Techniques Used: Mouse Assay, Irradiation, Western Blot, Ex Vivo, Expressing, Quantitative RT-PCR

Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot. (B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. (D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours. (E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type. .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot. (B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. (D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours. (E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type. .

Techniques Used: Activity Assay, Mouse Assay, Irradiation, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation

Phosphorylation of Mdm2 Ser394 does not significantly impact Mdm2 destabilization after DNA damage (A) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated time points (n=3 per genotype). Relative expression levels of Mdm2 were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (C) Ex vivo .
Figure Legend Snippet: Phosphorylation of Mdm2 Ser394 does not significantly impact Mdm2 destabilization after DNA damage (A) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated time points (n=3 per genotype). Relative expression levels of Mdm2 were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (C) Ex vivo .

Techniques Used: Ex Vivo, Irradiation, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation

Phosphorylation of Mdm2 Ser394 is necessary but not sufficient to stabilize and activate p53 (A) DNA sequence of both the WT and S394D allele at Mdm2 codon 394. The mutations in S394D also introduce a BclI restriction digest site. (B) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.10) and Mdm2 (p=0.88) relative to PI3K were normalized to wild-type. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. (D) Ex vivo thymocytes were either untreated or irradiated with 2Gy for 2 or 4 hours (n=3 per genotype). Relative expression of Mdm2 and Puma was determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (E) Ex vivo thymocytes were isolated and were either left untreated or irradiated with 2Gy for 12 hours (n=3 per genotype). Apoptotic cells were quantified by flow cytometry. Standard deviation indicated by error bars. (F) Primary MEF proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (G) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were analyzed by western blot. .
Figure Legend Snippet: Phosphorylation of Mdm2 Ser394 is necessary but not sufficient to stabilize and activate p53 (A) DNA sequence of both the WT and S394D allele at Mdm2 codon 394. The mutations in S394D also introduce a BclI restriction digest site. (B) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.10) and Mdm2 (p=0.88) relative to PI3K were normalized to wild-type. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. (D) Ex vivo thymocytes were either untreated or irradiated with 2Gy for 2 or 4 hours (n=3 per genotype). Relative expression of Mdm2 and Puma was determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (E) Ex vivo thymocytes were isolated and were either left untreated or irradiated with 2Gy for 12 hours (n=3 per genotype). Apoptotic cells were quantified by flow cytometry. Standard deviation indicated by error bars. (F) Primary MEF proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (G) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were analyzed by western blot. .

Techniques Used: Sequencing, Introduce, Mouse Assay, Western Blot, Irradiation, Ex Vivo, Expressing, Quantitative RT-PCR, Standard Deviation, Isolation, Flow Cytometry, Cytometry

13) Product Images from "Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2"

Article Title: Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

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

doi: 10.1073/pnas.1800431115

Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.
Figure Legend Snippet: Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.

Techniques Used: Activity Assay, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.
Figure Legend Snippet: Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.

Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Confocal Microscopy

14) Product Images from "ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice"

Article Title: ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice

Journal: Cancer Cell

doi: 10.1016/j.ccr.2012.04.011

Mdm2 Ser394 phosphorylation is necessary for full p53 stabilization and function in MEFs (A) Primary MEFs proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (B) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were then analyzed by western blot. (C) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times. (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation is necessary for full p53 stabilization and function in MEFs (A) Primary MEFs proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (B) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were then analyzed by western blot. (C) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times. (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. .

Techniques Used: Standard Deviation, Irradiation, Western Blot, Expressing, Quantitative RT-PCR

Mdm2 Ser394 phosphorylation regulates the attenuation of the p53 response to DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested either 4 or 8 hours later. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for either 12 or 18 hours. Protein levels were then analyzed by western blot. (C) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation regulates the attenuation of the p53 response to DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested either 4 or 8 hours later. Protein levels were then analyzed by western blot. (B) Ex vivo thymocytes were either untreated or irradiated with 2Gy for either 12 or 18 hours. Protein levels were then analyzed by western blot. (C) Ex vivo thymocytes were either untreated or irradiated with 2Gy for the indicated times (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh .

Techniques Used: Mouse Assay, Irradiation, Western Blot, Ex Vivo, Expressing, Quantitative RT-PCR

Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot. (B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. (D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours. (E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type. .
Figure Legend Snippet: Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage (A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot. (B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. (D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours. (E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type. .

Techniques Used: Activity Assay, Mouse Assay, Irradiation, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation

Phosphorylation of Mdm2 Ser394 is necessary but not sufficient to stabilize and activate p53 (A) DNA sequence of both the WT and S394D allele at Mdm2 codon 394. The mutations in S394D also introduce a BclI restriction digest site. (B) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.10) and Mdm2 (p=0.88) relative to PI3K were normalized to wild-type. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. (D) Ex vivo thymocytes were either untreated or irradiated with 2Gy for 2 or 4 hours (n=3 per genotype). Relative expression of Mdm2 and Puma was determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (E) Ex vivo thymocytes were isolated and were either left untreated or irradiated with 2Gy for 12 hours (n=3 per genotype). Apoptotic cells were quantified by flow cytometry. Standard deviation indicated by error bars. (F) Primary MEF proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (G) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were analyzed by western blot. .
Figure Legend Snippet: Phosphorylation of Mdm2 Ser394 is necessary but not sufficient to stabilize and activate p53 (A) DNA sequence of both the WT and S394D allele at Mdm2 codon 394. The mutations in S394D also introduce a BclI restriction digest site. (B) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.10) and Mdm2 (p=0.88) relative to PI3K were normalized to wild-type. (C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. (D) Ex vivo thymocytes were either untreated or irradiated with 2Gy for 2 or 4 hours (n=3 per genotype). Relative expression of Mdm2 and Puma was determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. (E) Ex vivo thymocytes were isolated and were either left untreated or irradiated with 2Gy for 12 hours (n=3 per genotype). Apoptotic cells were quantified by flow cytometry. Standard deviation indicated by error bars. (F) Primary MEF proliferation was counted each day (n=3 per genotype). Standard deviation indicated by error bars. (G) Primary MEFs were either untreated or irradiated with 4Gy for the indicated times, and protein levels were analyzed by western blot. .

Techniques Used: Sequencing, Introduce, Mouse Assay, Western Blot, Irradiation, Ex Vivo, Expressing, Quantitative RT-PCR, Standard Deviation, Isolation, Flow Cytometry, Cytometry

Phosphorylation of Mdm2 Ser394 regulates radiosensitivity in mice (A) DNA sequence of the WT and S394A alleles at Mdm2 codon 394. The mutations in S394A also introduce a BcgI restriction digest site. (B) PCR- BcgI analysis of F 2 generation mice. Primers flanking Mdm2 codon 394 were used to amplify DNA samples from WT, A/+ , and A/A F 2 generation mice, followed by BcgI digestion. (C) Mice that are either heterozygous and homozygous for the S394A allele are viable and were observed at Mendelian ratios after heterozygous intercrosses (p=0.322). (D) Cohorts of wild-type (n=14), A/A (n=14), and p53 −/− (n=8) mice were irradiated with 8Gy and survival was observed over 26 days. (E) WT, A/A , and p53 −/− .
Figure Legend Snippet: Phosphorylation of Mdm2 Ser394 regulates radiosensitivity in mice (A) DNA sequence of the WT and S394A alleles at Mdm2 codon 394. The mutations in S394A also introduce a BcgI restriction digest site. (B) PCR- BcgI analysis of F 2 generation mice. Primers flanking Mdm2 codon 394 were used to amplify DNA samples from WT, A/+ , and A/A F 2 generation mice, followed by BcgI digestion. (C) Mice that are either heterozygous and homozygous for the S394A allele are viable and were observed at Mendelian ratios after heterozygous intercrosses (p=0.322). (D) Cohorts of wild-type (n=14), A/A (n=14), and p53 −/− (n=8) mice were irradiated with 8Gy and survival was observed over 26 days. (E) WT, A/A , and p53 −/− .

Techniques Used: Mouse Assay, Sequencing, Introduce, Polymerase Chain Reaction, Irradiation

15) Product Images from "Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model"

Article Title: Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model

Journal: Scientific Reports

doi: 10.1038/s41598-017-02915-6

Quantitative PCR analysis (qPCR) for Tlr4 ( A ), Nlrp3 ( B ), Casp1 ( C ) and Il1b ( D ) gene expression. Results are presented as median plus range. The number of analyzed animals is represented next to the name of each group. Differences among groups were analyzed by one-way ANOVA with Newman-Keuls post test. a p
Figure Legend Snippet: Quantitative PCR analysis (qPCR) for Tlr4 ( A ), Nlrp3 ( B ), Casp1 ( C ) and Il1b ( D ) gene expression. Results are presented as median plus range. The number of analyzed animals is represented next to the name of each group. Differences among groups were analyzed by one-way ANOVA with Newman-Keuls post test. a p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

16) Product Images from "Toll-Like Receptors 4, 5, 6 and 7 are Constitutively Expressed in Non-Human Primate Retinal Neurons"

Article Title: Toll-Like Receptors 4, 5, 6 and 7 are Constitutively Expressed in Non-Human Primate Retinal Neurons

Journal: Journal of neuroimmunology

doi: 10.1016/j.jneuroim.2018.06.007

TLR7 co-localizes with the Muller cell marker vimentin A) Arrows indicate TLR7 staining of filamentous retinal cells. B) Arrows indicate vimentin staining of Muller cells. C) Merged image with arrows indicating co-localization of TLR7 and vimentin positive cells. Outer plexiform layer (OPL), inner nuclear layer (INL), ganglion cell layer (GCL). Scale bar= 20 μm.
Figure Legend Snippet: TLR7 co-localizes with the Muller cell marker vimentin A) Arrows indicate TLR7 staining of filamentous retinal cells. B) Arrows indicate vimentin staining of Muller cells. C) Merged image with arrows indicating co-localization of TLR7 and vimentin positive cells. Outer plexiform layer (OPL), inner nuclear layer (INL), ganglion cell layer (GCL). Scale bar= 20 μm.

Techniques Used: Marker, Staining

17) Product Images from "Activation of Melatonin Signaling Promotes β-Cell Survival and Function"

Article Title: Activation of Melatonin Signaling Promotes β-Cell Survival and Function

Journal: Molecular Endocrinology

doi: 10.1210/me.2014-1293

Persistent activation of β-cell MT receptor signaling modulates apoptotic and oxidative stress gene expression in response to β-cell proteotoxicity. Apoptosis RT 2 Profiler PCR (A) and oxidative stress RT 2 Profiler PCR (B) arrays demonstrating relative mRNA expression of 146 genes purported to mediate cellular apoptotic and oxidative stress pathways in INS 832/13 cells transduced with h-IAPP adenovirus for 48 hours and exposed to either MT (10 nM) or vehicle (DMSO). The graph represents mRNA levels expressed as fold change for MT (10 nM) vs vehicle (DMSO)-treated cells (n = 3 independent experiments for each array). Data are expressed as mean ± SEM. Dashed red line represents mRNA expression in vehicle (DMSO)-treated cells. Genes demonstrating notably significant changes vs vehicle are highlighted in red. DMSO vehicle treatment was administered to all non-MT conditions to correct for potential confounding effects of DMSO, which was used to prepare MT solutions.
Figure Legend Snippet: Persistent activation of β-cell MT receptor signaling modulates apoptotic and oxidative stress gene expression in response to β-cell proteotoxicity. Apoptosis RT 2 Profiler PCR (A) and oxidative stress RT 2 Profiler PCR (B) arrays demonstrating relative mRNA expression of 146 genes purported to mediate cellular apoptotic and oxidative stress pathways in INS 832/13 cells transduced with h-IAPP adenovirus for 48 hours and exposed to either MT (10 nM) or vehicle (DMSO). The graph represents mRNA levels expressed as fold change for MT (10 nM) vs vehicle (DMSO)-treated cells (n = 3 independent experiments for each array). Data are expressed as mean ± SEM. Dashed red line represents mRNA expression in vehicle (DMSO)-treated cells. Genes demonstrating notably significant changes vs vehicle are highlighted in red. DMSO vehicle treatment was administered to all non-MT conditions to correct for potential confounding effects of DMSO, which was used to prepare MT solutions.

Techniques Used: Activation Assay, Expressing, Polymerase Chain Reaction, Transduction

18) Product Images from "Feedback Mechanisms for Cardiac-Specific microRNAs and cAMP Signaling in Electrical Remodeling"

Article Title: Feedback Mechanisms for Cardiac-Specific microRNAs and cAMP Signaling in Electrical Remodeling

Journal: Circulation. Arrhythmia and electrophysiology

doi: 10.1161/CIRCEP.114.002162

Dicer Knockout Leads to Electrical Remodeling in Neonatal Cardiomyocytes (a) Immunofluorescence confocal images of neonatal cardiomyocytes transduced with Ad-GFP and Ad-GFP-Cre. Cre recombinase (red) is seen in the nucleus of Ad-GFP-Cre cells. GFP expression
Figure Legend Snippet: Dicer Knockout Leads to Electrical Remodeling in Neonatal Cardiomyocytes (a) Immunofluorescence confocal images of neonatal cardiomyocytes transduced with Ad-GFP and Ad-GFP-Cre. Cre recombinase (red) is seen in the nucleus of Ad-GFP-Cre cells. GFP expression

Techniques Used: Knock-Out, Immunofluorescence, Transduction, Expressing

miR-1/133a Delivery Preserves Left Ventricular Function and Prevents Hypertrophy after MI (a) Experimental time course for echocardiography and miR-1/133a delivery relative to MI surgery. (b) Cardiomyocytes isolated from adult mice 3 days after tail vein
Figure Legend Snippet: miR-1/133a Delivery Preserves Left Ventricular Function and Prevents Hypertrophy after MI (a) Experimental time course for echocardiography and miR-1/133a delivery relative to MI surgery. (b) Cardiomyocytes isolated from adult mice 3 days after tail vein

Techniques Used: Isolation, Mouse Assay

Dicer Knockout Leads to Electrical Remodeling in Adult Cardiomyocytes (a) qRT-PCR results normalized to GAPDH expression from sham and MI mice for Dicer1 mRNA ( n=3, *p
Figure Legend Snippet: Dicer Knockout Leads to Electrical Remodeling in Adult Cardiomyocytes (a) qRT-PCR results normalized to GAPDH expression from sham and MI mice for Dicer1 mRNA ( n=3, *p

Techniques Used: Knock-Out, Quantitative RT-PCR, Expressing, Mouse Assay

19) Product Images from "Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription"

Article Title: Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription

Journal: Nature Communications

doi: 10.1038/ncomms12237

JMJD1a regulates YAP/TAZ transcription ( a ) Representative western blot showing YAP/TAZ expression in MDA-MB-231 on CDM and on plastic. ( b ) Taqman qRT–PCR of CTGF ( n =4) and THBS1 ( n =5) mRNA levels in MDA-MB-231 cells grown on CDM or on plastic. ( c ) Quantification of JMJD1a and YAP/TAZ staining intensity from immunofluorescence images of MDA-MB-231 cells on plastic. Intensity (int den) was quantified using the CellProfiler software. n (cells)=146. R -value indicates correlation. ( d ) ChIP showing the binding of JMJD1a to the TAZ promoter. Analysis was performed by SYBR green-based detection and fold increase in signal relative to the background signal (IgG control antibody) is shown. n =3 (mean±s.d.). Paired t -test was used to calculate P value. ( e ) ChIP showing H3K9me2 levels on TAZ promoter of siControl and siJMJD1a_3-transfected MDA-MB-231 cells. ChIP was performed with two independent H3K9me2 antibodies. Analysis was performed by SYBR green-based detection and fold increase in signal relative to the background signal (IgG control antibody) is shown. Representative results from two independent experiments. ( f , g ) A representative western blot ( e ) and quantification ( f ) showing YAP/TAZ expression in JMJD1a-silenced MDA-MB-231 cells after 3 days of silencing, n =7. ( h ) Taqman qRT–PCR of JMJD1a ( KDM3A ), CTGF and THBS1 mRNA levels in JMJD1a-silenced MDA-MB-231 cells, n =4. ( i ) Taqman qRT–PCR of YAP and TAZ mRNA levels in JMJD1a siRNA-transfected MDA-MB-231 cells. n (JMJD1a and TAZ)=4, n (YAP)=3. ( j , k ) Representative western blot ( i ) and quantification ( j ) showing YAP/TAZ expression in JMJD1a-overexpressing MDA-MB-231 cells normalized to loading control, n =4. ( l ) Taqman qRT–PCR of JMJD1a ( KDM3A) , CTGF and THBS1 mRNA levels in JMJD1a-overexpressing MDA-MB-231 cells, n =4. ( m ) A representative western blot and quantification of YAP/TAZ expression on TIFF-derived CDM in GFP control and JMJD1a-GFP-overexpressing cells after 4 days on CDM. ( n ) Representative western blot of JMJD1a and YAP/TAZ expression in GFP control and JMJD1a-overexpressing MDA-MB-231 cells plated on 4 or 50 kPa hydrogels or on PL.
Figure Legend Snippet: JMJD1a regulates YAP/TAZ transcription ( a ) Representative western blot showing YAP/TAZ expression in MDA-MB-231 on CDM and on plastic. ( b ) Taqman qRT–PCR of CTGF ( n =4) and THBS1 ( n =5) mRNA levels in MDA-MB-231 cells grown on CDM or on plastic. ( c ) Quantification of JMJD1a and YAP/TAZ staining intensity from immunofluorescence images of MDA-MB-231 cells on plastic. Intensity (int den) was quantified using the CellProfiler software. n (cells)=146. R -value indicates correlation. ( d ) ChIP showing the binding of JMJD1a to the TAZ promoter. Analysis was performed by SYBR green-based detection and fold increase in signal relative to the background signal (IgG control antibody) is shown. n =3 (mean±s.d.). Paired t -test was used to calculate P value. ( e ) ChIP showing H3K9me2 levels on TAZ promoter of siControl and siJMJD1a_3-transfected MDA-MB-231 cells. ChIP was performed with two independent H3K9me2 antibodies. Analysis was performed by SYBR green-based detection and fold increase in signal relative to the background signal (IgG control antibody) is shown. Representative results from two independent experiments. ( f , g ) A representative western blot ( e ) and quantification ( f ) showing YAP/TAZ expression in JMJD1a-silenced MDA-MB-231 cells after 3 days of silencing, n =7. ( h ) Taqman qRT–PCR of JMJD1a ( KDM3A ), CTGF and THBS1 mRNA levels in JMJD1a-silenced MDA-MB-231 cells, n =4. ( i ) Taqman qRT–PCR of YAP and TAZ mRNA levels in JMJD1a siRNA-transfected MDA-MB-231 cells. n (JMJD1a and TAZ)=4, n (YAP)=3. ( j , k ) Representative western blot ( i ) and quantification ( j ) showing YAP/TAZ expression in JMJD1a-overexpressing MDA-MB-231 cells normalized to loading control, n =4. ( l ) Taqman qRT–PCR of JMJD1a ( KDM3A) , CTGF and THBS1 mRNA levels in JMJD1a-overexpressing MDA-MB-231 cells, n =4. ( m ) A representative western blot and quantification of YAP/TAZ expression on TIFF-derived CDM in GFP control and JMJD1a-GFP-overexpressing cells after 4 days on CDM. ( n ) Representative western blot of JMJD1a and YAP/TAZ expression in GFP control and JMJD1a-overexpressing MDA-MB-231 cells plated on 4 or 50 kPa hydrogels or on PL.

Techniques Used: Western Blot, Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Staining, Immunofluorescence, Software, Chromatin Immunoprecipitation, Binding Assay, SYBR Green Assay, Transfection, Derivative Assay

20) Product Images from "SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved"

Article Title: SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved

Journal: Biology of Sex Differences

doi: 10.1186/s13293-017-0158-2

Effect of O 3 in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O 3 exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold ( p
Figure Legend Snippet: Effect of O 3 in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O 3 exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold ( p

Techniques Used: Mouse Assay, Quantitative RT-PCR

21) Product Images from "Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2"

Article Title: Circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

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

doi: 10.1073/pnas.1800431115

Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.
Figure Legend Snippet: Rhythms in NRF2 levels and activity are disrupted with Bmal1 deletion. Bmal1 +/+ and Bmal1 −/− BMDMs were analyzed by TF-Seq. Bar charts reveal the relative activity of NRF2 in Bmal1 +/+ and Bmal1 −/− BMDMs following ( A ) 1 or ( B ) 4 h of LPS stimulation ( n = 3). ( C ) Wild-type BMDMs were lysed and the samples were exposed to biotinylated primer sequences for the E-box site located in the Nrf2 promoter (WT E-box) or a mutated control of the E-box. The sequences were then isolated using streptavidin beads before performing an immunoblot for BMAL1. ( D ) Peritoneal cells were isolated from wild-type mice at 4-h intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm (n= 3–6). ( E ) Bmal1 +/+ and Bmal1 −/− BMDMs were synchronized using 50% horse serum. RNA was extracted at 4-h intervals for 36 h and Nrf2 mRNA was measured by qPCR. A cosinor regression model was used to test the null hypothesis that the amplitude of expression = 0. The presence of the red line indicates the presence of a significant rhythm ( n = 3). ( F ) Nrf2 mRNA was measured by qPCR in Bmal1 +/+ and Bmal1 −/− BMDMs following 24 h of LPS (100 ng/mL) ( n = 4). ( G ) Immunoblot of NRF2 levels following 24 h of LPS (100 ng/mL) in Bmal1 +/+ and Bmal1 −/− BMDMs. Immunoblots are a representative of at least three independent experiments. Values provided below each lane indicate relative densitometry of each band. Statistical significance in graphs A and B was determined by false discovery rate (FDR); in D and E , by establishing a cosinor regression model; and in graph F , by one-way ANOVA. *** P ≤ 0.001.

Techniques Used: Activity Assay, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.
Figure Legend Snippet: Diurnal rhythms in ROS regulation. ( A ) Peritoneal cells were isolated from wild-type mice at ZT8 and ZT20. Cells were stained with a CD11b + antibody and CellROX stain. ROS levels were then measured by mean fluoresence intensity (MFI) in the myeloid population by flow cytometry ( n = 4–6). ( B ) Peritoneal cells were isolated from wild-type mice at ZT0 and ZT12. RNA was extracted and gene expression panels were used to measure fold change of oxidative stress pathway genes at ZT12 versus ZT0 ( n = 3). ( C ) Bmal1 +/+ and Bmal1 −/− BMDMs were treated with LPS (100 ng/mL) for 24 h before staining with CellROX. ROS was then measured via flow cytometry ( n = 4). ( D ) Bmal1 +/+ and Bmal1 −/− BMDMs were untreated or treated with LPS (100 ng/mL) for 24 h before staining with CellROX and MitoTracker green. The cells were imaged using confocal microscopy. Image shown is a representative of at least three independent experiments. Statistical significance of graph A was determined by unpaired Student’s t test. Statistical significance of C was determined by one-way ANOVA. * P ≤ 0.05 and ** P ≤ 0.01.

Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Confocal Microscopy

Related Articles

Polymerase Chain Reaction:

Article Title: Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model
Article Snippet: .. The cDNAs were mixed with the appropriate PCR master mix buffer (Rt² Syber Green ROX qPCR Primer Assay QiagenN® #330523) and analyzed on specific array plates (Rat Innate & Adaptive Immune Responses Rt² Profiler™ PCR, Qiagen®, #330231-PARN052Zc). .. We further analyzed the expression of selected genes involved in the innate immune response by individual qPCR (10 animals/group).

Article Title: Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery
Article Snippet: .. For the study of NSC gene expression, the 96-well QIAGEN PCR array for neurogenesis was used in combination with the RT2 SYBR Green qPCR Mastermix (QIAGEN), using 10 ng of cDNA per well. .. In brief, 5 d in vitro (DIV) secondary neurospheres were dissociated as described above and plated on 0.25 mg/mL MCM Gel 2D Cultrex (TREVIGEN, Helgerman Court, Gaithersburg, MD USA) in 24-well plates at a density of 1 × 104 cells/cm2 .

Article Title: IGF-1R and Leptin Expression Profile and the Effects of Metformin Treatment on Metabolic and Endocrine Parameters in PCOS Mice
Article Snippet: .. Subsequently, the reverse transcriptase (RT) was performed using the RT2 First Strand Kit (Ref: 330404; Qiagen, Hilden, Germany); and the quantitative Real-Time PCR array was carried out using the RT2 Profiler™ PCR Array Mouse Insulin Signaling Pathway (PAMM-030A; Qiagen, Hilden, Germany), 96-well plate, and RT2 SYBR® Green qPCR Mastermix (Qiagen, Hilden, Germany). .. All data were normalized with the Gusb , Hprt , Hprt1 , Hsp90ab1 , Gapdh , and ACTb genes and analyzed by ΔΔC t method in the http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php software, following the manufacturer's instructions.

Article Title: Sex differences after chronic stress in the expression of opioid-, stress- and neuroplasticity-related genes in the rat hippocampus
Article Snippet: .. 2.4 RT2 profiler array Gene expression levels were measured using RT2 SYBR® Green qPCR Mastermix and a custom RT2 PCR Array (both from Qiagen, Inc.) following the manufacturer's recommended protocol. ..

Article Title: Myeloid cell deletion of Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) induces non-alcoholic steatohepatitis
Article Snippet: .. As described previously [ ] expression of 86 genes related to fatty liver was measured using Mouse Fatty Liver RT2 Profiler PCR Arrays (#330231 PAMM-157ZA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Doncaster, VIC, Australia). .. Results were analysed using RT2 Profiler software, and expression was normalised to B2m (beta-2 macroglubulin), Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Gusb (beta glucuronidase) as housekeeping genes.

Real-time Polymerase Chain Reaction:

Article Title: Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model
Article Snippet: .. The cDNAs were mixed with the appropriate PCR master mix buffer (Rt² Syber Green ROX qPCR Primer Assay QiagenN® #330523) and analyzed on specific array plates (Rat Innate & Adaptive Immune Responses Rt² Profiler™ PCR, Qiagen®, #330231-PARN052Zc). .. We further analyzed the expression of selected genes involved in the innate immune response by individual qPCR (10 animals/group).

Article Title: Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery
Article Snippet: .. For the study of NSC gene expression, the 96-well QIAGEN PCR array for neurogenesis was used in combination with the RT2 SYBR Green qPCR Mastermix (QIAGEN), using 10 ng of cDNA per well. .. In brief, 5 d in vitro (DIV) secondary neurospheres were dissociated as described above and plated on 0.25 mg/mL MCM Gel 2D Cultrex (TREVIGEN, Helgerman Court, Gaithersburg, MD USA) in 24-well plates at a density of 1 × 104 cells/cm2 .

Article Title: The Aurora A-HP1γ pathway regulates gene expression and mitosis in cells from the sperm lineage
Article Snippet: .. Real time PCR was performed using RT2 SYBR® Green qPCR Mastermix (Qiagen) and RT2 qPCR Primers (Qiagen) on the Bio-Rad CFX96 system. .. Fold changes and standard error of the means (S.E.M.) were calculated using Bio-Rad CFX manager or SABioscience’s RT2 Profiler PCR Array Data Analysis software.

Article Title: Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism
Article Snippet: .. Real-time PCR reactions were carried out on ViiA™ 7 Real Time PCR System (Applied Biosystems) using the RT2 SYBR® Green qPCR Mastermix (Qiagen) according to manufacturer’s instructions. .. Data analysis was performed using the Qiagen’s integrated web-based software package for the PCR Array System, which automatically performs all ΔΔCt based fold-change calculations from raw threshold cycle data.

Article Title: IGF-1R and Leptin Expression Profile and the Effects of Metformin Treatment on Metabolic and Endocrine Parameters in PCOS Mice
Article Snippet: .. Subsequently, the reverse transcriptase (RT) was performed using the RT2 First Strand Kit (Ref: 330404; Qiagen, Hilden, Germany); and the quantitative Real-Time PCR array was carried out using the RT2 Profiler™ PCR Array Mouse Insulin Signaling Pathway (PAMM-030A; Qiagen, Hilden, Germany), 96-well plate, and RT2 SYBR® Green qPCR Mastermix (Qiagen, Hilden, Germany). .. All data were normalized with the Gusb , Hprt , Hprt1 , Hsp90ab1 , Gapdh , and ACTb genes and analyzed by ΔΔC t method in the http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php software, following the manufacturer's instructions.

Article Title: Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development, et al. Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development
Article Snippet: .. Each cDNA was synthesized and mixed with RT2 SYBR Green qPCR mastermix (Qiagen) and an equal volume was added into each well of a 96‐well array plate. .. Real‐time PCR was performed using the CFX 96 Real‐Time System (Bio‐Rad).

Article Title: Myeloid cell deletion of Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) induces non-alcoholic steatohepatitis
Article Snippet: .. As described previously [ ] expression of 86 genes related to fatty liver was measured using Mouse Fatty Liver RT2 Profiler PCR Arrays (#330231 PAMM-157ZA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Doncaster, VIC, Australia). .. Results were analysed using RT2 Profiler software, and expression was normalised to B2m (beta-2 macroglubulin), Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Gusb (beta glucuronidase) as housekeeping genes.

SYBR Green Assay:

Article Title: Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery
Article Snippet: .. For the study of NSC gene expression, the 96-well QIAGEN PCR array for neurogenesis was used in combination with the RT2 SYBR Green qPCR Mastermix (QIAGEN), using 10 ng of cDNA per well. .. In brief, 5 d in vitro (DIV) secondary neurospheres were dissociated as described above and plated on 0.25 mg/mL MCM Gel 2D Cultrex (TREVIGEN, Helgerman Court, Gaithersburg, MD USA) in 24-well plates at a density of 1 × 104 cells/cm2 .

Article Title: The Aurora A-HP1γ pathway regulates gene expression and mitosis in cells from the sperm lineage
Article Snippet: .. Real time PCR was performed using RT2 SYBR® Green qPCR Mastermix (Qiagen) and RT2 qPCR Primers (Qiagen) on the Bio-Rad CFX96 system. .. Fold changes and standard error of the means (S.E.M.) were calculated using Bio-Rad CFX manager or SABioscience’s RT2 Profiler PCR Array Data Analysis software.

Article Title: Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism
Article Snippet: .. Real-time PCR reactions were carried out on ViiA™ 7 Real Time PCR System (Applied Biosystems) using the RT2 SYBR® Green qPCR Mastermix (Qiagen) according to manufacturer’s instructions. .. Data analysis was performed using the Qiagen’s integrated web-based software package for the PCR Array System, which automatically performs all ΔΔCt based fold-change calculations from raw threshold cycle data.

Article Title: IGF-1R and Leptin Expression Profile and the Effects of Metformin Treatment on Metabolic and Endocrine Parameters in PCOS Mice
Article Snippet: .. Subsequently, the reverse transcriptase (RT) was performed using the RT2 First Strand Kit (Ref: 330404; Qiagen, Hilden, Germany); and the quantitative Real-Time PCR array was carried out using the RT2 Profiler™ PCR Array Mouse Insulin Signaling Pathway (PAMM-030A; Qiagen, Hilden, Germany), 96-well plate, and RT2 SYBR® Green qPCR Mastermix (Qiagen, Hilden, Germany). .. All data were normalized with the Gusb , Hprt , Hprt1 , Hsp90ab1 , Gapdh , and ACTb genes and analyzed by ΔΔC t method in the http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php software, following the manufacturer's instructions.

Article Title: Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development, et al. Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development
Article Snippet: .. Each cDNA was synthesized and mixed with RT2 SYBR Green qPCR mastermix (Qiagen) and an equal volume was added into each well of a 96‐well array plate. .. Real‐time PCR was performed using the CFX 96 Real‐Time System (Bio‐Rad).

Article Title: Myeloid cell deletion of Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) induces non-alcoholic steatohepatitis
Article Snippet: .. As described previously [ ] expression of 86 genes related to fatty liver was measured using Mouse Fatty Liver RT2 Profiler PCR Arrays (#330231 PAMM-157ZA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Doncaster, VIC, Australia). .. Results were analysed using RT2 Profiler software, and expression was normalised to B2m (beta-2 macroglubulin), Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Gusb (beta glucuronidase) as housekeeping genes.

Expressing:

Article Title: Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery
Article Snippet: .. For the study of NSC gene expression, the 96-well QIAGEN PCR array for neurogenesis was used in combination with the RT2 SYBR Green qPCR Mastermix (QIAGEN), using 10 ng of cDNA per well. .. In brief, 5 d in vitro (DIV) secondary neurospheres were dissociated as described above and plated on 0.25 mg/mL MCM Gel 2D Cultrex (TREVIGEN, Helgerman Court, Gaithersburg, MD USA) in 24-well plates at a density of 1 × 104 cells/cm2 .

Article Title: Sex differences after chronic stress in the expression of opioid-, stress- and neuroplasticity-related genes in the rat hippocampus
Article Snippet: .. 2.4 RT2 profiler array Gene expression levels were measured using RT2 SYBR® Green qPCR Mastermix and a custom RT2 PCR Array (both from Qiagen, Inc.) following the manufacturer's recommended protocol. ..

Article Title: Myeloid cell deletion of Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) induces non-alcoholic steatohepatitis
Article Snippet: .. As described previously [ ] expression of 86 genes related to fatty liver was measured using Mouse Fatty Liver RT2 Profiler PCR Arrays (#330231 PAMM-157ZA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Doncaster, VIC, Australia). .. Results were analysed using RT2 Profiler software, and expression was normalised to B2m (beta-2 macroglubulin), Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Gusb (beta glucuronidase) as housekeeping genes.

Synthesized:

Article Title: Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development, et al. Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development
Article Snippet: .. Each cDNA was synthesized and mixed with RT2 SYBR Green qPCR mastermix (Qiagen) and an equal volume was added into each well of a 96‐well array plate. .. Real‐time PCR was performed using the CFX 96 Real‐Time System (Bio‐Rad).

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  • 99
    Qiagen rt² sybr green rox qpcr mastermix
    Quantitative <t>PCR</t> analysis (qPCR) for Tlr4 ( A ), Nlrp3 ( B ), Casp1 ( C ) and Il1b ( D ) gene expression. Results are presented as median plus range. The number of analyzed animals is represented next to the name of each group. Differences among groups were analyzed by one-way ANOVA with Newman-Keuls post test. a p
    Rt² Sybr Green Rox Qpcr Mastermix, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rt² sybr green fluor qpcr mastermix
    Oct4 inhibition in organoid cultures. (A) Timeline for Oct4 inhibition using adenovirus in organoid culture. (B) Enhanced <t>green</t> fluorescent protein (eGFP) in hepatocytes infected with adenovirus containing short hairpin RNA (shRNA) for Oct4, multiplicity of infection (MOI) 5, > 70% infection observed at day 14 in culture. Original magnification: 100×. (C–G) Analyzed from D21 samples treated with shRNA for Oct4 (AD-Oct4) or shRNA for scramble control (AD-Scr). (C) mRNA levels of Oct4 assessed by <t>qRT-PCR</t> and expressed as fold change relative to GAPDH. (D) Oct4 protein assessed by sELISA. mRNA levels as assessed by qRT-PCR and expressed as fold change relative to GAPDH of (E) biliary-specific marker HNF-1β, (F) hepatocyte-specific marker HNF-4α, and (G) biliary-specific marker CK19. Significantly different from AD-Scr control: * p ≤ 0.05, ** p ≤ 0.01.
    Rt² Sybr Green Fluor Qpcr Mastermix, supplied by Qiagen, used in various techniques. Bioz Stars score: 97/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rt² sybr green fluor qpcr mastermix/product/Qiagen
    Average 97 stars, based on 22 article reviews
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    Image Search Results


    Quantitative PCR analysis (qPCR) for Tlr4 ( A ), Nlrp3 ( B ), Casp1 ( C ) and Il1b ( D ) gene expression. Results are presented as median plus range. The number of analyzed animals is represented next to the name of each group. Differences among groups were analyzed by one-way ANOVA with Newman-Keuls post test. a p

    Journal: Scientific Reports

    Article Title: Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model

    doi: 10.1038/s41598-017-02915-6

    Figure Lengend Snippet: Quantitative PCR analysis (qPCR) for Tlr4 ( A ), Nlrp3 ( B ), Casp1 ( C ) and Il1b ( D ) gene expression. Results are presented as median plus range. The number of analyzed animals is represented next to the name of each group. Differences among groups were analyzed by one-way ANOVA with Newman-Keuls post test. a p

    Article Snippet: The cDNAs were mixed with the appropriate PCR master mix buffer (Rt² Syber Green ROX qPCR Primer Assay QiagenN® #330523) and analyzed on specific array plates (Rat Innate & Adaptive Immune Responses Rt² Profiler™ PCR, Qiagen®, #330231-PARN052Zc).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Gene regulation of neferine-mediated autophagy induction. ( A ) RT 2 profiler autophagy PCR array analysis of neferine. HeLa cells were with treated with 10 μM of neferine for 24 h. The total RNA was extracted and reverse-transcripted as cDNA. Real-time PCR reactions were performed using the RT2 SYBR® Green qPCR Mastermix and data analysis was determined using the Qiagen’s integrated web-based software package for the PCR Array System. Scatter plot highlighted the up-regulation and down-regulation of genes in response to neferine treatment. Inner panel: quantification of PCR array analysis. ( B ) Neferine-mediated genes regulation was confirmed by western blotting. Upper panel, HeLa cells were treated with neferine (10 μM) for the indicated time. Cell lysates were analyzed with antibodies against CXCR4, P-PERK, PERK, p62, ULK-1 and actin respectively. Lower panel, HeLa cells were treated with neferine (10 μM) for the indicated time and rapamycin (300 nM) for 24 h. Cell lysates were analyzed with antibodies against P-eIF-2α, eIF-2α and actin respectively. The quantification graphs and full-length blots/gels are presented in Supplementary Figs. S2 and S10 ( A ), respectively. ( C ) Activation of PERK and ULK-1 is required for neferine-induced autophagy. HeLa cells were transfected with control si RNA, PERK or ULK-1 si RNA together with EGFP-LC3 plasmid for 48 h, cells were treated with neferine (10 μM) for 4 h and then fixed for fluorescence imaging and cells counting. Western blot images indicate the gene knock down efficiency. Bar chart represents the quantitation of autophagic cells. The full-length blots/gels are presented in Supplementary Fig. S10 ( B ). ( D ) Effect of CXCR4 in Nef-induced autophagy. EGFP-LC3 transfected HeLa cells were treated with 10 μM neferine in the presence or absence of CXCR4 specific inhibitor, AMD 3100 (25 mg/mL) for 4 h. The cells were then fixed for fluorescence imaging and cells counting. Bar chart represents the quantitation of autophagic cells. Western blot image indicates the LC3-II conversion in HeLa cells in response to neferine and AMD 3100 treatment. The full-length blots/gels are presented in Supplementary Fig. S10 ( C ). Percentages of autophagic cells demonstrated by the increased number of cells with EGFP-LC3 dots signal (≥10 dots/cell) over the total number of EGFP-positive cells in the same field. More than 1000 EGFP-positive cells were scored for each treatment. Error bars, S.D. **P

    Journal: Scientific Reports

    Article Title: Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism

    doi: 10.1038/s41598-019-56675-6

    Figure Lengend Snippet: Gene regulation of neferine-mediated autophagy induction. ( A ) RT 2 profiler autophagy PCR array analysis of neferine. HeLa cells were with treated with 10 μM of neferine for 24 h. The total RNA was extracted and reverse-transcripted as cDNA. Real-time PCR reactions were performed using the RT2 SYBR® Green qPCR Mastermix and data analysis was determined using the Qiagen’s integrated web-based software package for the PCR Array System. Scatter plot highlighted the up-regulation and down-regulation of genes in response to neferine treatment. Inner panel: quantification of PCR array analysis. ( B ) Neferine-mediated genes regulation was confirmed by western blotting. Upper panel, HeLa cells were treated with neferine (10 μM) for the indicated time. Cell lysates were analyzed with antibodies against CXCR4, P-PERK, PERK, p62, ULK-1 and actin respectively. Lower panel, HeLa cells were treated with neferine (10 μM) for the indicated time and rapamycin (300 nM) for 24 h. Cell lysates were analyzed with antibodies against P-eIF-2α, eIF-2α and actin respectively. The quantification graphs and full-length blots/gels are presented in Supplementary Figs. S2 and S10 ( A ), respectively. ( C ) Activation of PERK and ULK-1 is required for neferine-induced autophagy. HeLa cells were transfected with control si RNA, PERK or ULK-1 si RNA together with EGFP-LC3 plasmid for 48 h, cells were treated with neferine (10 μM) for 4 h and then fixed for fluorescence imaging and cells counting. Western blot images indicate the gene knock down efficiency. Bar chart represents the quantitation of autophagic cells. The full-length blots/gels are presented in Supplementary Fig. S10 ( B ). ( D ) Effect of CXCR4 in Nef-induced autophagy. EGFP-LC3 transfected HeLa cells were treated with 10 μM neferine in the presence or absence of CXCR4 specific inhibitor, AMD 3100 (25 mg/mL) for 4 h. The cells were then fixed for fluorescence imaging and cells counting. Bar chart represents the quantitation of autophagic cells. Western blot image indicates the LC3-II conversion in HeLa cells in response to neferine and AMD 3100 treatment. The full-length blots/gels are presented in Supplementary Fig. S10 ( C ). Percentages of autophagic cells demonstrated by the increased number of cells with EGFP-LC3 dots signal (≥10 dots/cell) over the total number of EGFP-positive cells in the same field. More than 1000 EGFP-positive cells were scored for each treatment. Error bars, S.D. **P

    Article Snippet: Real-time PCR reactions were carried out on ViiA™ 7 Real Time PCR System (Applied Biosystems) using the RT2 SYBR® Green qPCR Mastermix (Qiagen) according to manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SYBR Green Assay, Software, Western Blot, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Imaging, Quantitation Assay

    Oct4 inhibition in organoid cultures. (A) Timeline for Oct4 inhibition using adenovirus in organoid culture. (B) Enhanced green fluorescent protein (eGFP) in hepatocytes infected with adenovirus containing short hairpin RNA (shRNA) for Oct4, multiplicity of infection (MOI) 5, > 70% infection observed at day 14 in culture. Original magnification: 100×. (C–G) Analyzed from D21 samples treated with shRNA for Oct4 (AD-Oct4) or shRNA for scramble control (AD-Scr). (C) mRNA levels of Oct4 assessed by qRT-PCR and expressed as fold change relative to GAPDH. (D) Oct4 protein assessed by sELISA. mRNA levels as assessed by qRT-PCR and expressed as fold change relative to GAPDH of (E) biliary-specific marker HNF-1β, (F) hepatocyte-specific marker HNF-4α, and (G) biliary-specific marker CK19. Significantly different from AD-Scr control: * p ≤ 0.05, ** p ≤ 0.01.

    Journal: Gene Expression

    Article Title: Oct4 Is Crucial for Transdifferentiation of Hepatocytes to Biliary Epithelial Cells in an In Vitro Organoid Culture Model

    doi: 10.3727/105221617X15124876321401

    Figure Lengend Snippet: Oct4 inhibition in organoid cultures. (A) Timeline for Oct4 inhibition using adenovirus in organoid culture. (B) Enhanced green fluorescent protein (eGFP) in hepatocytes infected with adenovirus containing short hairpin RNA (shRNA) for Oct4, multiplicity of infection (MOI) 5, > 70% infection observed at day 14 in culture. Original magnification: 100×. (C–G) Analyzed from D21 samples treated with shRNA for Oct4 (AD-Oct4) or shRNA for scramble control (AD-Scr). (C) mRNA levels of Oct4 assessed by qRT-PCR and expressed as fold change relative to GAPDH. (D) Oct4 protein assessed by sELISA. mRNA levels as assessed by qRT-PCR and expressed as fold change relative to GAPDH of (E) biliary-specific marker HNF-1β, (F) hepatocyte-specific marker HNF-4α, and (G) biliary-specific marker CK19. Significantly different from AD-Scr control: * p ≤ 0.05, ** p ≤ 0.01.

    Article Snippet: Reverse-transcribed samples were amplified in parallel on an ABI PRISM 7000 SDS instrument (Applied Biosystems, Foster City, CA, USA). qRT-PCR for each sample was performed in triplicate in a 20-μl reaction with 50 ng of cDNA, 5 pmol of each primer, and 1× SYBR GREEN PCR mix (#330510, Qiagen).

    Techniques: Inhibition, Infection, shRNA, Quantitative RT-PCR, Marker