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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 "Sex-Specific Regulation of Gene Expression Networks by Surfactant Protein A (SP-A) Variants in Alveolar Macrophages in Response to Klebsiella pneumoniae"

Article Title: Sex-Specific Regulation of Gene Expression Networks by Surfactant Protein A (SP-A) Variants in Alveolar Macrophages in Response to Klebsiella pneumoniae

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01290

Effect of infection on the expression of genes involved in the Cell Cycle-node in SP-A1 (6A 2 , 6A 4 ), SP-A2 (1A 0 , 1A 3 ), and KO male and female. The expression level of genes involved in direct and indirect interactions with Cell Cycle shown in Figure 5A and Supplementary Figure 3 (CCNA1, CCND1, CDK1, CDK2, CDKN1A, CKDN1B, CTNNB1, IRF1, MARCO, MMP12, MYC, NKX3-1, PPARA, RAG1, STAT5a/b, TAP1, TAP2, TCF4, and TIE1) were measured in KO (A) , SP-A2 (1A 0 ) (B) , SP-A2 (1A 3 ) (C) , SP-A1 (6A 2 ) (D) and SP-A1 (6A 4 ) (E) . The expression levels were normalized to GAPDH and the significant differences ( P
Figure Legend Snippet: Effect of infection on the expression of genes involved in the Cell Cycle-node in SP-A1 (6A 2 , 6A 4 ), SP-A2 (1A 0 , 1A 3 ), and KO male and female. The expression level of genes involved in direct and indirect interactions with Cell Cycle shown in Figure 5A and Supplementary Figure 3 (CCNA1, CCND1, CDK1, CDK2, CDKN1A, CKDN1B, CTNNB1, IRF1, MARCO, MMP12, MYC, NKX3-1, PPARA, RAG1, STAT5a/b, TAP1, TAP2, TCF4, and TIE1) were measured in KO (A) , SP-A2 (1A 0 ) (B) , SP-A2 (1A 3 ) (C) , SP-A1 (6A 2 ) (D) and SP-A1 (6A 4 ) (E) . The expression levels were normalized to GAPDH and the significant differences ( P

Techniques Used: Infection, Expressing

18) Product Images from "Resolvin D-series regulates Phospholipase D both during inflammation and resolution by modulating phagocyte functions"

Article Title: Resolvin D-series regulates Phospholipase D both during inflammation and resolution by modulating phagocyte functions

Journal: bioRxiv

doi: 10.1101/827295

M1 to M2 macrophage reprogramming induced by PLD. (A,B) Flow cytometry analysis of surface expression of polarization markers for CD80 and CD86-positive cells (M1 markers) or CD163 and CD206-positive cells (M2 markers). Experiments were performed in M’zero’ (M0) macrophages overexpressing either PLD1 or PLD2 (1.5 ug DNA plasmid per condition) after 24 hr transfection (A) or silenced with shPLD1 or shPLD2 (100 nM per condition) after 72 hr transfection with shRNAs (“Scrbl”, scrambled RNA control) (B). Shown in (C) are the transfection controls to show efficiency of either overexpression or silencing of PLD1 and PLD2 plasmids or shRNA, respectively. (D) Flow cytometry analysis of surface expression of polarization markers for CD38/CD86-positive cells (M1 markers) or CD23/CD206-positive cells (M2 markers) in M1 macrophages upon ectopic overexpression of PLD1 or PLD2 plasmids. See Supplementary Figure S9 for flow cytometry plots. (E) Gene expression of CD206 using qRT-PCR with or without RvD5 (10 nM) treatment in culture for 24 hrs. (F) Phagocytosis of M1 macrophages silenced with PLD1 or PLD2 shRNA. Fluorescence intensity of phagocytosis of BacLightGFP-labeled E. coli was measured by real-time microscopy of M1 macrophages upon silencing PLD1 or PLD2 with 100 nM shRNA (“Scrbl”, scrambled RNA control) for 72 hr, followed by incubation with 10 nM RvD5 for 1 hr. A cell to E. coli ratio was 1:50 with 3×10 5 cells. (inset=example of fluorescent-field view from the microscope). (G) Efferocytosis of M1 macrophages after transfection of PLD1 or PLD2 expression plasmids. Fluorescence intensity of green fluorescence intensity from efferocytosis of green fluorescence (CellTraceTM-CFDA)-labeled apoptotic neutrophils (PMNs) was measured by flow cytometry, upon transfection of PLD1 or PLD2 expression plasmids for 48 hr, followed by incubation for 1 hr with 10 nM RvD5. A macrophage to CFDA-labeled apoptotic PMN was added to a 1:5 ratio with 3×10‘ cells/well. (H) Effect of ResolvinsD1-5 on surface expression of polarization markers on M1 and M2 macrophages, measured by flow cytometry. Data in bar graphs are mean ± SEM (n > 3 independent experiments; for real-time microscopy, sample size n∼60 cells read for each condition); statistical significance (*p
Figure Legend Snippet: M1 to M2 macrophage reprogramming induced by PLD. (A,B) Flow cytometry analysis of surface expression of polarization markers for CD80 and CD86-positive cells (M1 markers) or CD163 and CD206-positive cells (M2 markers). Experiments were performed in M’zero’ (M0) macrophages overexpressing either PLD1 or PLD2 (1.5 ug DNA plasmid per condition) after 24 hr transfection (A) or silenced with shPLD1 or shPLD2 (100 nM per condition) after 72 hr transfection with shRNAs (“Scrbl”, scrambled RNA control) (B). Shown in (C) are the transfection controls to show efficiency of either overexpression or silencing of PLD1 and PLD2 plasmids or shRNA, respectively. (D) Flow cytometry analysis of surface expression of polarization markers for CD38/CD86-positive cells (M1 markers) or CD23/CD206-positive cells (M2 markers) in M1 macrophages upon ectopic overexpression of PLD1 or PLD2 plasmids. See Supplementary Figure S9 for flow cytometry plots. (E) Gene expression of CD206 using qRT-PCR with or without RvD5 (10 nM) treatment in culture for 24 hrs. (F) Phagocytosis of M1 macrophages silenced with PLD1 or PLD2 shRNA. Fluorescence intensity of phagocytosis of BacLightGFP-labeled E. coli was measured by real-time microscopy of M1 macrophages upon silencing PLD1 or PLD2 with 100 nM shRNA (“Scrbl”, scrambled RNA control) for 72 hr, followed by incubation with 10 nM RvD5 for 1 hr. A cell to E. coli ratio was 1:50 with 3×10 5 cells. (inset=example of fluorescent-field view from the microscope). (G) Efferocytosis of M1 macrophages after transfection of PLD1 or PLD2 expression plasmids. Fluorescence intensity of green fluorescence intensity from efferocytosis of green fluorescence (CellTraceTM-CFDA)-labeled apoptotic neutrophils (PMNs) was measured by flow cytometry, upon transfection of PLD1 or PLD2 expression plasmids for 48 hr, followed by incubation for 1 hr with 10 nM RvD5. A macrophage to CFDA-labeled apoptotic PMN was added to a 1:5 ratio with 3×10‘ cells/well. (H) Effect of ResolvinsD1-5 on surface expression of polarization markers on M1 and M2 macrophages, measured by flow cytometry. Data in bar graphs are mean ± SEM (n > 3 independent experiments; for real-time microscopy, sample size n∼60 cells read for each condition); statistical significance (*p

Techniques Used: Flow Cytometry, Expressing, Plasmid Preparation, Transfection, Over Expression, shRNA, Quantitative RT-PCR, Fluorescence, Labeling, Microscopy, Incubation

19) Product Images from "Exosomal miR-1298 and lncRNA-RP11-583F2.2 Expression in Hepato-cellular Carcinoma"

Article Title: Exosomal miR-1298 and lncRNA-RP11-583F2.2 Expression in Hepato-cellular Carcinoma

Journal: Current Genomics

doi: 10.2174/1389202920666191210111849

Bar chart showing the positivity rates for serum exosomal lncRNA-RP11-583F2.2, and exosomal Hsa‐miR‐1298 expression among the HCC, the CHC, and healthy control groups. The data are presented as percentages, and * indicates P
Figure Legend Snippet: Bar chart showing the positivity rates for serum exosomal lncRNA-RP11-583F2.2, and exosomal Hsa‐miR‐1298 expression among the HCC, the CHC, and healthy control groups. The data are presented as percentages, and * indicates P

Techniques Used: Expressing

BOXPLOT: Serum exosomal lncRNA-RP11-583F2.2 and exosomal RAB11A mRNA as determined by qRT‐PCR among the HCC, CHC, and healthy control groups. ( A ) LncRNA‐RP11‐513I15.6; ( B ) Has‐miR‐1298; The data are presented as the median fold changes (P
Figure Legend Snippet: BOXPLOT: Serum exosomal lncRNA-RP11-583F2.2 and exosomal RAB11A mRNA as determined by qRT‐PCR among the HCC, CHC, and healthy control groups. ( A ) LncRNA‐RP11‐513I15.6; ( B ) Has‐miR‐1298; The data are presented as the median fold changes (P

Techniques Used: Quantitative RT-PCR

20) Product Images from "Sex-Specific Regulation of Gene Expression Networks by Surfactant Protein A (SP-A) Variants in Alveolar Macrophages in Response to Klebsiella pneumoniae"

Article Title: Sex-Specific Regulation of Gene Expression Networks by Surfactant Protein A (SP-A) Variants in Alveolar Macrophages in Response to Klebsiella pneumoniae

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01290

Effect of infection on the expression of genes involved in the Cell Cycle-node in SP-A1 (6A 2 , 6A 4 ), SP-A2 (1A 0 , 1A 3 ), and KO male and female. The expression level of genes involved in direct and indirect interactions with Cell Cycle shown in Figure 5A and Supplementary Figure 3 (CCNA1, CCND1, CDK1, CDK2, CDKN1A, CKDN1B, CTNNB1, IRF1, MARCO, MMP12, MYC, NKX3-1, PPARA, RAG1, STAT5a/b, TAP1, TAP2, TCF4, and TIE1) were measured in KO (A) , SP-A2 (1A 0 ) (B) , SP-A2 (1A 3 ) (C) , SP-A1 (6A 2 ) (D) and SP-A1 (6A 4 ) (E) . The expression levels were normalized to GAPDH and the significant differences ( P
Figure Legend Snippet: Effect of infection on the expression of genes involved in the Cell Cycle-node in SP-A1 (6A 2 , 6A 4 ), SP-A2 (1A 0 , 1A 3 ), and KO male and female. The expression level of genes involved in direct and indirect interactions with Cell Cycle shown in Figure 5A and Supplementary Figure 3 (CCNA1, CCND1, CDK1, CDK2, CDKN1A, CKDN1B, CTNNB1, IRF1, MARCO, MMP12, MYC, NKX3-1, PPARA, RAG1, STAT5a/b, TAP1, TAP2, TCF4, and TIE1) were measured in KO (A) , SP-A2 (1A 0 ) (B) , SP-A2 (1A 3 ) (C) , SP-A1 (6A 2 ) (D) and SP-A1 (6A 4 ) (E) . The expression levels were normalized to GAPDH and the significant differences ( P

Techniques Used: Infection, Expressing

21) 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

22) 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

23) 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

24) 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

25) 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

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Amplification:

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Purification:

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SYBR Green Assay:

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    Qiagen sybr green qpcr master mix
    Box-and-whisker plots of bacterial groups quantified by <t>qPCR.</t> Notes: Bacterial groups quantified by <t>SYBR</t> Green qPCR and expressed as Log10 bacteria per gram stool in hospitalized patients with CDI and non-CDI group. Outlier point were shown by *.
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    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
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    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.
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    Box-and-whisker plots of bacterial groups quantified by qPCR. Notes: Bacterial groups quantified by SYBR Green qPCR and expressed as Log10 bacteria per gram stool in hospitalized patients with CDI and non-CDI group. Outlier point were shown by *.

    Journal: Infection and Drug Resistance

    Article Title: Intestinal Microbiota in Elderly Inpatients with Clostridioides difficile Infection

    doi: 10.2147/IDR.S262019

    Figure Lengend Snippet: Box-and-whisker plots of bacterial groups quantified by qPCR. Notes: Bacterial groups quantified by SYBR Green qPCR and expressed as Log10 bacteria per gram stool in hospitalized patients with CDI and non-CDI group. Outlier point were shown by *.

    Article Snippet: Samples were run in duplicate in a volume of 25 μL containing 1 × SYBR green qPCR master mix, 0.5 μM of each primer and 50 ng of purified fecal DNA.

    Techniques: Whisker Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay

    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

    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