Structured Review

Santa Cruz Biotechnology antibody against iκbα
SENP3 deficiency selectively attenuates MAPK signaling and JNK phosphorylation in macrophages. A, RAW 264.7 cells transfected with si-Cont or si-SENP3 were stimulated with LPS (100 ng/ml) for the indicated time. <t>IκBα</t> degradation was assessed by IB. B, NF-κB-luciferase ( left panel ) or AP-1-luciferase ( right panel ) and Renilla were transfected into RAW 264.7 cells together with the indicated siRNA. 48 h after transfection, cells were stimulated with LPS (100 ng/ml) for 6 h followed by luciferase reporter assays. Graphs show the mean ± S.D. and data shown are representative of three independent experiments. ns, no statistical difference; *, p
Antibody Against Iκbα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 5248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/antibody against iκbα/product/Santa Cruz Biotechnology
Average 88 stars, based on 5248 article reviews
Price from $9.99 to $1999.99
antibody against iκbα - by Bioz Stars, 2020-10
88/100 stars

Images

1) Product Images from "DeSUMOylation of MKK7 kinase by the SUMO2/3 protease SENP3 potentiates lipopolysaccharide-induced inflammatory signaling in macrophages"

Article Title: DeSUMOylation of MKK7 kinase by the SUMO2/3 protease SENP3 potentiates lipopolysaccharide-induced inflammatory signaling in macrophages

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.816769

SENP3 deficiency selectively attenuates MAPK signaling and JNK phosphorylation in macrophages. A, RAW 264.7 cells transfected with si-Cont or si-SENP3 were stimulated with LPS (100 ng/ml) for the indicated time. IκBα degradation was assessed by IB. B, NF-κB-luciferase ( left panel ) or AP-1-luciferase ( right panel ) and Renilla were transfected into RAW 264.7 cells together with the indicated siRNA. 48 h after transfection, cells were stimulated with LPS (100 ng/ml) for 6 h followed by luciferase reporter assays. Graphs show the mean ± S.D. and data shown are representative of three independent experiments. ns, no statistical difference; *, p
Figure Legend Snippet: SENP3 deficiency selectively attenuates MAPK signaling and JNK phosphorylation in macrophages. A, RAW 264.7 cells transfected with si-Cont or si-SENP3 were stimulated with LPS (100 ng/ml) for the indicated time. IκBα degradation was assessed by IB. B, NF-κB-luciferase ( left panel ) or AP-1-luciferase ( right panel ) and Renilla were transfected into RAW 264.7 cells together with the indicated siRNA. 48 h after transfection, cells were stimulated with LPS (100 ng/ml) for 6 h followed by luciferase reporter assays. Graphs show the mean ± S.D. and data shown are representative of three independent experiments. ns, no statistical difference; *, p

Techniques Used: Transfection, Luciferase

2) Product Images from "Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway"

Article Title: Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0787-2

ERK/p38 is downstream of RhoA/ROCK in supervillin-mediated cell metastasis during hypoxia. a. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assaying for phosphorylated p38, phosphorylated ERK, Snail1, and Vimentin by immunoblotting. β-tubulin was used as the loading control. b, c. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assay for cell migration ( b ) and invasion ( c ). The number of migrated SV1, SV4, or SV5 cells treated with PD0325901 was compared to those control cells treated with PBS. D. MHCC-97H and Huh-7 cells co-transfected with control or supervillin-specific siRNA and a RhoA(WT), RhoA(V14), or RhoA(N19) plasmids for 48 h were treated with PBS or the ROCK inhibitor Y27632 2HCl (10 μM) for 16 h during hypoxia, and then assayed for phosphorylated p38 and ERK by immunoblotting. β-actin was used as the loading control
Figure Legend Snippet: ERK/p38 is downstream of RhoA/ROCK in supervillin-mediated cell metastasis during hypoxia. a. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assaying for phosphorylated p38, phosphorylated ERK, Snail1, and Vimentin by immunoblotting. β-tubulin was used as the loading control. b, c. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assay for cell migration ( b ) and invasion ( c ). The number of migrated SV1, SV4, or SV5 cells treated with PD0325901 was compared to those control cells treated with PBS. D. MHCC-97H and Huh-7 cells co-transfected with control or supervillin-specific siRNA and a RhoA(WT), RhoA(V14), or RhoA(N19) plasmids for 48 h were treated with PBS or the ROCK inhibitor Y27632 2HCl (10 μM) for 16 h during hypoxia, and then assayed for phosphorylated p38 and ERK by immunoblotting. β-actin was used as the loading control

Techniques Used: Transfection, Migration

Supervillin regulates hypoxia-induced epithelial-mesenchymal transition (EMT) of HCC. a. Bright-field microscopy showing the morphological changes that occur when HepG2 and Huh-7 cells are cultured for 16 h in hypoxia. b. Immunoblots showing the changes in E-cadherin, Vimentin, Snail1, and supervillin isoforms during hypoxia after treatment with supervillin-specific siRNA. HepG2 and Huh-7 cells were transfected with a control or supervillin-specific siRNA and incubated under normoxia for 48 h followed by being scratched and exposed to normoxia or hypoxia for 16 h; β-tubulin was used as the loading control. c. Immunofluorescence staining showing that the relative amounts and localization of E-cadherin, Snail1, and Vimentin under normal and hypoxic conditions in Huh-7 cells treated with control or supervillin-specific siRNA. Scale bar = 50 μm
Figure Legend Snippet: Supervillin regulates hypoxia-induced epithelial-mesenchymal transition (EMT) of HCC. a. Bright-field microscopy showing the morphological changes that occur when HepG2 and Huh-7 cells are cultured for 16 h in hypoxia. b. Immunoblots showing the changes in E-cadherin, Vimentin, Snail1, and supervillin isoforms during hypoxia after treatment with supervillin-specific siRNA. HepG2 and Huh-7 cells were transfected with a control or supervillin-specific siRNA and incubated under normoxia for 48 h followed by being scratched and exposed to normoxia or hypoxia for 16 h; β-tubulin was used as the loading control. c. Immunofluorescence staining showing that the relative amounts and localization of E-cadherin, Snail1, and Vimentin under normal and hypoxic conditions in Huh-7 cells treated with control or supervillin-specific siRNA. Scale bar = 50 μm

Techniques Used: Microscopy, Cell Culture, Western Blot, Transfection, Incubation, Immunofluorescence, Staining

3) Product Images from "Spicatoside A in red Liriope platyphylla displays a laxative effect in a constipation rat model via regulating mAChRs and ER stress signaling"

Article Title: Spicatoside A in red Liriope platyphylla displays a laxative effect in a constipation rat model via regulating mAChRs and ER stress signaling

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3960

Detection of the ER stress response. (A) Expression levels of ER stress-related proteins IRE1α, IRE1β, p-IRE1, JNK, p-JNK, eIF2α and p-eIF2α were measured by western blot analysis. Band intensities were evaluated relative to the intensity of the actin bands. Data are presented as mean ± standard deviation from three replicates (N=5-6 rats per treatment group). * P
Figure Legend Snippet: Detection of the ER stress response. (A) Expression levels of ER stress-related proteins IRE1α, IRE1β, p-IRE1, JNK, p-JNK, eIF2α and p-eIF2α were measured by western blot analysis. Band intensities were evaluated relative to the intensity of the actin bands. Data are presented as mean ± standard deviation from three replicates (N=5-6 rats per treatment group). * P

Techniques Used: Expressing, Western Blot, Standard Deviation

4) Product Images from "Mechanisms underlying differential expression of interleukin-8 in breast cancer cells"

Article Title: Mechanisms underlying differential expression of interleukin-8 in breast cancer cells

Journal: Oncogene

doi: 10.1038/sj.onc.1207815

NF-κB, AP-1 and C/EBP transcription factors are acting together to control IL-8 gene expression A. MDA-MB-231 cells were infected at MOI 100 with backbone Ad5 or Ad-IκB viruses. 24h after infection, the medium was collected and assayed for IL-8 levels by ELISA. Results represent the mean ± SD of three independent experiments. B. RNA were collected from the same cells and used to measure IL-8 RNA levels by quantitative PCR. Results are expressed as arbitrary units corresponding to the ratio of IL-8 levels normalized by rS9 levels (n=3). C. Reduction of IL-8 expression using RNA interference against Fra-1, Fra-2 or c-Jun. MDA-MB-231 cells were transfected with siFra-1, siFRa-2, sic-Jun or siGFP (as control). The extent of silencing of was determined by western blot using antibodies against Fra-1, Fra-2, c-Jun or β-actin (left panel). Secreted IL-8 levels were measured by ELISA 48h afer transfection. Results represent the mean ± SD of three independent experiments. D. xp2-IL8 reporter was cotransfected along with 100 ng of expression vectors of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPα or C/EBPβ in MCF-7 cells. Results show relative luciferase activities (n=3).
Figure Legend Snippet: NF-κB, AP-1 and C/EBP transcription factors are acting together to control IL-8 gene expression A. MDA-MB-231 cells were infected at MOI 100 with backbone Ad5 or Ad-IκB viruses. 24h after infection, the medium was collected and assayed for IL-8 levels by ELISA. Results represent the mean ± SD of three independent experiments. B. RNA were collected from the same cells and used to measure IL-8 RNA levels by quantitative PCR. Results are expressed as arbitrary units corresponding to the ratio of IL-8 levels normalized by rS9 levels (n=3). C. Reduction of IL-8 expression using RNA interference against Fra-1, Fra-2 or c-Jun. MDA-MB-231 cells were transfected with siFra-1, siFRa-2, sic-Jun or siGFP (as control). The extent of silencing of was determined by western blot using antibodies against Fra-1, Fra-2, c-Jun or β-actin (left panel). Secreted IL-8 levels were measured by ELISA 48h afer transfection. Results represent the mean ± SD of three independent experiments. D. xp2-IL8 reporter was cotransfected along with 100 ng of expression vectors of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPα or C/EBPβ in MCF-7 cells. Results show relative luciferase activities (n=3).

Techniques Used: Expressing, Multiple Displacement Amplification, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Luciferase

AP-1 pathway is important for IL-8 gene activity A. AP-1 relative activity was assessed in MDA-MB-231 and MCF-7 cells by transfection of an AP-1 reporter. Results show relative CAT activities (n=3) after normalization for β-galactosidase activity. B. The importance of AP-1 pathway in IL-8 promoter activity regulation was evaluated by transfection of 100 ng of empty vector (C) or expression vectors of JDP1, JDP2 and Tam67 AP-1 inhibitors in MDA-MB-231 and MCF-7 cells, along with xp2-IL8 construct. Results show relative luciferase activities (n=3).
Figure Legend Snippet: AP-1 pathway is important for IL-8 gene activity A. AP-1 relative activity was assessed in MDA-MB-231 and MCF-7 cells by transfection of an AP-1 reporter. Results show relative CAT activities (n=3) after normalization for β-galactosidase activity. B. The importance of AP-1 pathway in IL-8 promoter activity regulation was evaluated by transfection of 100 ng of empty vector (C) or expression vectors of JDP1, JDP2 and Tam67 AP-1 inhibitors in MDA-MB-231 and MCF-7 cells, along with xp2-IL8 construct. Results show relative luciferase activities (n=3).

Techniques Used: Activity Assay, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Expressing, Construct, Luciferase

NF-κB site is crucial for the high activity of IL-8 promoter A . IL-8 promoter constructs harboring single, double or triple mutations of AP-1, C/EBP or NF-κB sites were transfected in MDA-MB-231 and MCF-7 cells. Results show relative luciferase activities (n=3). B. Binding of nuclear factors to AP-1, C/EBP and NF-κB sites was examined by gel shift assays using 2 μg of nuclear extracts of MDA-MB-231 and MCF-7 cells. The right panel is a control of nuclear extract loading which corresponds to a western blotting of HDAC-1 antibody against MCF-7 and MDA-MB-231 nuclear extracts.
Figure Legend Snippet: NF-κB site is crucial for the high activity of IL-8 promoter A . IL-8 promoter constructs harboring single, double or triple mutations of AP-1, C/EBP or NF-κB sites were transfected in MDA-MB-231 and MCF-7 cells. Results show relative luciferase activities (n=3). B. Binding of nuclear factors to AP-1, C/EBP and NF-κB sites was examined by gel shift assays using 2 μg of nuclear extracts of MDA-MB-231 and MCF-7 cells. The right panel is a control of nuclear extract loading which corresponds to a western blotting of HDAC-1 antibody against MCF-7 and MDA-MB-231 nuclear extracts.

Techniques Used: Activity Assay, Construct, Transfection, Multiple Displacement Amplification, Luciferase, Binding Assay, Electrophoretic Mobility Shift Assay, Western Blot

NF-κB factors are the primary regulators of in IL-8 promoter activity A. NF-κB relative activity was evaluated in MDA-MB-231 and MCF-7 cells by transfection of a NF-κB reporter. Results show relative luciferase activities (n=3). B. The importance of NF-κB in IL-8 promoter activity regulation was evaluated by infection of MDA-MB-231 and MCF-7 cells with Ad5 and Ad-IκB viruses (MOI 100) and transfection of xp2-IL8 construct. Results show relative luciferase activities (n=3). C. xp2-IL-8, NF-κB and AP-1 reporters were transfected in MDA-MB-231 cells along with CMV-GAL and control CMV5 or CMV-hERα expression vectors. Cells were treated or not with estradiol (10-8M) for 24h. Results show relative luciferase and CAT activities (n=3).
Figure Legend Snippet: NF-κB factors are the primary regulators of in IL-8 promoter activity A. NF-κB relative activity was evaluated in MDA-MB-231 and MCF-7 cells by transfection of a NF-κB reporter. Results show relative luciferase activities (n=3). B. The importance of NF-κB in IL-8 promoter activity regulation was evaluated by infection of MDA-MB-231 and MCF-7 cells with Ad5 and Ad-IκB viruses (MOI 100) and transfection of xp2-IL8 construct. Results show relative luciferase activities (n=3). C. xp2-IL-8, NF-κB and AP-1 reporters were transfected in MDA-MB-231 cells along with CMV-GAL and control CMV5 or CMV-hERα expression vectors. Cells were treated or not with estradiol (10-8M) for 24h. Results show relative luciferase and CAT activities (n=3).

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

Identification of the factors of MDA-MB-231 cells bound to IL-8 promoter A. Factors bound to C/EBP site of IL-8 promoter in MDA-MB-231 cells were identified by gel shift assay using C/EBPα, C/EBPδ and C/EBPβ antibodies. Competition with a 10 fold excess of cold consensus C/EBP or NF-κB oligonucleotides confirms the specificity of the complexes. B. The identification of AP-1 members of MDA-MB-231 cel extracts bound to AP-1 site was performed by gel shift assay using c-fos, c-Jun, Fra-1 and Fra-2 antibodies. Competition with a 10 fold excess of cold C/EBP or consensus AP-1 oligonucleotides confirms the specificity of the complexes. C. NF-κB proteins present in MDA-MB-231 cells which bound to the NF-κB site were identified by gel shift assay using p50, p65, c-Rel, p52 and RelB antibodies. Oligonucleotides competition were done with a 10 fold excess of cold AP-1 or consensus NF-κB probes. D . Protein levels of Fra-1, Fra-2, c-jun, p50, p65, C/EBPα and C/EBPβ were determined by western blot using 30 μg of MDA-MB-231 and MCF-7 whole cell extracts. The upper band observed on Fra-1 blot corresponds to the phosphorylated form of the protein.
Figure Legend Snippet: Identification of the factors of MDA-MB-231 cells bound to IL-8 promoter A. Factors bound to C/EBP site of IL-8 promoter in MDA-MB-231 cells were identified by gel shift assay using C/EBPα, C/EBPδ and C/EBPβ antibodies. Competition with a 10 fold excess of cold consensus C/EBP or NF-κB oligonucleotides confirms the specificity of the complexes. B. The identification of AP-1 members of MDA-MB-231 cel extracts bound to AP-1 site was performed by gel shift assay using c-fos, c-Jun, Fra-1 and Fra-2 antibodies. Competition with a 10 fold excess of cold C/EBP or consensus AP-1 oligonucleotides confirms the specificity of the complexes. C. NF-κB proteins present in MDA-MB-231 cells which bound to the NF-κB site were identified by gel shift assay using p50, p65, c-Rel, p52 and RelB antibodies. Oligonucleotides competition were done with a 10 fold excess of cold AP-1 or consensus NF-κB probes. D . Protein levels of Fra-1, Fra-2, c-jun, p50, p65, C/EBPα and C/EBPβ were determined by western blot using 30 μg of MDA-MB-231 and MCF-7 whole cell extracts. The upper band observed on Fra-1 blot corresponds to the phosphorylated form of the protein.

Techniques Used: Multiple Displacement Amplification, Electrophoretic Mobility Shift Assay, Western Blot

5) Product Images from "The ATP-binding cassette transporter-2 (ABCA2) overexpression modulates sphingosine levels and transcription of the amyloid precursor protein (APP) gene"

Article Title: The ATP-binding cassette transporter-2 (ABCA2) overexpression modulates sphingosine levels and transcription of the amyloid precursor protein (APP) gene

Journal: Current Alzheimer research

doi:

Model of proposed mechanism for the elevation of APP mRNA in ABCA2-overexpressing N2a cells ABCA2 overexpression reduces the in vivo binding of JDP2 and HDAC3 at the AP-1 site in the APP promoter, which decreases formation of a transcription repressor complex. APP transcription is positively regulated in ABCA2 overexpressing cells by increased in vivo binding of the c-jun transcriptional activator at the AP-1 site, as well as by the increased in vivo binding of the USF-1 and USF-2 transcriptional activators at the E-box site. APP transcriptional regulation is mediated through PKC. ABCA2 overexpression increases the activity of alkaline and acidic ceramidase enzymes as well as the mass of sphingosine, which is a physiological inhibitor of PKC. Ceramidase inhibition with ceranib-1 or PKC activation with PMA or DAG decreases APP transcription, while inhibition of PKC with GF109203x elevates APP transcription.
Figure Legend Snippet: Model of proposed mechanism for the elevation of APP mRNA in ABCA2-overexpressing N2a cells ABCA2 overexpression reduces the in vivo binding of JDP2 and HDAC3 at the AP-1 site in the APP promoter, which decreases formation of a transcription repressor complex. APP transcription is positively regulated in ABCA2 overexpressing cells by increased in vivo binding of the c-jun transcriptional activator at the AP-1 site, as well as by the increased in vivo binding of the USF-1 and USF-2 transcriptional activators at the E-box site. APP transcriptional regulation is mediated through PKC. ABCA2 overexpression increases the activity of alkaline and acidic ceramidase enzymes as well as the mass of sphingosine, which is a physiological inhibitor of PKC. Ceramidase inhibition with ceranib-1 or PKC activation with PMA or DAG decreases APP transcription, while inhibition of PKC with GF109203x elevates APP transcription.

Techniques Used: Over Expression, In Vivo, Binding Assay, Activity Assay, Inhibition, Activation Assay

AP-1 and USF proteins bind the endogenous APP promoter in vivo Chromatin immunoprecipitation for determination of in vivo binding of AP-1 and USF proteins to the endogenous APP promoter was performed as described in Methods. (A) CHIP of c-jun, c-fos, JDP2 and HDAC3. (B) CHIP of USF-1 and USF-2. The experiments were performed ≥ 2 times.
Figure Legend Snippet: AP-1 and USF proteins bind the endogenous APP promoter in vivo Chromatin immunoprecipitation for determination of in vivo binding of AP-1 and USF proteins to the endogenous APP promoter was performed as described in Methods. (A) CHIP of c-jun, c-fos, JDP2 and HDAC3. (B) CHIP of USF-1 and USF-2. The experiments were performed ≥ 2 times.

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Binding Assay

(A) USF proteins positively regulate APP promoter activity through an E-box element in the APP promoter The E-box element, CAGCTGA (consensus CANNTG) at position −49 base pairs upstream of the transcription start site was mutated to CGTGCTAC by site-directed mutagenesis. Cells were co-transfected with the wild-type APP promoter or APP E-box mutant promoter constructs and USF-1, USF-2 or dominant-negative A-USF expression constructs. Cell homogenates were assayed for fold-change in APP promoter activity as described in Methods. Results are the mean ± SD of six determinations. Statistical significance was determined using the Student’s t test, p
Figure Legend Snippet: (A) USF proteins positively regulate APP promoter activity through an E-box element in the APP promoter The E-box element, CAGCTGA (consensus CANNTG) at position −49 base pairs upstream of the transcription start site was mutated to CGTGCTAC by site-directed mutagenesis. Cells were co-transfected with the wild-type APP promoter or APP E-box mutant promoter constructs and USF-1, USF-2 or dominant-negative A-USF expression constructs. Cell homogenates were assayed for fold-change in APP promoter activity as described in Methods. Results are the mean ± SD of six determinations. Statistical significance was determined using the Student’s t test, p

Techniques Used: Activity Assay, Mutagenesis, Transfection, Construct, Dominant Negative Mutation, Expressing

6) Product Images from "ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †"

Article Title: ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00974-07

Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Concentration Assay, Incubation, Cell Counting, Flow Cytometry, Cytometry, Sampling, Apoptosis Assay

Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Luciferase, Plasmid Preparation, Cell Culture, Incubation, DNA Extraction, Staining, Flow Cytometry, Cytometry

Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).
Figure Legend Snippet: Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).

Techniques Used: Binding Assay, Activity Assay, In Vitro, Protein Binding, Concentration Assay, Incubation, Sequencing, Electrophoresis

Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Infection, Expressing, Plasmid Preparation, Cell Culture, Selection, Incubation

7) Product Images from "ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †"

Article Title: ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00974-07

Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Concentration Assay, Incubation, Cell Counting, Flow Cytometry, Cytometry, Sampling, Apoptosis Assay

Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Luciferase, Plasmid Preparation, Cell Culture, Incubation, DNA Extraction, Staining, Flow Cytometry, Cytometry

Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).
Figure Legend Snippet: Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).

Techniques Used: Binding Assay, Activity Assay, In Vitro, Protein Binding, Concentration Assay, Incubation, Sequencing, Electrophoresis

Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Infection, Expressing, Plasmid Preparation, Cell Culture, Selection, Incubation

8) Product Images from "ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †"

Article Title: ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00974-07

Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Concentration Assay, Incubation, Cell Counting, Flow Cytometry, Cytometry, Sampling, Apoptosis Assay

Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Luciferase, Plasmid Preparation, Cell Culture, Incubation, DNA Extraction, Staining, Flow Cytometry, Cytometry

Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).
Figure Legend Snippet: Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).

Techniques Used: Binding Assay, Activity Assay, In Vitro, Protein Binding, Concentration Assay, Incubation, Sequencing, Electrophoresis

Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Infection, Expressing, Plasmid Preparation, Cell Culture, Selection, Incubation

9) Product Images from "ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †"

Article Title: ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00974-07

Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Arsenite-induced apoptosis and cytotoxicity in ATF4-deficient cells. Cells (10 7 ) preinfected with siRNA to LUC (control [Con]), mAPE1 (S-mAPE1), hAPE1 (S-hAPE1), ATF4 (S-ATF4), or hApe1 or mApe1 retroviral expression vectors (hApe1 and mApe1, respectively) were challenged with the indicated concentration of sodium arsenite for 30 min. The cells were washed twice, and the incubation was continued in fresh medium for 4 h. 10T½ (A) or TK6 (B) cells were then reseeded into 96-well plates (10 3 cells per well) and incubated with fresh medium for 96 h. The cell number was determined by using Cell Counting kit 8. The results were confirmed via a parallel traditional colony-forming assay for 10T½ cells or trypan blue exclusion for TK6 cells (data not shown). (C) Apoptosis determined by the annexin V-FITC-propidium iodide assay using flow cytometry. Arsenite-challenged cells were incubated for 24 h in fresh medium before sampling for the apoptosis assay. The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Concentration Assay, Incubation, Cell Counting, Flow Cytometry, Cytometry, Sampling, Apoptosis Assay

Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Abasic DNA damage and apoptosis in ATF4-deficient cells. The 10T½ cells preinfected with vectors expressing siRNA against luciferase (sLUC) or ATF4 (sATF4) or with sATF4 and an mApe1 retroviral expression vector (sATF4+mApe1) were cultured to confluence. The cells were then treated with 50 μM sodium arsenite for 30 min. The cells were washed twice and incubated in fresh medium for the indica ted times. The cells were subjected to DNA extraction to determine abasic damage using an aldehyde-reactive probe assay (A) or collected to determine apoptosis by annexin V-FITC-propidium iodide staining and flow cytometry (B). The data were quantified from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Expressing, Luciferase, Plasmid Preparation, Cell Culture, Incubation, DNA Extraction, Staining, Flow Cytometry, Cytometry

Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).
Figure Legend Snippet: Identification of arsenite-activated DNA-binding activity using an EMSA. (A) Arsenite treatment of 10T½ cells activates in vitro protein binding for the 123-bp APE1 promoter fragment. The cells were treated without arsenite or with the indicated arsenite concentration for 30 min, after which the cells were washed and incubated in fresh medium for the time shown. Preparation of cell extracts and EMSA reactions were carried out as described in Materials and Methods. (B) Competition with excess (5- to 100-fold) concentrations of unlabeled wild-type DNA sequence (w). The presence (+) or absence (−) of sodium arsenite or cold competitor is shown. (C) Competition with oligonucleotides containing altered factor binding sites. Competitor oligonucleotides were used at a 50-fold (wild type [w] only) or 100-fold molar excess. Following electrophoresis, the gels were autoradiographed for 16 h. (D) Competitor oligonucleotide sequences. The altered sites are underlined. (E) EMSA supershift only by ATF4 or c-Jun antibodies. Extracts prepared from 10T½ cells treated for 30 min with or without 50 μM sodium arsenite were used in EMSA reactions. Supershift analysis using rabbit immunoglobulin G control (Con Ig) or antibodies directed against the indicated transcription factors was carried out as described in Materials and Methods. The gels were autoradiographed for 16 to 48 h. Only the top portion of the autoradiograms is shown. Separate panels are shown for control (a) and arsenite-treated cells (b).

Techniques Used: Binding Assay, Activity Assay, In Vitro, Protein Binding, Concentration Assay, Incubation, Sequencing, Electrophoresis

Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
Figure Legend Snippet: Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

Techniques Used: Infection, Expressing, Plasmid Preparation, Cell Culture, Selection, Incubation

10) Product Images from "Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages"

Article Title: Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0705533

Effects of RTX and capsazepine on LPS- and IFN- γ -induced NO release, iNOS expression, PGE 2 release and/or COX-2 expression. RAW264.7 cells were treated with LPS (100 ng ml −1 ), IFN- γ (3 ng ml −1 ), RTX and/or capsazepine at the concentrations indicated for 24 h, then the culture medium was collected for NO assay (a, b), PGE 2 assay (d), and cell lysate, as described above, was subjected to SDS–PAGE and measured and quantified for iNOS (c) or COX-2 immunoreactivities (d). The data represent the mean±s.e.m. from at least three independent experiments. * P
Figure Legend Snippet: Effects of RTX and capsazepine on LPS- and IFN- γ -induced NO release, iNOS expression, PGE 2 release and/or COX-2 expression. RAW264.7 cells were treated with LPS (100 ng ml −1 ), IFN- γ (3 ng ml −1 ), RTX and/or capsazepine at the concentrations indicated for 24 h, then the culture medium was collected for NO assay (a, b), PGE 2 assay (d), and cell lysate, as described above, was subjected to SDS–PAGE and measured and quantified for iNOS (c) or COX-2 immunoreactivities (d). The data represent the mean±s.e.m. from at least three independent experiments. * P

Techniques Used: Expressing, SDS Page

Vanilloids reduced iNOS and COX-2 mRNA expression. Following the treatment with drugs as indicated for 12 h, changes in iNOS (a) and COX-2 (b) mRNA levels were measured by the PCR products. RNA isolation and the RT–PCR process were carried out as described. The β -actin mRNA level was considered the internal control.
Figure Legend Snippet: Vanilloids reduced iNOS and COX-2 mRNA expression. Following the treatment with drugs as indicated for 12 h, changes in iNOS (a) and COX-2 (b) mRNA levels were measured by the PCR products. RNA isolation and the RT–PCR process were carried out as described. The β -actin mRNA level was considered the internal control.

Techniques Used: Expressing, Polymerase Chain Reaction, Isolation, Reverse Transcription Polymerase Chain Reaction

11) Product Images from "Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants ▿ Mutants ▿ §"

Article Title: Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants ▿ Mutants ▿ §

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01330-08

(A) Coimmunoprecipitation assays were performed by immunoprecipitation (IP) with anti-c-Jun and blotting with anti-PU.1 and anti-c-Jun. To control for nuclear protein extract concentrations, levels of the nuclear protein TATA binding protein (TBP) were examined. (B) For ChIP assays, isolated lysates were immunoprecipitated with either anti-PU.1 or anti-c-Jun, followed by amplification of purified DNA fragments using primers specific for the c- Jun promoter or Mcsfr promoter or by antiacetylated H4, followed by amplification of purified DNA fragments using primers specific for the HPRT promoter.
Figure Legend Snippet: (A) Coimmunoprecipitation assays were performed by immunoprecipitation (IP) with anti-c-Jun and blotting with anti-PU.1 and anti-c-Jun. To control for nuclear protein extract concentrations, levels of the nuclear protein TATA binding protein (TBP) were examined. (B) For ChIP assays, isolated lysates were immunoprecipitated with either anti-PU.1 or anti-c-Jun, followed by amplification of purified DNA fragments using primers specific for the c- Jun promoter or Mcsfr promoter or by antiacetylated H4, followed by amplification of purified DNA fragments using primers specific for the HPRT promoter.

Techniques Used: Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Isolation, Amplification, Purification

c- JUN , GATA2 , PU.1 , and C/EBP α expression levels from CD34 + spleen cells of JMML patients were compared to those of CD34 + cells from the cord blood (CB) of healthy newborns, using three cord blood samples and seven JMML samples. *, P = 0.0001; **, P = 0.014; ***, P = 0.01. c- JUN expression levels from peripheral blood mononuclear cells (PB MNC) of JMML patients were compared to those from peripheral blood mononuclear cells of healthy controls, using four healthy controls assayed in triplicate and four JMML samples assayed in triplicate. #, P = 0.03.
Figure Legend Snippet: c- JUN , GATA2 , PU.1 , and C/EBP α expression levels from CD34 + spleen cells of JMML patients were compared to those of CD34 + cells from the cord blood (CB) of healthy newborns, using three cord blood samples and seven JMML samples. *, P = 0.0001; **, P = 0.014; ***, P = 0.01. c- JUN expression levels from peripheral blood mononuclear cells (PB MNC) of JMML patients were compared to those from peripheral blood mononuclear cells of healthy controls, using four healthy controls assayed in triplicate and four JMML samples assayed in triplicate. #, P = 0.03.

Techniques Used: Expressing

12) Product Images from "Genome-wide analysis of EGR2/SOX10 binding in myelinating peripheral nerve"

Article Title: Genome-wide analysis of EGR2/SOX10 binding in myelinating peripheral nerve

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks313

ChIP-Seq analysis of SOX10 binding. Binding profiles of SOX10 at selected loci are shown. Those peaks marked with an asterisk were identified as statistically significant relative to sequenced ChIP input data. Peak coordinates are listed in Supplementary Table S2 . All genes shown are reduced in microarray analysis of S16 cells treated with Sox10 siRNA ( Supplementary Table S5 ).
Figure Legend Snippet: ChIP-Seq analysis of SOX10 binding. Binding profiles of SOX10 at selected loci are shown. Those peaks marked with an asterisk were identified as statistically significant relative to sequenced ChIP input data. Peak coordinates are listed in Supplementary Table S2 . All genes shown are reduced in microarray analysis of S16 cells treated with Sox10 siRNA ( Supplementary Table S5 ).

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Microarray

In vivo ChIP-Seq analysis of EGR2 and SOX10 binding in peripheral nerve. ChIP assays were performed from pooled rat sciatic nerves at P15, using two independent pools. Fragments associated with EGR2 and SOX10 were sequenced, and mapped to the Rn4 build of the rat genome. Logos display enriched motifs that correspond to previously published consensus sequences for EGR2 and SOX10 ( 43 , 44 ).
Figure Legend Snippet: In vivo ChIP-Seq analysis of EGR2 and SOX10 binding in peripheral nerve. ChIP assays were performed from pooled rat sciatic nerves at P15, using two independent pools. Fragments associated with EGR2 and SOX10 were sequenced, and mapped to the Rn4 build of the rat genome. Logos display enriched motifs that correspond to previously published consensus sequences for EGR2 and SOX10 ( 43 , 44 ).

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Binding Assay

Analysis of SOX10-regulated genes. ( A ) Sox10 siRNA was introduced into the rat S16 cell line, and microarray analysis of three independent replicates was used to identify SOX10-regulated genes. Comparison with array data from the Egr2 hypomorph allele at P7 revealed a subset of genes that are positively regulated by both EGR2 and SOX10, and another set of genes that are apparently SOX10 induced, but repressed by EGR2 activity in post-natal Schwann cells. Functional annotation clustering performed by DAVID revealed clusters associated with the indicated terms (see Supplementary Table S8 for complete listing). ( B ) Peak location analysis was performed for those peaks located within 100 kb of genes that are repressed or activated by EGR2 or SOX10, as determined by microarray analysis of Egr2 hypomorphic mice ( 5 , 46 ) or siRNA analysis of SOX10-regulated genes in the S16 cell line ( Supplementary Table S6 ). Pie charts represent relative proportions of peaks located within the gene body itself (gene), or 0–2, 2–10 and 10–100 kb away from the gene on either the 5′ or 3′ side. Peaks > 100 kb are not included in the analysis. SOX10 peaks in SOX10 repressed genes are not included because of the low number of such peaks.
Figure Legend Snippet: Analysis of SOX10-regulated genes. ( A ) Sox10 siRNA was introduced into the rat S16 cell line, and microarray analysis of three independent replicates was used to identify SOX10-regulated genes. Comparison with array data from the Egr2 hypomorph allele at P7 revealed a subset of genes that are positively regulated by both EGR2 and SOX10, and another set of genes that are apparently SOX10 induced, but repressed by EGR2 activity in post-natal Schwann cells. Functional annotation clustering performed by DAVID revealed clusters associated with the indicated terms (see Supplementary Table S8 for complete listing). ( B ) Peak location analysis was performed for those peaks located within 100 kb of genes that are repressed or activated by EGR2 or SOX10, as determined by microarray analysis of Egr2 hypomorphic mice ( 5 , 46 ) or siRNA analysis of SOX10-regulated genes in the S16 cell line ( Supplementary Table S6 ). Pie charts represent relative proportions of peaks located within the gene body itself (gene), or 0–2, 2–10 and 10–100 kb away from the gene on either the 5′ or 3′ side. Peaks > 100 kb are not included in the analysis. SOX10 peaks in SOX10 repressed genes are not included because of the low number of such peaks.

Techniques Used: Microarray, Activity Assay, Functional Assay, Mouse Assay

SOX10 binding within Sox10 locus. Binding pattern of SOX10 within its own locus is shown. The SOX10 MCS track indicates previously identified multispecies-conserved sequences numbered ( 2–7 ), as in ref. ( 57 ). Peaks marked with an asterisk are statistically significant relative to sequenced ChIP input DNA.
Figure Legend Snippet: SOX10 binding within Sox10 locus. Binding pattern of SOX10 within its own locus is shown. The SOX10 MCS track indicates previously identified multispecies-conserved sequences numbered ( 2–7 ), as in ref. ( 57 ). Peaks marked with an asterisk are statistically significant relative to sequenced ChIP input DNA.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

13) Product Images from "Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation"

Article Title: Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation

Journal: BMC Cancer

doi: 10.1186/1471-2407-12-156

Suggested pathway initiated by Cas III-ia leading to autophagy and apoptosis in C6 glioma cells. Cas III-ia may cause oxidative stress by JNK activation. JNK can phosphorylate Bcl-2 and release Beclin 1 enhancing autophagy. Alternatively, JNK can induce phosphorylation of c-jun; phospho-c-jun favors the production of AP-1 which, in turn, increases the expression of many genes among which are c-jun itself, Beclin, Atg7, Bax and FasL. Beclin 1 and Atg 7 may induce autophagy and FasL can stimulate Fas-R inducing the activation of caspase 8, which then degrades Bid into Bidt inducing Bax oligomerization and the depolarization of the mitochondrial membrane with release of cyt c and the consequent activation of caspase 3. Autophagy and apoptosis can thus induce cell death.
Figure Legend Snippet: Suggested pathway initiated by Cas III-ia leading to autophagy and apoptosis in C6 glioma cells. Cas III-ia may cause oxidative stress by JNK activation. JNK can phosphorylate Bcl-2 and release Beclin 1 enhancing autophagy. Alternatively, JNK can induce phosphorylation of c-jun; phospho-c-jun favors the production of AP-1 which, in turn, increases the expression of many genes among which are c-jun itself, Beclin, Atg7, Bax and FasL. Beclin 1 and Atg 7 may induce autophagy and FasL can stimulate Fas-R inducing the activation of caspase 8, which then degrades Bid into Bidt inducing Bax oligomerization and the depolarization of the mitochondrial membrane with release of cyt c and the consequent activation of caspase 3. Autophagy and apoptosis can thus induce cell death.

Techniques Used: IA, Activation Assay, Expressing

14) Product Images from "Distinct transcriptional regulatory modules underlie STAT3's cell type-independent and cell type-specific functions"

Article Title: Distinct transcriptional regulatory modules underlie STAT3's cell type-independent and cell type-specific functions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1300

Discrete transcriptional regulatory modules (TRM) determine STAT3 cell type-specific functions. TRMs for each cell type were generated by collecting de novo motifs generated by HOMER and excluding motifs resembling STAT3 or a STAT3 half-site. Motifs were then scanned again and compared to a random background generated from the control background libraries to generate a z -score. Motifs with a z -score
Figure Legend Snippet: Discrete transcriptional regulatory modules (TRM) determine STAT3 cell type-specific functions. TRMs for each cell type were generated by collecting de novo motifs generated by HOMER and excluding motifs resembling STAT3 or a STAT3 half-site. Motifs were then scanned again and compared to a random background generated from the control background libraries to generate a z -score. Motifs with a z -score

Techniques Used: Time-resolved Mass Spectrometry, Generated

STAT3 biological specificity is found within the cell type-specific lists of genomic binding sites. Gene Ontology term enrichment analysis was done using GREAT with default parameters. Over-represented terms displayed here are from the ‘Biological Process’ category ( A ) and the Mouse genome informatics phenotype::genotype category ( B ). A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line.
Figure Legend Snippet: STAT3 biological specificity is found within the cell type-specific lists of genomic binding sites. Gene Ontology term enrichment analysis was done using GREAT with default parameters. Over-represented terms displayed here are from the ‘Biological Process’ category ( A ) and the Mouse genome informatics phenotype::genotype category ( B ). A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line.

Techniques Used: Binding Assay

STAT3 binds to a small but significant ‘shared overlap’ of binding sites in divergent cell types, but is otherwise strongly cell type specific. ( A ) Summary of the distances between the overlapping peaks for each pair of STAT3 ChIP-seq libraries. The distance was measured as the number of base pairs between the summits of the two overlapping peaks. ( B ) Overlap of the binding peaks in three STAT3 libraries culled from mouse ESCs, CD4 + T cells, macrophages and AtT-20 cells. Peaks were considered to overlap if their peak summits were within 200 bp of one another. The overlap was simulated by generating lists of faux ChIP-seq peaks followed by the assessment of their overlap (this was performed 1000 times to generate the number of overlaps expected by chance-value listed in brackets). ( C ) The ‘shared overlap’ sites are more likely found in the most highly ranked STAT3-binding sites. STAT3-binding sites were ranked by fold enrichment and then the cumulative overlap of STAT3 peaks appearing in all four cell types was plotted for ESCs, CD4 + T cells, macrophages and AtT-20 cells. ( D ) Genome distributions of STAT3-binding sites orientated with respect to the nearest gene to the STAT3-binding site. Coloured bars describe the distance from the STAT3-binding sites to the nearest TSS, as described in the key. The gray regions denote a random background and represent the expected distribution of peaks were the binding sites randomly distributed across the sequenceable genome.
Figure Legend Snippet: STAT3 binds to a small but significant ‘shared overlap’ of binding sites in divergent cell types, but is otherwise strongly cell type specific. ( A ) Summary of the distances between the overlapping peaks for each pair of STAT3 ChIP-seq libraries. The distance was measured as the number of base pairs between the summits of the two overlapping peaks. ( B ) Overlap of the binding peaks in three STAT3 libraries culled from mouse ESCs, CD4 + T cells, macrophages and AtT-20 cells. Peaks were considered to overlap if their peak summits were within 200 bp of one another. The overlap was simulated by generating lists of faux ChIP-seq peaks followed by the assessment of their overlap (this was performed 1000 times to generate the number of overlaps expected by chance-value listed in brackets). ( C ) The ‘shared overlap’ sites are more likely found in the most highly ranked STAT3-binding sites. STAT3-binding sites were ranked by fold enrichment and then the cumulative overlap of STAT3 peaks appearing in all four cell types was plotted for ESCs, CD4 + T cells, macrophages and AtT-20 cells. ( D ) Genome distributions of STAT3-binding sites orientated with respect to the nearest gene to the STAT3-binding site. Coloured bars describe the distance from the STAT3-binding sites to the nearest TSS, as described in the key. The gray regions denote a random background and represent the expected distribution of peaks were the binding sites randomly distributed across the sequenceable genome.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

The shared overlap of STAT3 binding in all four cell types forms a cell type-independent regulatory network with MYC and E2F1. ( A ) HOMER was used to generate de novo motifs from the list of 35 STAT3-binding sites common to all four cell types. Motifs resembling STAT3 or a STAT3 half-site were removed and over-represented motifs were collected and annotated to genes. Interaction networks were constructed by interrogating the PPI network for proteins interacting with those representing the enriched motifs. TFs were clustered together by motif similarity and coloured by the cluster they belong to: white-colored nodes do not have a representative motif in the databases or do not bind to DNA directly; proteins with a bold circle have a motif enriched in that cell type, while proteins with no bolded circle have no discovered motif but were linked to STAT3 through the PPI network. Proteins in the network were filtered by gene expression and here we present the union of the network in all four cell types (the separate networks are presented in Supplementary Figure S6 ) ( B ) ChIP-seq data from ESCs (GSE11431) were re-analyzed and binding sites overlapping within 400 bp were collected. The heatmap shows the 35 STAT3-binding sites together with the other TFs bound in the vicinity of STAT3 . ( C ) We designed primers for 14 STAT3-binding sites shared between all four cell types and performed ChIP-qPCR. Macrophages were treated for 4 h with IL-10 (black bars) or left untreated (white bars) and chromatin was harvested. ChIP was performed using antibodies against STAT3 (left panel), E2F1 (right panel) or GFP (as a control in both panels). Each group of bars represents a STAT3-binding site and is labeled with the name of the nearest gene.
Figure Legend Snippet: The shared overlap of STAT3 binding in all four cell types forms a cell type-independent regulatory network with MYC and E2F1. ( A ) HOMER was used to generate de novo motifs from the list of 35 STAT3-binding sites common to all four cell types. Motifs resembling STAT3 or a STAT3 half-site were removed and over-represented motifs were collected and annotated to genes. Interaction networks were constructed by interrogating the PPI network for proteins interacting with those representing the enriched motifs. TFs were clustered together by motif similarity and coloured by the cluster they belong to: white-colored nodes do not have a representative motif in the databases or do not bind to DNA directly; proteins with a bold circle have a motif enriched in that cell type, while proteins with no bolded circle have no discovered motif but were linked to STAT3 through the PPI network. Proteins in the network were filtered by gene expression and here we present the union of the network in all four cell types (the separate networks are presented in Supplementary Figure S6 ) ( B ) ChIP-seq data from ESCs (GSE11431) were re-analyzed and binding sites overlapping within 400 bp were collected. The heatmap shows the 35 STAT3-binding sites together with the other TFs bound in the vicinity of STAT3 . ( C ) We designed primers for 14 STAT3-binding sites shared between all four cell types and performed ChIP-qPCR. Macrophages were treated for 4 h with IL-10 (black bars) or left untreated (white bars) and chromatin was harvested. ChIP was performed using antibodies against STAT3 (left panel), E2F1 (right panel) or GFP (as a control in both panels). Each group of bars represents a STAT3-binding site and is labeled with the name of the nearest gene.

Techniques Used: Binding Assay, Construct, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Labeling

The shared overlap STAT3-binding sites co-regulate a set of key genes important for STAT3 function in multiple cell types. ( A ) Significantly over-represented terms from the ‘Pathway Commons’ category for the genes associated with the shared overlap. Shown here are the top five terms only. A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line. ( B ) The expression of the closest genes within 200 kb of the shared overlap was measured in a series of gene expression microarray data sets which show the activation of STAT3 or the loss of STAT3 activity (LIF withdrawal from ESCs). Expression data are reversed in ESCs for clarity, but is otherwise down-regulated upon removal of LIF whereas all other treatments show up-regulation in response to cytokine stimulation. P -values are from a Wilcoxon test between the treated and untreated conditions: CD4 + T cells treated with IL-21 ( P = 5.59 × 10 −5 ) (GSE19198), naïve CD4 + T cells treated with IL-6 ( P = 2.45 × 10 −3 ) (GSE21671), ESCs upon withdrawal of LIF from the medium ( P = 0.027) (GSE27708), peritoneal macrophages stimulated with IL-10 ( P = 2.14 × 10 −4 ) (GSE31529), AtT-20 cells treated with LIF ( P = 1.00 × 10 −4 ) (GSE19042) and liver cells stimulated with IL-6 ( P = 0.032) (GSE21060). Genes marked in green do not have a corresponding probe on the microarray.
Figure Legend Snippet: The shared overlap STAT3-binding sites co-regulate a set of key genes important for STAT3 function in multiple cell types. ( A ) Significantly over-represented terms from the ‘Pathway Commons’ category for the genes associated with the shared overlap. Shown here are the top five terms only. A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line. ( B ) The expression of the closest genes within 200 kb of the shared overlap was measured in a series of gene expression microarray data sets which show the activation of STAT3 or the loss of STAT3 activity (LIF withdrawal from ESCs). Expression data are reversed in ESCs for clarity, but is otherwise down-regulated upon removal of LIF whereas all other treatments show up-regulation in response to cytokine stimulation. P -values are from a Wilcoxon test between the treated and untreated conditions: CD4 + T cells treated with IL-21 ( P = 5.59 × 10 −5 ) (GSE19198), naïve CD4 + T cells treated with IL-6 ( P = 2.45 × 10 −3 ) (GSE21671), ESCs upon withdrawal of LIF from the medium ( P = 0.027) (GSE27708), peritoneal macrophages stimulated with IL-10 ( P = 2.14 × 10 −4 ) (GSE31529), AtT-20 cells treated with LIF ( P = 1.00 × 10 −4 ) (GSE19042) and liver cells stimulated with IL-6 ( P = 0.032) (GSE21060). Genes marked in green do not have a corresponding probe on the microarray.

Techniques Used: Binding Assay, Expressing, Microarray, Activation Assay, Activity Assay

A putative model explaining how STAT3 can perform both cell type-independent and cell type-specific functions by assembling around distinct TRMs. STAT3 binding to the genome occurs in two distinct ways: (i) a cell type-independent mode that is primarily concerned with the regulation of STAT3’s own activity and (ii) a number of cell type-specific modes that execute distinct transcriptional programmes in various cell types.
Figure Legend Snippet: A putative model explaining how STAT3 can perform both cell type-independent and cell type-specific functions by assembling around distinct TRMs. STAT3 binding to the genome occurs in two distinct ways: (i) a cell type-independent mode that is primarily concerned with the regulation of STAT3’s own activity and (ii) a number of cell type-specific modes that execute distinct transcriptional programmes in various cell types.

Techniques Used: Time-resolved Mass Spectrometry, Binding Assay, Activity Assay

STAT3 uses alternative (non-canonical) modes of binding to DNA. ( A ) de novo generated motifs from HOMER are very similar in the six lists of STAT3 binding. Motifs were generated from the entire lists of STAT3-binding sites for each category, except for CD4 + T cells where the top 1000 sites were used (as for the entire list we could only identify a STAT3 half-site). ( B ) z -score heatmap to show over representation of variant STAT3 motifs in the STAT3-binding sites in the various cell types. Variant motifs are presented here as all single base pair mutations of one-half of the STAT3 heterodimeric motif. ( C ) Pie charts showing the frequency of the DNA words TTCnnnGAA (canonical STAT3) or a non-canonical STAT3 binding: TTAnnnGGA and TGCnnnGGA for ESCs, CD4 + T cells, any two cell types and any three cell types; TGCnnnGAA for macrophages and the shared overlap; and TTAnnnGGA, TGCnnnGGA and CTCnnnGAA for AtT-20 cells. ( D ) Cartoon representation of the Asn466 amino acid of STAT3 making contact with the DNA base pairs [PDB entry 1bg1 ( 46 )].
Figure Legend Snippet: STAT3 uses alternative (non-canonical) modes of binding to DNA. ( A ) de novo generated motifs from HOMER are very similar in the six lists of STAT3 binding. Motifs were generated from the entire lists of STAT3-binding sites for each category, except for CD4 + T cells where the top 1000 sites were used (as for the entire list we could only identify a STAT3 half-site). ( B ) z -score heatmap to show over representation of variant STAT3 motifs in the STAT3-binding sites in the various cell types. Variant motifs are presented here as all single base pair mutations of one-half of the STAT3 heterodimeric motif. ( C ) Pie charts showing the frequency of the DNA words TTCnnnGAA (canonical STAT3) or a non-canonical STAT3 binding: TTAnnnGGA and TGCnnnGGA for ESCs, CD4 + T cells, any two cell types and any three cell types; TGCnnnGAA for macrophages and the shared overlap; and TTAnnnGGA, TGCnnnGGA and CTCnnnGAA for AtT-20 cells. ( D ) Cartoon representation of the Asn466 amino acid of STAT3 making contact with the DNA base pairs [PDB entry 1bg1 ( 46 )].

Techniques Used: Binding Assay, Generated, Variant Assay

The shared overlap of STAT3-binding sites controls the expression of a core set of genes important for STAT3 function. ( A ) Genomic views of STAT3 ChIP-seq sequence tag densities for four STAT3-binding sites common to ESCs, CD4 + T cells, macrophages and AtT-20 cells. STAT3 binding is shown around the TSS of Stat3 and Socs3, within the second intron of Bcl3 and ∼6 kb 5′ of the TSS of Ptpn1. ( B ) Average evolutionary conservation for the different categories of STAT3-binding events. STAT3 peak summits were extended by 1 kb either side and the Euarchontoglires evolutionary conservation scores were annotated as determined by phastCons in the UCSC Genome Browser ( http://genome.ucsc.edu/ ).
Figure Legend Snippet: The shared overlap of STAT3-binding sites controls the expression of a core set of genes important for STAT3 function. ( A ) Genomic views of STAT3 ChIP-seq sequence tag densities for four STAT3-binding sites common to ESCs, CD4 + T cells, macrophages and AtT-20 cells. STAT3 binding is shown around the TSS of Stat3 and Socs3, within the second intron of Bcl3 and ∼6 kb 5′ of the TSS of Ptpn1. ( B ) Average evolutionary conservation for the different categories of STAT3-binding events. STAT3 peak summits were extended by 1 kb either side and the Euarchontoglires evolutionary conservation scores were annotated as determined by phastCons in the UCSC Genome Browser ( http://genome.ucsc.edu/ ).

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Sequencing

15) Product Images from "Distinct transcriptional regulatory modules underlie STAT3's cell type-independent and cell type-specific functions"

Article Title: Distinct transcriptional regulatory modules underlie STAT3's cell type-independent and cell type-specific functions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1300

Discrete transcriptional regulatory modules (TRM) determine STAT3 cell type-specific functions. TRMs for each cell type were generated by collecting de novo motifs generated by HOMER and excluding motifs resembling STAT3 or a STAT3 half-site. Motifs were then scanned again and compared to a random background generated from the control background libraries to generate a z -score. Motifs with a z -score
Figure Legend Snippet: Discrete transcriptional regulatory modules (TRM) determine STAT3 cell type-specific functions. TRMs for each cell type were generated by collecting de novo motifs generated by HOMER and excluding motifs resembling STAT3 or a STAT3 half-site. Motifs were then scanned again and compared to a random background generated from the control background libraries to generate a z -score. Motifs with a z -score

Techniques Used: Time-resolved Mass Spectrometry, Generated

STAT3 biological specificity is found within the cell type-specific lists of genomic binding sites. Gene Ontology term enrichment analysis was done using GREAT with default parameters. Over-represented terms displayed here are from the ‘Biological Process’ category ( A ) and the Mouse genome informatics phenotype::genotype category ( B ). A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line.
Figure Legend Snippet: STAT3 biological specificity is found within the cell type-specific lists of genomic binding sites. Gene Ontology term enrichment analysis was done using GREAT with default parameters. Over-represented terms displayed here are from the ‘Biological Process’ category ( A ) and the Mouse genome informatics phenotype::genotype category ( B ). A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line.

Techniques Used: Binding Assay

STAT3 binds to a small but significant ‘shared overlap’ of binding sites in divergent cell types, but is otherwise strongly cell type specific. ( A ) Summary of the distances between the overlapping peaks for each pair of STAT3 ChIP-seq libraries. The distance was measured as the number of base pairs between the summits of the two overlapping peaks. ( B ) Overlap of the binding peaks in three STAT3 libraries culled from mouse ESCs, CD4 + T cells, macrophages and AtT-20 cells. Peaks were considered to overlap if their peak summits were within 200 bp of one another. The overlap was simulated by generating lists of faux ChIP-seq peaks followed by the assessment of their overlap (this was performed 1000 times to generate the number of overlaps expected by chance-value listed in brackets). ( C ) The ‘shared overlap’ sites are more likely found in the most highly ranked STAT3-binding sites. STAT3-binding sites were ranked by fold enrichment and then the cumulative overlap of STAT3 peaks appearing in all four cell types was plotted for ESCs, CD4 + T cells, macrophages and AtT-20 cells. ( D ) Genome distributions of STAT3-binding sites orientated with respect to the nearest gene to the STAT3-binding site. Coloured bars describe the distance from the STAT3-binding sites to the nearest TSS, as described in the key. The gray regions denote a random background and represent the expected distribution of peaks were the binding sites randomly distributed across the sequenceable genome.
Figure Legend Snippet: STAT3 binds to a small but significant ‘shared overlap’ of binding sites in divergent cell types, but is otherwise strongly cell type specific. ( A ) Summary of the distances between the overlapping peaks for each pair of STAT3 ChIP-seq libraries. The distance was measured as the number of base pairs between the summits of the two overlapping peaks. ( B ) Overlap of the binding peaks in three STAT3 libraries culled from mouse ESCs, CD4 + T cells, macrophages and AtT-20 cells. Peaks were considered to overlap if their peak summits were within 200 bp of one another. The overlap was simulated by generating lists of faux ChIP-seq peaks followed by the assessment of their overlap (this was performed 1000 times to generate the number of overlaps expected by chance-value listed in brackets). ( C ) The ‘shared overlap’ sites are more likely found in the most highly ranked STAT3-binding sites. STAT3-binding sites were ranked by fold enrichment and then the cumulative overlap of STAT3 peaks appearing in all four cell types was plotted for ESCs, CD4 + T cells, macrophages and AtT-20 cells. ( D ) Genome distributions of STAT3-binding sites orientated with respect to the nearest gene to the STAT3-binding site. Coloured bars describe the distance from the STAT3-binding sites to the nearest TSS, as described in the key. The gray regions denote a random background and represent the expected distribution of peaks were the binding sites randomly distributed across the sequenceable genome.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

The shared overlap of STAT3 binding in all four cell types forms a cell type-independent regulatory network with MYC and E2F1. ( A ) HOMER was used to generate de novo motifs from the list of 35 STAT3-binding sites common to all four cell types. Motifs resembling STAT3 or a STAT3 half-site were removed and over-represented motifs were collected and annotated to genes. Interaction networks were constructed by interrogating the PPI network for proteins interacting with those representing the enriched motifs. TFs were clustered together by motif similarity and coloured by the cluster they belong to: white-colored nodes do not have a representative motif in the databases or do not bind to DNA directly; proteins with a bold circle have a motif enriched in that cell type, while proteins with no bolded circle have no discovered motif but were linked to STAT3 through the PPI network. Proteins in the network were filtered by gene expression and here we present the union of the network in all four cell types (the separate networks are presented in Supplementary Figure S6 ) ( B ) ChIP-seq data from ESCs (GSE11431) were re-analyzed and binding sites overlapping within 400 bp were collected. The heatmap shows the 35 STAT3-binding sites together with the other TFs bound in the vicinity of STAT3 . ( C ) We designed primers for 14 STAT3-binding sites shared between all four cell types and performed ChIP-qPCR. Macrophages were treated for 4 h with IL-10 (black bars) or left untreated (white bars) and chromatin was harvested. ChIP was performed using antibodies against STAT3 (left panel), E2F1 (right panel) or GFP (as a control in both panels). Each group of bars represents a STAT3-binding site and is labeled with the name of the nearest gene.
Figure Legend Snippet: The shared overlap of STAT3 binding in all four cell types forms a cell type-independent regulatory network with MYC and E2F1. ( A ) HOMER was used to generate de novo motifs from the list of 35 STAT3-binding sites common to all four cell types. Motifs resembling STAT3 or a STAT3 half-site were removed and over-represented motifs were collected and annotated to genes. Interaction networks were constructed by interrogating the PPI network for proteins interacting with those representing the enriched motifs. TFs were clustered together by motif similarity and coloured by the cluster they belong to: white-colored nodes do not have a representative motif in the databases or do not bind to DNA directly; proteins with a bold circle have a motif enriched in that cell type, while proteins with no bolded circle have no discovered motif but were linked to STAT3 through the PPI network. Proteins in the network were filtered by gene expression and here we present the union of the network in all four cell types (the separate networks are presented in Supplementary Figure S6 ) ( B ) ChIP-seq data from ESCs (GSE11431) were re-analyzed and binding sites overlapping within 400 bp were collected. The heatmap shows the 35 STAT3-binding sites together with the other TFs bound in the vicinity of STAT3 . ( C ) We designed primers for 14 STAT3-binding sites shared between all four cell types and performed ChIP-qPCR. Macrophages were treated for 4 h with IL-10 (black bars) or left untreated (white bars) and chromatin was harvested. ChIP was performed using antibodies against STAT3 (left panel), E2F1 (right panel) or GFP (as a control in both panels). Each group of bars represents a STAT3-binding site and is labeled with the name of the nearest gene.

Techniques Used: Binding Assay, Construct, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Labeling

The shared overlap STAT3-binding sites co-regulate a set of key genes important for STAT3 function in multiple cell types. ( A ) Significantly over-represented terms from the ‘Pathway Commons’ category for the genes associated with the shared overlap. Shown here are the top five terms only. A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line. ( B ) The expression of the closest genes within 200 kb of the shared overlap was measured in a series of gene expression microarray data sets which show the activation of STAT3 or the loss of STAT3 activity (LIF withdrawal from ESCs). Expression data are reversed in ESCs for clarity, but is otherwise down-regulated upon removal of LIF whereas all other treatments show up-regulation in response to cytokine stimulation. P -values are from a Wilcoxon test between the treated and untreated conditions: CD4 + T cells treated with IL-21 ( P = 5.59 × 10 −5 ) (GSE19198), naïve CD4 + T cells treated with IL-6 ( P = 2.45 × 10 −3 ) (GSE21671), ESCs upon withdrawal of LIF from the medium ( P = 0.027) (GSE27708), peritoneal macrophages stimulated with IL-10 ( P = 2.14 × 10 −4 ) (GSE31529), AtT-20 cells treated with LIF ( P = 1.00 × 10 −4 ) (GSE19042) and liver cells stimulated with IL-6 ( P = 0.032) (GSE21060). Genes marked in green do not have a corresponding probe on the microarray.
Figure Legend Snippet: The shared overlap STAT3-binding sites co-regulate a set of key genes important for STAT3 function in multiple cell types. ( A ) Significantly over-represented terms from the ‘Pathway Commons’ category for the genes associated with the shared overlap. Shown here are the top five terms only. A significant q -value of 0.05 is represented by a solid black line and a q -value of 0.01 by a dotted gray line. ( B ) The expression of the closest genes within 200 kb of the shared overlap was measured in a series of gene expression microarray data sets which show the activation of STAT3 or the loss of STAT3 activity (LIF withdrawal from ESCs). Expression data are reversed in ESCs for clarity, but is otherwise down-regulated upon removal of LIF whereas all other treatments show up-regulation in response to cytokine stimulation. P -values are from a Wilcoxon test between the treated and untreated conditions: CD4 + T cells treated with IL-21 ( P = 5.59 × 10 −5 ) (GSE19198), naïve CD4 + T cells treated with IL-6 ( P = 2.45 × 10 −3 ) (GSE21671), ESCs upon withdrawal of LIF from the medium ( P = 0.027) (GSE27708), peritoneal macrophages stimulated with IL-10 ( P = 2.14 × 10 −4 ) (GSE31529), AtT-20 cells treated with LIF ( P = 1.00 × 10 −4 ) (GSE19042) and liver cells stimulated with IL-6 ( P = 0.032) (GSE21060). Genes marked in green do not have a corresponding probe on the microarray.

Techniques Used: Binding Assay, Expressing, Microarray, Activation Assay, Activity Assay

A putative model explaining how STAT3 can perform both cell type-independent and cell type-specific functions by assembling around distinct TRMs. STAT3 binding to the genome occurs in two distinct ways: (i) a cell type-independent mode that is primarily concerned with the regulation of STAT3’s own activity and (ii) a number of cell type-specific modes that execute distinct transcriptional programmes in various cell types.
Figure Legend Snippet: A putative model explaining how STAT3 can perform both cell type-independent and cell type-specific functions by assembling around distinct TRMs. STAT3 binding to the genome occurs in two distinct ways: (i) a cell type-independent mode that is primarily concerned with the regulation of STAT3’s own activity and (ii) a number of cell type-specific modes that execute distinct transcriptional programmes in various cell types.

Techniques Used: Time-resolved Mass Spectrometry, Binding Assay, Activity Assay

STAT3 uses alternative (non-canonical) modes of binding to DNA. ( A ) de novo generated motifs from HOMER are very similar in the six lists of STAT3 binding. Motifs were generated from the entire lists of STAT3-binding sites for each category, except for CD4 + T cells where the top 1000 sites were used (as for the entire list we could only identify a STAT3 half-site). ( B ) z -score heatmap to show over representation of variant STAT3 motifs in the STAT3-binding sites in the various cell types. Variant motifs are presented here as all single base pair mutations of one-half of the STAT3 heterodimeric motif. ( C ) Pie charts showing the frequency of the DNA words TTCnnnGAA (canonical STAT3) or a non-canonical STAT3 binding: TTAnnnGGA and TGCnnnGGA for ESCs, CD4 + T cells, any two cell types and any three cell types; TGCnnnGAA for macrophages and the shared overlap; and TTAnnnGGA, TGCnnnGGA and CTCnnnGAA for AtT-20 cells. ( D ) Cartoon representation of the Asn466 amino acid of STAT3 making contact with the DNA base pairs [PDB entry 1bg1 ( 46 )].
Figure Legend Snippet: STAT3 uses alternative (non-canonical) modes of binding to DNA. ( A ) de novo generated motifs from HOMER are very similar in the six lists of STAT3 binding. Motifs were generated from the entire lists of STAT3-binding sites for each category, except for CD4 + T cells where the top 1000 sites were used (as for the entire list we could only identify a STAT3 half-site). ( B ) z -score heatmap to show over representation of variant STAT3 motifs in the STAT3-binding sites in the various cell types. Variant motifs are presented here as all single base pair mutations of one-half of the STAT3 heterodimeric motif. ( C ) Pie charts showing the frequency of the DNA words TTCnnnGAA (canonical STAT3) or a non-canonical STAT3 binding: TTAnnnGGA and TGCnnnGGA for ESCs, CD4 + T cells, any two cell types and any three cell types; TGCnnnGAA for macrophages and the shared overlap; and TTAnnnGGA, TGCnnnGGA and CTCnnnGAA for AtT-20 cells. ( D ) Cartoon representation of the Asn466 amino acid of STAT3 making contact with the DNA base pairs [PDB entry 1bg1 ( 46 )].

Techniques Used: Binding Assay, Generated, Variant Assay

The shared overlap of STAT3-binding sites controls the expression of a core set of genes important for STAT3 function. ( A ) Genomic views of STAT3 ChIP-seq sequence tag densities for four STAT3-binding sites common to ESCs, CD4 + T cells, macrophages and AtT-20 cells. STAT3 binding is shown around the TSS of Stat3 and Socs3, within the second intron of Bcl3 and ∼6 kb 5′ of the TSS of Ptpn1. ( B ) Average evolutionary conservation for the different categories of STAT3-binding events. STAT3 peak summits were extended by 1 kb either side and the Euarchontoglires evolutionary conservation scores were annotated as determined by phastCons in the UCSC Genome Browser ( http://genome.ucsc.edu/ ).
Figure Legend Snippet: The shared overlap of STAT3-binding sites controls the expression of a core set of genes important for STAT3 function. ( A ) Genomic views of STAT3 ChIP-seq sequence tag densities for four STAT3-binding sites common to ESCs, CD4 + T cells, macrophages and AtT-20 cells. STAT3 binding is shown around the TSS of Stat3 and Socs3, within the second intron of Bcl3 and ∼6 kb 5′ of the TSS of Ptpn1. ( B ) Average evolutionary conservation for the different categories of STAT3-binding events. STAT3 peak summits were extended by 1 kb either side and the Euarchontoglires evolutionary conservation scores were annotated as determined by phastCons in the UCSC Genome Browser ( http://genome.ucsc.edu/ ).

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Sequencing

16) Product Images from "IL-1? Promotes Corneal Epithelial Cell Migration by Increasing MMP-9 Expression through NF-?B- and AP-1-Dependent Pathways"

Article Title: IL-1? Promotes Corneal Epithelial Cell Migration by Increasing MMP-9 Expression through NF-?B- and AP-1-Dependent Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057955

IL-1β induces MMP-9 expression via p42/p44 MAPK. (A) Cells were pretreated with U0126 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with U0126, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with siRNA of scrambled or p42, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. The protein levels of p42 were determined by Western blotting. (E) Cells were treated with IL-1β for the indicated time intervals or pretreated with U0126, and then treated with IL-1β for 30 min. The phospho-p42/p44 MAPK protein expression was determined by Western blotting. (F, G) Cells were treated with IL-1β for the indicated time intervals. The nuclear fractions were prepared and subjected to Western blotting using an anti-phospho-p42/p44 MAPK antibody. Lamin A was used as a marker protein for nuclear fractions. The translocation of p42/p44 MAPK was observed using a fluorescence microscope. Data are expressed as mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: IL-1β induces MMP-9 expression via p42/p44 MAPK. (A) Cells were pretreated with U0126 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with U0126, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with siRNA of scrambled or p42, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. The protein levels of p42 were determined by Western blotting. (E) Cells were treated with IL-1β for the indicated time intervals or pretreated with U0126, and then treated with IL-1β for 30 min. The phospho-p42/p44 MAPK protein expression was determined by Western blotting. (F, G) Cells were treated with IL-1β for the indicated time intervals. The nuclear fractions were prepared and subjected to Western blotting using an anti-phospho-p42/p44 MAPK antibody. Lamin A was used as a marker protein for nuclear fractions. The translocation of p42/p44 MAPK was observed using a fluorescence microscope. Data are expressed as mean±S.E.M. of three independent experiments. * P

Techniques Used: Expressing, Incubation, Activity Assay, Transfection, Western Blot, Marker, Translocation Assay, Fluorescence, Microscopy

NF-κB is required for IL-1β-induced MMP-9 expression. (A) Cells were pretreated with Bay11-7082 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with Bay11-7082, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with wild-type MMP-9 promoter and NF-κB-mutated MMP-9 promoter, and then incubated with IL-1β for 12 h. The promoter activity of MMP-9 was determined. (E) Cells were treated with IL-1β for the indicated times. The translocation of p65 was observed using a fluorescence microscope. (F) Cells were treated with IL-1β for the indicated times. The nuclear and cytosolic fractions were prepared and subjected to Western blotting using an anti-IκBα or anti-p65 antibody. Lamin A and GAPDH were used as a marker protein for nuclear and cytosolic fractions, respectively. (G) Cells were treated with IL-1β for the indicated times. The NF-κB promoter activity was measured. Data are expressed as mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: NF-κB is required for IL-1β-induced MMP-9 expression. (A) Cells were pretreated with Bay11-7082 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with Bay11-7082, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with wild-type MMP-9 promoter and NF-κB-mutated MMP-9 promoter, and then incubated with IL-1β for 12 h. The promoter activity of MMP-9 was determined. (E) Cells were treated with IL-1β for the indicated times. The translocation of p65 was observed using a fluorescence microscope. (F) Cells were treated with IL-1β for the indicated times. The nuclear and cytosolic fractions were prepared and subjected to Western blotting using an anti-IκBα or anti-p65 antibody. Lamin A and GAPDH were used as a marker protein for nuclear and cytosolic fractions, respectively. (G) Cells were treated with IL-1β for the indicated times. The NF-κB promoter activity was measured. Data are expressed as mean±S.E.M. of three independent experiments. * P

Techniques Used: Expressing, Incubation, Activity Assay, Transfection, Translocation Assay, Fluorescence, Microscopy, Western Blot, Marker

17) Product Images from "Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro"

Article Title: Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

Journal: Experimental Cell Research

doi: 10.1016/j.yexcr.2013.03.029

Silencing of PRKD2 impacts on U87MG gene expression. PRKD2 expression was silenced with siPRKD2_5. Two and four days post transfection target gene expression was analyzed by qPCR using validated primer pairs. Hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as housekeeping gene. Relative gene expression of target genes is presented in relation to mock transfection. Results represent mean±SD from three independent experiments. Gene expression ratios were calculated by REST as described in Materials and methods .
Figure Legend Snippet: Silencing of PRKD2 impacts on U87MG gene expression. PRKD2 expression was silenced with siPRKD2_5. Two and four days post transfection target gene expression was analyzed by qPCR using validated primer pairs. Hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as housekeeping gene. Relative gene expression of target genes is presented in relation to mock transfection. Results represent mean±SD from three independent experiments. Gene expression ratios were calculated by REST as described in Materials and methods .

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

Pharmacological inhibition and silencing of PRKD2 impairs MAPK signaling and c-Jun regulation but does not affect the NFκB pathway. Cells were incubated with (A) CRT0066101 at the indicated concentrations overnight or (B) transfected with scrambled siRNA (S) and siPRKD2_5 (P5) for two days and activated with 1 µM BSA-complexed S1P for the indicated times. Protein levels of PRKD2 and MAPK family member activation were analyzed as described in Fig. 4 . Results of one experiment out of three (that provided similar results) are shown. (C) c-Jun expression and activation was analyzed in nontransfected and transfected (siSCR, siPRKD2_5) cells in the absence or presence of S1P (1 µM, 5 min). Nuclear lysates were separated by SDS-PAGE (10 µg protein/lane) and transferred to PVDF membrane. Total c-Jun expression and c-Jun protein activated by phosphorylation at Ser63 or Ser73 were analyzed using specific antibodies. Results of one experiment out of three are shown. (D) Cytosolic and nuclear PRKD2, pPRKD2, and NFκB (p50 and p65) expression was analyzed in nontransfected and transfected (siSCR, siPRKD2_5) cells under unstimulated and stimulated (1 µM S1P for 5 min) conditions. Cytosolic and nuclear protein fractions were separated by SDS-PAGE (10 µg protein/lane) and transferred to PVDF membrane. Immunoreactive bands were detected using rabbit antibodies. One experiment out of two is shown.
Figure Legend Snippet: Pharmacological inhibition and silencing of PRKD2 impairs MAPK signaling and c-Jun regulation but does not affect the NFκB pathway. Cells were incubated with (A) CRT0066101 at the indicated concentrations overnight or (B) transfected with scrambled siRNA (S) and siPRKD2_5 (P5) for two days and activated with 1 µM BSA-complexed S1P for the indicated times. Protein levels of PRKD2 and MAPK family member activation were analyzed as described in Fig. 4 . Results of one experiment out of three (that provided similar results) are shown. (C) c-Jun expression and activation was analyzed in nontransfected and transfected (siSCR, siPRKD2_5) cells in the absence or presence of S1P (1 µM, 5 min). Nuclear lysates were separated by SDS-PAGE (10 µg protein/lane) and transferred to PVDF membrane. Total c-Jun expression and c-Jun protein activated by phosphorylation at Ser63 or Ser73 were analyzed using specific antibodies. Results of one experiment out of three are shown. (D) Cytosolic and nuclear PRKD2, pPRKD2, and NFκB (p50 and p65) expression was analyzed in nontransfected and transfected (siSCR, siPRKD2_5) cells under unstimulated and stimulated (1 µM S1P for 5 min) conditions. Cytosolic and nuclear protein fractions were separated by SDS-PAGE (10 µg protein/lane) and transferred to PVDF membrane. Immunoreactive bands were detected using rabbit antibodies. One experiment out of two is shown.

Techniques Used: Inhibition, Incubation, Transfection, Activation Assay, Expressing, SDS Page

Efficacy of PRKD2 silencing. Knockdown of PRKD2 expression in U87MG cells was performed by RNA interference using two different siRNA constructs (Oligofectamine was used to transfect 20 nM siRNA). Silencing efficacy was analyzed by (A) qPCR two and four days post transfection and (B) Western blotting three and five days post transfection. Results in (A) represent mean±SD of three different experiments. Results in (B) show one representative Western blot out of five independent experiments. Numbers represent relative optical densities of PRKD2 protein normalized to loading controls.
Figure Legend Snippet: Efficacy of PRKD2 silencing. Knockdown of PRKD2 expression in U87MG cells was performed by RNA interference using two different siRNA constructs (Oligofectamine was used to transfect 20 nM siRNA). Silencing efficacy was analyzed by (A) qPCR two and four days post transfection and (B) Western blotting three and five days post transfection. Results in (A) represent mean±SD of three different experiments. Results in (B) show one representative Western blot out of five independent experiments. Numbers represent relative optical densities of PRKD2 protein normalized to loading controls.

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

Interference with PRKD2 expression reduces chemotactic migration and the invasive potential of U87MG cells. Untreated cells, cells transfected with siSCR and siPRKD2_5 and cells treated with 1 µM CRT0066101 were allowed to migrate across uncoated or matrigel-coated Transwell inserts. The lower chamber was loaded with medium containing FCS (10 or 0.5%; v/v) or S1P (1 µM or 2.5 µM; serum free). Cells that migrated to the bottom side of the membrane were fixed with methanol, stained with toluidine blue for 2 min and the membrane was washed twice with water. Cells in six fields from each membrane were counted using an optical microscope (20x magnifications). (A) Representative images of migrated cells. Quantitative analysis of cell migration across (B) uncoated or (C) matrigel-coated Transwell inserts. Data are presented as mean±SEM. Assays were performed in quadruplicate for each condition. *** p
Figure Legend Snippet: Interference with PRKD2 expression reduces chemotactic migration and the invasive potential of U87MG cells. Untreated cells, cells transfected with siSCR and siPRKD2_5 and cells treated with 1 µM CRT0066101 were allowed to migrate across uncoated or matrigel-coated Transwell inserts. The lower chamber was loaded with medium containing FCS (10 or 0.5%; v/v) or S1P (1 µM or 2.5 µM; serum free). Cells that migrated to the bottom side of the membrane were fixed with methanol, stained with toluidine blue for 2 min and the membrane was washed twice with water. Cells in six fields from each membrane were counted using an optical microscope (20x magnifications). (A) Representative images of migrated cells. Quantitative analysis of cell migration across (B) uncoated or (C) matrigel-coated Transwell inserts. Data are presented as mean±SEM. Assays were performed in quadruplicate for each condition. *** p

Techniques Used: Expressing, Migration, Transfection, Staining, Microscopy

Interference with PRKD2 expression impairs random migration of U87MG cells. For time-lapse microscopy untreated cells and cells transfected with siSCR or siPRKD2_5 were seeded on 24-well tissues. Two days post transfection images were acquired every 20 min for 24 h at five different positions of each well. Cell motility of 50 cells per treatment group was assessed using the Manual Tracking Macro and the Chemotaxis plugin of ImageJ. The (A) mean velocity, (B) mean accumulated distance and (C) the mean Euclidian distance was calculated. * p
Figure Legend Snippet: Interference with PRKD2 expression impairs random migration of U87MG cells. For time-lapse microscopy untreated cells and cells transfected with siSCR or siPRKD2_5 were seeded on 24-well tissues. Two days post transfection images were acquired every 20 min for 24 h at five different positions of each well. Cell motility of 50 cells per treatment group was assessed using the Manual Tracking Macro and the Chemotaxis plugin of ImageJ. The (A) mean velocity, (B) mean accumulated distance and (C) the mean Euclidian distance was calculated. * p

Techniques Used: Expressing, Migration, Time-lapse Microscopy, Transfection, Chemotaxis Assay

S1P activates mitogenic signaling cascades in U87MG cells. Cells were grown in 6 well plates and activated with BSA-complexed S1P in a concentration (A; 1 nM–10 µM; 10 min) or time-dependent (B; 1 µM; 5–60 min) manner. Whole cell lysates were separated by SDS-PAGE (10 or 30 µg protein/lane) and transferred to PVDF membrane. Protein levels of phospho-PRKD2 (Ser876), PRKD2, phospho-p44/42 MAPK (pp44/42), p44/42 MAPK, phospho-p38 MAPK (pp38), p38, phospho-p54/46 JNK (p54/46 JNK), and JNK were determined using specific antibodies. Results of one experiment out of three (that provided similar results) are shown.
Figure Legend Snippet: S1P activates mitogenic signaling cascades in U87MG cells. Cells were grown in 6 well plates and activated with BSA-complexed S1P in a concentration (A; 1 nM–10 µM; 10 min) or time-dependent (B; 1 µM; 5–60 min) manner. Whole cell lysates were separated by SDS-PAGE (10 or 30 µg protein/lane) and transferred to PVDF membrane. Protein levels of phospho-PRKD2 (Ser876), PRKD2, phospho-p44/42 MAPK (pp44/42), p44/42 MAPK, phospho-p38 MAPK (pp38), p38, phospho-p54/46 JNK (p54/46 JNK), and JNK were determined using specific antibodies. Results of one experiment out of three (that provided similar results) are shown.

Techniques Used: Concentration Assay, SDS Page

18) Product Images from "Curcumin analogue T83 exhibits potent antitumor activity and induces radiosensitivity through inactivation of Jab1 in nasopharyngeal carcinoma"

Article Title: Curcumin analogue T83 exhibits potent antitumor activity and induces radiosensitivity through inactivation of Jab1 in nasopharyngeal carcinoma

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-323

T83 inhibited Jab1 in a dose- and time-dependent manner in NPC cells. Cells from NPC cell lines were treated with and without T83 at the specified concentrations for 24 h (A) or exposed to 2 μM T83 for various time points (B) . Expression of Jab1,p53, p27 and β-actin were examined by Western blotting (C) . DMSO was used as control in “0” groups.
Figure Legend Snippet: T83 inhibited Jab1 in a dose- and time-dependent manner in NPC cells. Cells from NPC cell lines were treated with and without T83 at the specified concentrations for 24 h (A) or exposed to 2 μM T83 for various time points (B) . Expression of Jab1,p53, p27 and β-actin were examined by Western blotting (C) . DMSO was used as control in “0” groups.

Techniques Used: Expressing, Western Blot

Jab1 knockdown enhanced the effects of T83 on cell viability inhibition and apoptosis induction. CNE2 cells (left) and CNE2R cells (right) were transfected with control siRNA (Cont-si) or Jab1 siRNA (Jab1-si) and then exposed to the indicated doses of T83 for 48 h; they were then examined for colony formation (A) , sub-G1 (B) , and cleaved caspase-3 (C-Caspase 3) (C) . Protein levels were quantified with use of ImageJ software. All data represent three independent experiments, mean ± s.d. DMSO was used as control in “0” groups.
Figure Legend Snippet: Jab1 knockdown enhanced the effects of T83 on cell viability inhibition and apoptosis induction. CNE2 cells (left) and CNE2R cells (right) were transfected with control siRNA (Cont-si) or Jab1 siRNA (Jab1-si) and then exposed to the indicated doses of T83 for 48 h; they were then examined for colony formation (A) , sub-G1 (B) , and cleaved caspase-3 (C-Caspase 3) (C) . Protein levels were quantified with use of ImageJ software. All data represent three independent experiments, mean ± s.d. DMSO was used as control in “0” groups.

Techniques Used: Inhibition, Transfection, Software

19) Product Images from "Dipeptidyl peptidase IV inhibitor attenuates kidney injury in rat remnant kidney"

Article Title: Dipeptidyl peptidase IV inhibitor attenuates kidney injury in rat remnant kidney

Journal: BMC Nephrology

doi: 10.1186/1471-2369-14-98

Effects of sitagliptin on apoptosis. Apoptotic cells detected in the kidneys stained with TUNEL ( A ). Representative western blot and group data showing cleaved caspase-3, cleaved caspase-9, and Bax protein abundance in the kidneys of rats from each group ( B ). The intensity of the bands corresponding to cleaved caspase-3, cleaved caspase-9, and Bax was corrected by β-actin levels. The results are expressed as the mean ± s.d. *Significantly different with respect to the sham-operated rats; # significantly different with respect to the nephrectomized rats; * # P
Figure Legend Snippet: Effects of sitagliptin on apoptosis. Apoptotic cells detected in the kidneys stained with TUNEL ( A ). Representative western blot and group data showing cleaved caspase-3, cleaved caspase-9, and Bax protein abundance in the kidneys of rats from each group ( B ). The intensity of the bands corresponding to cleaved caspase-3, cleaved caspase-9, and Bax was corrected by β-actin levels. The results are expressed as the mean ± s.d. *Significantly different with respect to the sham-operated rats; # significantly different with respect to the nephrectomized rats; * # P

Techniques Used: Staining, TUNEL Assay, Western Blot

20) Product Images from "The p38 SAPK Is Recruited to Chromatin via Its Interaction with Transcription Factors *"

Article Title: The p38 SAPK Is Recruited to Chromatin via Its Interaction with Transcription Factors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.155846

p38 SAPK recruitment at the c-Fos promoter requires the transcription factor Elk1. A , HeLa cells were transfected with either control or Elk1 siRNAs and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.
Figure Legend Snippet: p38 SAPK recruitment at the c-Fos promoter requires the transcription factor Elk1. A , HeLa cells were transfected with either control or Elk1 siRNAs and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.

Techniques Used: Transfection, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

21) Product Images from "Interleukin-10 Prevents Diet-Induced Insulin Resistance by Attenuating Macrophage and Cytokine Response in Skeletal Muscle"

Article Title: Interleukin-10 Prevents Diet-Induced Insulin Resistance by Attenuating Macrophage and Cytokine Response in Skeletal Muscle

Journal: Diabetes

doi: 10.2337/db08-1261

HFD increases macrophage levels in WT skeletal muscle, and diet-induced inflammatory response in muscle is attenuated in MCK-IL10 mice. A : Macrophage-specific CD68 levels in skeletal muscle. B : Immunohistochemistry analysis using anti-CD68 and anti-F4/80 ( arrows ) in skeletal muscle. C : Skeletal muscle F4/80 levels in immunofluorescence staining. * P
Figure Legend Snippet: HFD increases macrophage levels in WT skeletal muscle, and diet-induced inflammatory response in muscle is attenuated in MCK-IL10 mice. A : Macrophage-specific CD68 levels in skeletal muscle. B : Immunohistochemistry analysis using anti-CD68 and anti-F4/80 ( arrows ) in skeletal muscle. C : Skeletal muscle F4/80 levels in immunofluorescence staining. * P

Techniques Used: Mouse Assay, Immunohistochemistry, Immunofluorescence, Staining

22) Product Images from "Programmed cell death 4 (PDCD4) suppresses metastastic potential of human hepatocellular carcinoma cells"

Article Title: Programmed cell death 4 (PDCD4) suppresses metastastic potential of human hepatocellular carcinoma cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-28-71

Effects of PDCD4 on MHCC-97H cell proliferation and apoptosis . A: Western blot analysis for identification of transfection efficiency. B: MTT assay for cell proliferation. C: Flow cytometric assay for cell apoptosis. D: Hoechst 33258 staining for cell apoptosis (×200). Morphological changes of cell apoptosis were shown as chromatin condensation and nuclear fragmentation. Representative images are shown from three individual experiments. In C and D, a or Group1, b or Group 2, and c or Group3 represents cells of MHCC-97H-PDCD4, MHCC-97H-vector and MHCC-97H, respectively; d shows statistical analysis for each assay. Bars represent the means ± SD. The difference between Group1 and Group2 or Group3 was significant (P
Figure Legend Snippet: Effects of PDCD4 on MHCC-97H cell proliferation and apoptosis . A: Western blot analysis for identification of transfection efficiency. B: MTT assay for cell proliferation. C: Flow cytometric assay for cell apoptosis. D: Hoechst 33258 staining for cell apoptosis (×200). Morphological changes of cell apoptosis were shown as chromatin condensation and nuclear fragmentation. Representative images are shown from three individual experiments. In C and D, a or Group1, b or Group 2, and c or Group3 represents cells of MHCC-97H-PDCD4, MHCC-97H-vector and MHCC-97H, respectively; d shows statistical analysis for each assay. Bars represent the means ± SD. The difference between Group1 and Group2 or Group3 was significant (P

Techniques Used: Western Blot, Transfection, MTT Assay, Flow Cytometry, Staining, Plasmid Preparation

Expression of PDCD4 in HCC cells . A: Immunocytochemical staining. The positive staining(×200) was brownish and localized in cytoplasm. D: Western blot assay. Representative figures are shown from one of three individual experiments. B, C or E shows statistical analysis for immunocytochemical staining, real – time PCR or western blot assay, respectively. In A, a, b or c represents cells of MHCC-97H, MHCC-97L or Hep3B, respectively; d shows cell staining without the primary antibody. In B, C and E, Group1, Group 2 or Group3 represents cells of MHCC-97H, MHCC-97L and Hep3B, respectively. Bars represent the means ± SD. The difference between Group1 and Group2 (P
Figure Legend Snippet: Expression of PDCD4 in HCC cells . A: Immunocytochemical staining. The positive staining(×200) was brownish and localized in cytoplasm. D: Western blot assay. Representative figures are shown from one of three individual experiments. B, C or E shows statistical analysis for immunocytochemical staining, real – time PCR or western blot assay, respectively. In A, a, b or c represents cells of MHCC-97H, MHCC-97L or Hep3B, respectively; d shows cell staining without the primary antibody. In B, C and E, Group1, Group 2 or Group3 represents cells of MHCC-97H, MHCC-97L and Hep3B, respectively. Bars represent the means ± SD. The difference between Group1 and Group2 (P

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

Effects of PDCD4 on MHCC-97H cell metastatic potential . B: Western blots for MTA1 expression. A and C: Statistical analysis for MTA1 expression with real-time PCR and western blot assay, respectively. D: Cell migration assay. E: Matrigel invasion assay. Representative images are shown from three individual experiments. In A, C, D and E, a or Group1, b or Group 2, and c or Group3 represents cells of MHCC-97H-PDCD4, MHCC-97H-vector and MHCC-97H, respectively. Bars represent the means ± SD. Difference between Group1 and Group2 or Group1 and Group3 was significant (P
Figure Legend Snippet: Effects of PDCD4 on MHCC-97H cell metastatic potential . B: Western blots for MTA1 expression. A and C: Statistical analysis for MTA1 expression with real-time PCR and western blot assay, respectively. D: Cell migration assay. E: Matrigel invasion assay. Representative images are shown from three individual experiments. In A, C, D and E, a or Group1, b or Group 2, and c or Group3 represents cells of MHCC-97H-PDCD4, MHCC-97H-vector and MHCC-97H, respectively. Bars represent the means ± SD. Difference between Group1 and Group2 or Group1 and Group3 was significant (P

Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Cell Migration Assay, Invasion Assay, Plasmid Preparation

23) Product Images from "Stressed-Induced TMEM135 Protein Is Part of a Conserved Genetic Network Involved in Fat Storage and Longevity Regulation in Caenorhabditis elegans"

Article Title: Stressed-Induced TMEM135 Protein Is Part of a Conserved Genetic Network Involved in Fat Storage and Longevity Regulation in Caenorhabditis elegans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014228

Nile Red staining and MitoTracker Red staining, survival differences and DAF-16 levels among the three C. elegans strains. Fig. 6A is representative staining with Nile Red in the worm. ( B ) Represents semi-quantitative assessment of Nile Red staining intensity, N = 4 per group. ( C ) Represents survival analysis among the three C. elegans strains at 20°C. ( D ) Represents survival analysis among the three C. elegans strains at 15°C. Wild-type = controls shown in red, tmem 135 (− / −) = tmem135 -deleted animals shown in black, TMEM135::GFP = C. elegans animal overexpressing TMEM135 shown in green. ( E ) Quantitative assessment of MitoTracker Red fluorescence. ( F ) Western blot analysis and quantification of DAF-16 levels in the three C. elegans strains, N = 6 per group, values are mean ± SEM.
Figure Legend Snippet: Nile Red staining and MitoTracker Red staining, survival differences and DAF-16 levels among the three C. elegans strains. Fig. 6A is representative staining with Nile Red in the worm. ( B ) Represents semi-quantitative assessment of Nile Red staining intensity, N = 4 per group. ( C ) Represents survival analysis among the three C. elegans strains at 20°C. ( D ) Represents survival analysis among the three C. elegans strains at 15°C. Wild-type = controls shown in red, tmem 135 (− / −) = tmem135 -deleted animals shown in black, TMEM135::GFP = C. elegans animal overexpressing TMEM135 shown in green. ( E ) Quantitative assessment of MitoTracker Red fluorescence. ( F ) Western blot analysis and quantification of DAF-16 levels in the three C. elegans strains, N = 6 per group, values are mean ± SEM.

Techniques Used: Staining, Fluorescence, Western Blot

24) Product Images from "Long descending cervical propriospinal neurons differ from thoracic propriospinal neurons in response to low thoracic spinal injury"

Article Title: Long descending cervical propriospinal neurons differ from thoracic propriospinal neurons in response to low thoracic spinal injury

Journal: BMC Neuroscience

doi: 10.1186/1471-2202-11-148

Post-injury expression of ATF-3 . One gene of interest that was examined using PCR was ATF-3. However due to the lack of PCR reaction product in the control animals and presence of PCR reaction product in the experimental animals, calculation of -ΔΔCp values and statistics was problematic. Therefore we probed for expression of ATF-3 protein immunohistochemically. A, C: In uninjured control animals a DTMR labelled LDPT neuron is clearly visible (red) and there is a lack of any ATF-3 labelling ( B ; green). D, E, F However, when an animal that received a complete T-9 spinal transection was examined 4-days post-injury, the FG labelled LDPT neuron ( D; in yellow) colocalizes with the nuclear ATF-3 labelling shown in green ( E, F ). G, H, I Moreover, when animals that received a T-9 spinal contusion injury are examined 1-week post-injury, the DTMR labelled LDPT neurons ( G; in red), again, colocalize with the ATF-3 labelling ( H; in green). These findings complement the PCR findings indicating an up-regulation of ATF-3 post-SCI. CNT = Uninjured Control, TXN = Transection, CON = Contusion. Scale Bars = 50 μm
Figure Legend Snippet: Post-injury expression of ATF-3 . One gene of interest that was examined using PCR was ATF-3. However due to the lack of PCR reaction product in the control animals and presence of PCR reaction product in the experimental animals, calculation of -ΔΔCp values and statistics was problematic. Therefore we probed for expression of ATF-3 protein immunohistochemically. A, C: In uninjured control animals a DTMR labelled LDPT neuron is clearly visible (red) and there is a lack of any ATF-3 labelling ( B ; green). D, E, F However, when an animal that received a complete T-9 spinal transection was examined 4-days post-injury, the FG labelled LDPT neuron ( D; in yellow) colocalizes with the nuclear ATF-3 labelling shown in green ( E, F ). G, H, I Moreover, when animals that received a T-9 spinal contusion injury are examined 1-week post-injury, the DTMR labelled LDPT neurons ( G; in red), again, colocalize with the ATF-3 labelling ( H; in green). These findings complement the PCR findings indicating an up-regulation of ATF-3 post-SCI. CNT = Uninjured Control, TXN = Transection, CON = Contusion. Scale Bars = 50 μm

Techniques Used: Expressing, Polymerase Chain Reaction

25) Product Images from "Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation"

Article Title: Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118473

S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of scrambled, c-Src, EGFR, or PDGFR, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, and GAPDH proteins were determined by Western blot.
Figure Legend Snippet: S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of scrambled, c-Src, EGFR, or PDGFR, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, and GAPDH proteins were determined by Western blot.

Techniques Used: Activation Assay, Incubation, Western Blot, Transfection

S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3).
Figure Legend Snippet: S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3).

Techniques Used: Activation Assay, Transfection, Incubation, Western Blot

26) Product Images from "Upregulation of Nuclear Factor-Related Kappa B Suggests a Disorder of Transcriptional Regulation in Minimal Change Nephrotic Syndrome"

Article Title: Upregulation of Nuclear Factor-Related Kappa B Suggests a Disorder of Transcriptional Regulation in Minimal Change Nephrotic Syndrome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030523

Post-translational modifications of NFRKB. A , representative experiment of N-glycosidase-F traitement. Total protein lysates were purified from PBMC, treated with the N-glycosidase-F, then immunoblotted with anti-NFRKB antibody. Note that the band size is reduced by ∼20 kDa. B , Immunoprecipitation of NFRKB from PBMC protein lysates followed by immunoblotting with anti-sumo-1 or anti-sumo-2/3. These data are representative of two independent experiments.
Figure Legend Snippet: Post-translational modifications of NFRKB. A , representative experiment of N-glycosidase-F traitement. Total protein lysates were purified from PBMC, treated with the N-glycosidase-F, then immunoblotted with anti-NFRKB antibody. Note that the band size is reduced by ∼20 kDa. B , Immunoprecipitation of NFRKB from PBMC protein lysates followed by immunoblotting with anti-sumo-1 or anti-sumo-2/3. These data are representative of two independent experiments.

Techniques Used: Purification, Immunoprecipitation

27) Product Images from "Participation of MCP-induced protein 1 in lipopolysaccharide preconditioning-induced ischemic stroke tolerance by regulating the expression of proinflammatory cytokines"

Article Title: Participation of MCP-induced protein 1 in lipopolysaccharide preconditioning-induced ischemic stroke tolerance by regulating the expression of proinflammatory cytokines

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-8-182

MCPIP1 knockout mice in higher mortality and exacerbated edema to brain ischemia/reperfusion caused injury. (A) Eight-week-old MCPIP1 KO and littermate WT mice were subjected to MCAO for 90 minutes followed by 72 hours of reperfusion; sham-operated mice had the same procedures except no MCA occlusion. Survival was monitored for 72 hours after the MCAO. MCPIP1 knock out I/R mice showed lower survival than wild type I/R ( p
Figure Legend Snippet: MCPIP1 knockout mice in higher mortality and exacerbated edema to brain ischemia/reperfusion caused injury. (A) Eight-week-old MCPIP1 KO and littermate WT mice were subjected to MCAO for 90 minutes followed by 72 hours of reperfusion; sham-operated mice had the same procedures except no MCA occlusion. Survival was monitored for 72 hours after the MCAO. MCPIP1 knock out I/R mice showed lower survival than wild type I/R ( p

Techniques Used: Knock-Out, Mouse Assay

JNK signaling plays a critical role in proinflammatory cytokines production . MCPIP1 knockout mice were treated with SP600125 (15 mg/kg, iv) 60 min prior to MCAO and proteins extracted from the ischemic hemisphere of MCPIP1 knockout mice undergoing ischemia 30 min followed by 30 and 90 min reperfusion separately. ( A ) A representative western blot shows protein levels of JNK phosphorylation. (B) Densitometric analysis was used to quantify p-JNK protein levels versus total JNK in 3 independent western blots and the data are expressed as the normalized folds with respect to sham. Values represent mean ± SD, * p
Figure Legend Snippet: JNK signaling plays a critical role in proinflammatory cytokines production . MCPIP1 knockout mice were treated with SP600125 (15 mg/kg, iv) 60 min prior to MCAO and proteins extracted from the ischemic hemisphere of MCPIP1 knockout mice undergoing ischemia 30 min followed by 30 and 90 min reperfusion separately. ( A ) A representative western blot shows protein levels of JNK phosphorylation. (B) Densitometric analysis was used to quantify p-JNK protein levels versus total JNK in 3 independent western blots and the data are expressed as the normalized folds with respect to sham. Values represent mean ± SD, * p

Techniques Used: Knock-Out, Mouse Assay, Western Blot

Activation of c-Jun N terminal kinase (JNK) signaling pathway . Proteins extracted from the ischemic hemisphere of wild type and MCPIP1 knockout mice undergoing ischemia 30 min followed by 30 and 90 min reperfusion separately. The samples were subjected to Western blot analysis with antibodies as indicated. ( A ) A representative western blot shows protein levels of JNK phosphorylation. (B) Densitometric analysis was used to quantify p-JNK protein levels versus total JNK in 3 independent western blots and the data are expressed as the normalized folds with respect to sham. Values represent mean ± SD, * p
Figure Legend Snippet: Activation of c-Jun N terminal kinase (JNK) signaling pathway . Proteins extracted from the ischemic hemisphere of wild type and MCPIP1 knockout mice undergoing ischemia 30 min followed by 30 and 90 min reperfusion separately. The samples were subjected to Western blot analysis with antibodies as indicated. ( A ) A representative western blot shows protein levels of JNK phosphorylation. (B) Densitometric analysis was used to quantify p-JNK protein levels versus total JNK in 3 independent western blots and the data are expressed as the normalized folds with respect to sham. Values represent mean ± SD, * p

Techniques Used: Activation Assay, Knock-Out, Mouse Assay, Western Blot

MCPIP1 mRNA and protein increase in human microglia treated with LPS . ( A ) MCPIP1 mRNA expression in human microglia treated with LPS (0.1 μg/ml) as measured by qRT-PCR. Values represent mean ± SD, § p
Figure Legend Snippet: MCPIP1 mRNA and protein increase in human microglia treated with LPS . ( A ) MCPIP1 mRNA expression in human microglia treated with LPS (0.1 μg/ml) as measured by qRT-PCR. Values represent mean ± SD, § p

Techniques Used: Expressing, Quantitative RT-PCR

MCPIP1 mRNA increases in ipsilateral brain after MCAO . MCPIP1 mRNA expression in mouse brain was determined at 3 h, 6 h and 24 h after MCAO by qRT-PCR. Values represent mean ± SD, * p
Figure Legend Snippet: MCPIP1 mRNA increases in ipsilateral brain after MCAO . MCPIP1 mRNA expression in mouse brain was determined at 3 h, 6 h and 24 h after MCAO by qRT-PCR. Values represent mean ± SD, * p

Techniques Used: Expressing, Quantitative RT-PCR

Loss of LPS-induced brain ischemia tolerance in MCPIP1 KO mice . Wild type and MCPIP1 knockout mice were pretreated with LPS (0.2 mg/kg) 24 hours before MCAO. (A) Infarct images obtained by TTC staining at 48 h after MCAO. The normal tissue was stained deep red and the infarct was stained milky. (B) Brain infarcts were assessed 48 hours after MCAO and quantified as percentage area of ischemic hemisphere. Values represent mean ± SD, * p
Figure Legend Snippet: Loss of LPS-induced brain ischemia tolerance in MCPIP1 KO mice . Wild type and MCPIP1 knockout mice were pretreated with LPS (0.2 mg/kg) 24 hours before MCAO. (A) Infarct images obtained by TTC staining at 48 h after MCAO. The normal tissue was stained deep red and the infarct was stained milky. (B) Brain infarcts were assessed 48 hours after MCAO and quantified as percentage area of ischemic hemisphere. Values represent mean ± SD, * p

Techniques Used: Mouse Assay, Knock-Out, Staining

MCPIP1 mRNA and protein levels are elevated in mouse microglia and brain upon treatment with LPS . ( A ) MCPIP1 mRNA expression in mouse microglia treated with LPS (0.1 μg/ml) as measured by qRT-PCR. Values represent mean ± SD, # p
Figure Legend Snippet: MCPIP1 mRNA and protein levels are elevated in mouse microglia and brain upon treatment with LPS . ( A ) MCPIP1 mRNA expression in mouse microglia treated with LPS (0.1 μg/ml) as measured by qRT-PCR. Values represent mean ± SD, # p

Techniques Used: Expressing, Quantitative RT-PCR

28) Product Images from "Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis"

Article Title: Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082033

EGb independently and individually blocked IL-1-mediated activation of JNK and induced degradation of c-Jun in chondrocytes. Binding of IL-1 to its receptor causes activation of MAPK upstream kinases (MKKs) and subsequently MAPKs, including JNK, p38 and ERK. Activated MAPKs then stimulate AP-1. EGb through inhibiting JNK activity and inducing ubiquitination-dependent degradation of c-Jun suppressed IL-1-mediated effects in chondrocytes.
Figure Legend Snippet: EGb independently and individually blocked IL-1-mediated activation of JNK and induced degradation of c-Jun in chondrocytes. Binding of IL-1 to its receptor causes activation of MAPK upstream kinases (MKKs) and subsequently MAPKs, including JNK, p38 and ERK. Activated MAPKs then stimulate AP-1. EGb through inhibiting JNK activity and inducing ubiquitination-dependent degradation of c-Jun suppressed IL-1-mediated effects in chondrocytes.

Techniques Used: Activation Assay, Binding Assay, Activity Assay

EGb inhibited IL-1-induced JNK but not p38 or ERK activity. Chondrocytes were pretreated with various concentrations of EGb for 72-1 for 15 min. The total cell lysates were analyzed by western blot using antibodies against un-phosphorylated or phosphorylated JNK (A), p38 (B) or ERK (C). The fold inductions of densitometric intensity of individual proteins equalized with β-actin, compared to those in un-stimulated samples, from more than 3 independent experiments using different donor cells (n = 5 for JNK and n = 3 for ERK and p38) are shown (Fig. 4D).
Figure Legend Snippet: EGb inhibited IL-1-induced JNK but not p38 or ERK activity. Chondrocytes were pretreated with various concentrations of EGb for 72-1 for 15 min. The total cell lysates were analyzed by western blot using antibodies against un-phosphorylated or phosphorylated JNK (A), p38 (B) or ERK (C). The fold inductions of densitometric intensity of individual proteins equalized with β-actin, compared to those in un-stimulated samples, from more than 3 independent experiments using different donor cells (n = 5 for JNK and n = 3 for ERK and p38) are shown (Fig. 4D).

Techniques Used: Activity Assay, Western Blot

29) Product Images from "Protective Effects of Hesperidin (Citrus Flavonone) on High Glucose Induced Oxidative Stress and Apoptosis in a Cellular Model for Diabetic Retinopathy"

Article Title: Protective Effects of Hesperidin (Citrus Flavonone) on High Glucose Induced Oxidative Stress and Apoptosis in a Cellular Model for Diabetic Retinopathy

Journal: Nutrients

doi: 10.3390/nu9121312

Effects of hesperidin on protein expression related to apoptosis in RGC-5 cells under high glucose conditions. The RGCs were cultured with normal (NG) or high glucose (HG) plus hesperidin at concentrations of 12.5 (Hesp 12.5), 25 (Hesp 25), or 50 μmol/L (Hesp 50) for 48 h. ( A ) Protein bands of cleaved caspase-9 and cleaved caspase-3 in RGC-5 cells detected by Western blotting; ( B ) Quantitative densitometric analysis of caspase-9 and cleaved caspase-3; ( C ) Protein bands of Bax and Bcl-2 in RGC-5 cells detected by Western blotting; ( D ) Changes of the ratios of Bax/Bcl-2 are displayed in the bottom panel. The results are presented as the mean ± SD of five independent experiments ( n = 5), each of which was performed in triplicate. a p
Figure Legend Snippet: Effects of hesperidin on protein expression related to apoptosis in RGC-5 cells under high glucose conditions. The RGCs were cultured with normal (NG) or high glucose (HG) plus hesperidin at concentrations of 12.5 (Hesp 12.5), 25 (Hesp 25), or 50 μmol/L (Hesp 50) for 48 h. ( A ) Protein bands of cleaved caspase-9 and cleaved caspase-3 in RGC-5 cells detected by Western blotting; ( B ) Quantitative densitometric analysis of caspase-9 and cleaved caspase-3; ( C ) Protein bands of Bax and Bcl-2 in RGC-5 cells detected by Western blotting; ( D ) Changes of the ratios of Bax/Bcl-2 are displayed in the bottom panel. The results are presented as the mean ± SD of five independent experiments ( n = 5), each of which was performed in triplicate. a p

Techniques Used: Expressing, Cell Culture, Western Blot

30) Product Images from "Chondroprotective Effects and Mechanisms of Dextromethorphan: Repurposing Antitussive Medication for Osteoarthritis Treatment"

Article Title: Chondroprotective Effects and Mechanisms of Dextromethorphan: Repurposing Antitussive Medication for Osteoarthritis Treatment

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19030825

Mechanisms and chondro-protective effects of DXM were verified in human OA chondrocytes. ( A ) Human OA chondrocytes were co-treated with TNF-α (5 ng/mL) and various doses of DXM for 24 h. Total cell lysate and supernatants were analyzed for pro-MMP-13 and Col II expression with Western blots. ( B ) Chondrocytes were treated with TNF-α (5 ng/mL) and various doses of DXM for 8 h. The mRNA levels of MMP-13 were determined by real-time RT-PCR. ( C ) Chondrocytes were pretreated with various doses of DXM for 2 h, and then the cells were stimulated with TNF-α for 15 min. Total cell lysates were analyzed with Western blots (for pIKKα/β, IKKα and β-actin expression). ( D ) In parallel, the expression of p-c-Jun, c-Jun, p-JNK, and JNK (c-Jun N-terminal kinase) in total cell lysates was determined. Data were shown as representative data from at least three independent experiments of different donors. Values are the means ± S.E.M. and significance (* p
Figure Legend Snippet: Mechanisms and chondro-protective effects of DXM were verified in human OA chondrocytes. ( A ) Human OA chondrocytes were co-treated with TNF-α (5 ng/mL) and various doses of DXM for 24 h. Total cell lysate and supernatants were analyzed for pro-MMP-13 and Col II expression with Western blots. ( B ) Chondrocytes were treated with TNF-α (5 ng/mL) and various doses of DXM for 8 h. The mRNA levels of MMP-13 were determined by real-time RT-PCR. ( C ) Chondrocytes were pretreated with various doses of DXM for 2 h, and then the cells were stimulated with TNF-α for 15 min. Total cell lysates were analyzed with Western blots (for pIKKα/β, IKKα and β-actin expression). ( D ) In parallel, the expression of p-c-Jun, c-Jun, p-JNK, and JNK (c-Jun N-terminal kinase) in total cell lysates was determined. Data were shown as representative data from at least three independent experiments of different donors. Values are the means ± S.E.M. and significance (* p

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

31) Product Images from "The Inhibitory Effect of Quercetin on Asymmetric Dimethylarginine-Induced Apoptosis Is Mediated by the Endoplasmic Reticulum Stress Pathway in Glomerular Endothelial Cells"

Article Title: The Inhibitory Effect of Quercetin on Asymmetric Dimethylarginine-Induced Apoptosis Is Mediated by the Endoplasmic Reticulum Stress Pathway in Glomerular Endothelial Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15010484

QC inhibited ADMA-induced ER stress. ( A ) QC prevented the ER stress pathway by down-regulating the expression of PERK-ATF4-CHOP. GEnCs were treated for 1 h in the presence or absence of 20 μM QC prior to incubation with 100 μM ADMA for 24 h. Cells were then lysed, and levels of Thr 981 phosphorylated PERK (p-PERK), ATF4, and CHOP were assayed by western blot analysis. β-Actin or PERK is shown as a loading control. Results are the mean ± SD ( n = 3); ( B ) Real-time PCR analysis of ATF4 and CHOP mRNA levels in cells exposed to ADMA (100 μM) for 24 h in the absence or presence of QC (means ± SD, n = 3); ( C ) Cells were transfected with graded concentrations of pcDNA3-GADD153 expression vectors or the control empty vector, and incubated for 24 h. Cells were pretreated with 20 μM QC for 1 h and then restimulated with 100 μM ADMA for a further 24 h. Apoptosis was assessed by flow cytometry (mean ± SD, n = 3); and ( D , E ) QC inhibited ADMA-induced IRE1 expression, JNK phosphorylation and c-Jun phosphorylation. GEnC were treated for 1 h in the presence or absence of 20 μM QC before incubation with 100 μM ADMA for 24 h. Cells were then lysed, and levels of IRE1, p-JNK and p-c-Jun were assayed by western blot analysis. Results are the mean ± SD ( n = 3). p
Figure Legend Snippet: QC inhibited ADMA-induced ER stress. ( A ) QC prevented the ER stress pathway by down-regulating the expression of PERK-ATF4-CHOP. GEnCs were treated for 1 h in the presence or absence of 20 μM QC prior to incubation with 100 μM ADMA for 24 h. Cells were then lysed, and levels of Thr 981 phosphorylated PERK (p-PERK), ATF4, and CHOP were assayed by western blot analysis. β-Actin or PERK is shown as a loading control. Results are the mean ± SD ( n = 3); ( B ) Real-time PCR analysis of ATF4 and CHOP mRNA levels in cells exposed to ADMA (100 μM) for 24 h in the absence or presence of QC (means ± SD, n = 3); ( C ) Cells were transfected with graded concentrations of pcDNA3-GADD153 expression vectors or the control empty vector, and incubated for 24 h. Cells were pretreated with 20 μM QC for 1 h and then restimulated with 100 μM ADMA for a further 24 h. Apoptosis was assessed by flow cytometry (mean ± SD, n = 3); and ( D , E ) QC inhibited ADMA-induced IRE1 expression, JNK phosphorylation and c-Jun phosphorylation. GEnC were treated for 1 h in the presence or absence of 20 μM QC before incubation with 100 μM ADMA for 24 h. Cells were then lysed, and levels of IRE1, p-JNK and p-c-Jun were assayed by western blot analysis. Results are the mean ± SD ( n = 3). p

Techniques Used: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

32) Product Images from "Role of areca nut induced JNK/ATF2/Jun axis in the activation of TGF-β pathway in precancerous Oral Submucous Fibrosis"

Article Title: Role of areca nut induced JNK/ATF2/Jun axis in the activation of TGF-β pathway in precancerous Oral Submucous Fibrosis

Journal: Scientific Reports

doi: 10.1038/srep34314

ATF2/Jun mediate areca nut induced TGF-β pathway activation in HaCaT cells. ( a ) Representative immunofluorescence images of areca nut induced nuclear localized pATF2 and p-c-Jun at 2 hours. Magnification: 63X; Zoom: 1.8; Scale bar = 10 μm. ( b ) Bar graph of qPCR results of chIP assay showing fold enrichment of pATF2 and p-c-Jun on TGF-β2 promoter at 2 hours upon areca nut treatment compared to untreated cells. ( c ) Bar graph showing fold change at 2 hour of areca nut induced TGF-β2 and its compromise upon transient knock down of ATF2 or c-Jun. ( d,e ) Immunoblots representing pSMAD2 levels at 2 hour areca nut treatment and its compromise upon transient knock down of ATF2 or c-Jun. β-actin is used as loading control in all the immunoblots. *** , ** represent p values ≤ 0.0001; 0.001 respectively. (5H; 5 μg/ml areca nut extract).
Figure Legend Snippet: ATF2/Jun mediate areca nut induced TGF-β pathway activation in HaCaT cells. ( a ) Representative immunofluorescence images of areca nut induced nuclear localized pATF2 and p-c-Jun at 2 hours. Magnification: 63X; Zoom: 1.8; Scale bar = 10 μm. ( b ) Bar graph of qPCR results of chIP assay showing fold enrichment of pATF2 and p-c-Jun on TGF-β2 promoter at 2 hours upon areca nut treatment compared to untreated cells. ( c ) Bar graph showing fold change at 2 hour of areca nut induced TGF-β2 and its compromise upon transient knock down of ATF2 or c-Jun. ( d,e ) Immunoblots representing pSMAD2 levels at 2 hour areca nut treatment and its compromise upon transient knock down of ATF2 or c-Jun. β-actin is used as loading control in all the immunoblots. *** , ** represent p values ≤ 0.0001; 0.001 respectively. (5H; 5 μg/ml areca nut extract).

Techniques Used: Activation Assay, Immunofluorescence, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Western Blot

pJNK, pATF2 and p-c-Jun are up regulated in OSF tissues. ( a ) Representative images of immunohistochemistry performed on normal (n = 8) and OSF (n = 8) tissues for evaluating activation of JNK (pJNK), ATF2 (pATF2) and c-Jun (p-c-Jun). pJNK, pATF2 and p-c-Jun levels were observed to be high in OSF tissues as compared to normal tissues. Magnification factor = 20X; scale bar = 50 μm. ( b ) Scatter graph depicting labeling index for each of the tissue samples scored for pJNK, pATF2 and p-c-Jun staining. Unpaired t-test was performed for statistical significance between the median labeling index of two groups (Normal and OSF tissues). ( c ) Pearson’s correlation graph for pATF2 (Y axis) and pJNK (X axis) staining in OSF tissues. ( d ) Pearson’s correlation graph for p-c-Jun (Y axis) and pJNK (X axis) staining in OSF tissues. *** represents p value ≤ 0.0001.
Figure Legend Snippet: pJNK, pATF2 and p-c-Jun are up regulated in OSF tissues. ( a ) Representative images of immunohistochemistry performed on normal (n = 8) and OSF (n = 8) tissues for evaluating activation of JNK (pJNK), ATF2 (pATF2) and c-Jun (p-c-Jun). pJNK, pATF2 and p-c-Jun levels were observed to be high in OSF tissues as compared to normal tissues. Magnification factor = 20X; scale bar = 50 μm. ( b ) Scatter graph depicting labeling index for each of the tissue samples scored for pJNK, pATF2 and p-c-Jun staining. Unpaired t-test was performed for statistical significance between the median labeling index of two groups (Normal and OSF tissues). ( c ) Pearson’s correlation graph for pATF2 (Y axis) and pJNK (X axis) staining in OSF tissues. ( d ) Pearson’s correlation graph for p-c-Jun (Y axis) and pJNK (X axis) staining in OSF tissues. *** represents p value ≤ 0.0001.

Techniques Used: Immunohistochemistry, Activation Assay, Labeling, Staining

Schematic representation of key findings from the study. Areca nut acts on muscarinic acid receptors to release calcium and activate CAMKII. It also induces intracellular reactive oxygen species (ROS). CAMKII and ROS together activate JNK which subsequently phosphorylates ATF2 and c-Jun transcription factors. The two transcription factors induce TGF-β2 promoter. The translated TGF-β protein can now activate the canonical SMAD signaling pathway and auto-induce TGF-β and other targets * 22 in epithelial cells.
Figure Legend Snippet: Schematic representation of key findings from the study. Areca nut acts on muscarinic acid receptors to release calcium and activate CAMKII. It also induces intracellular reactive oxygen species (ROS). CAMKII and ROS together activate JNK which subsequently phosphorylates ATF2 and c-Jun transcription factors. The two transcription factors induce TGF-β2 promoter. The translated TGF-β protein can now activate the canonical SMAD signaling pathway and auto-induce TGF-β and other targets * 22 in epithelial cells.

Techniques Used:

Areca nut activates ATF2 and Jun in HaCaT cells. ( a ) Protein expression data for phospho and total proteins of ATF2 and c-Jun upon areca nut treatment for the indicated time points. ( b,c ) Immunoblots depicting induction of phospho ATF2 and phospho c-Jun by areca nut is independent of TβR-I ( b ) but dependent on JNK ( c ) activity. ( d–f ) Immunoblots representing compromise in areca nut induced pATF2/p-c-Jun upon treatment with ( d ) atropine; ( e ) KN93 and ( f ) DPI. β-actin is used as loading control in all the immunoblots.
Figure Legend Snippet: Areca nut activates ATF2 and Jun in HaCaT cells. ( a ) Protein expression data for phospho and total proteins of ATF2 and c-Jun upon areca nut treatment for the indicated time points. ( b,c ) Immunoblots depicting induction of phospho ATF2 and phospho c-Jun by areca nut is independent of TβR-I ( b ) but dependent on JNK ( c ) activity. ( d–f ) Immunoblots representing compromise in areca nut induced pATF2/p-c-Jun upon treatment with ( d ) atropine; ( e ) KN93 and ( f ) DPI. β-actin is used as loading control in all the immunoblots.

Techniques Used: Expressing, Western Blot, Activity Assay

33) Product Images from "In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth"

Article Title: In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

Journal: Cancers

doi: 10.3390/cancers8050049

Modulation of WNT/beta-catenin activity impacts the response of CRC cells to hyperthermia. ( A , C ) WNT/beta-catenin transcriptional activity was measured with the luciferase reporter pair TOP-Flash and FOP-Flash. Treatments (16 h) with lithium chloride were performed at 20 mM, or with AZD6244 (Ei) at 0.5 μM. The standard deviations are based upon three experiments with duplicate samples per treatment and condition (temperature) were performed; ( B ) Representative Western blot analyses with nuclear or total cell lysates of HCT-116 cells exposed to 42 °C for 0, 7, or 16 h. Active and total beta-catenin were detected in nuclear lysates and equal loading was ascertained by Ponceau staining. Additional markers of WNT/beta-catenin activity were analyzed with total cell lysates. Vertical lines indicate same Western blot analyses; ( D ) Clonal growth assays as described in Figure 1 were performed with 20 mM lithium chloride. Standard deviation was calculated based upon three independent experiments with triplicate samples.
Figure Legend Snippet: Modulation of WNT/beta-catenin activity impacts the response of CRC cells to hyperthermia. ( A , C ) WNT/beta-catenin transcriptional activity was measured with the luciferase reporter pair TOP-Flash and FOP-Flash. Treatments (16 h) with lithium chloride were performed at 20 mM, or with AZD6244 (Ei) at 0.5 μM. The standard deviations are based upon three experiments with duplicate samples per treatment and condition (temperature) were performed; ( B ) Representative Western blot analyses with nuclear or total cell lysates of HCT-116 cells exposed to 42 °C for 0, 7, or 16 h. Active and total beta-catenin were detected in nuclear lysates and equal loading was ascertained by Ponceau staining. Additional markers of WNT/beta-catenin activity were analyzed with total cell lysates. Vertical lines indicate same Western blot analyses; ( D ) Clonal growth assays as described in Figure 1 were performed with 20 mM lithium chloride. Standard deviation was calculated based upon three independent experiments with triplicate samples.

Techniques Used: Activity Assay, Luciferase, Western Blot, Staining, Standard Deviation

Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through HGFR, a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to EGFR signaling, and to increasing in time signaling amplifying mechanism.
Figure Legend Snippet: Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through HGFR, a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to EGFR signaling, and to increasing in time signaling amplifying mechanism.

Techniques Used: Mutagenesis, Activity Assay

Increase of WNT/beta-catenin activity in HCT-116 cells results in higher levels of ERK1/2 activation and decreased ability for clonal growth at 42 °C. ( A , C ) WNT/beta-catenin transcriptional activity was measured as in Figure 5 after exposure of the cells to mock treatment (M), 5 μM of the GSK-beta kinase inhibitor (GSKi) TWS119, or 0.5 μM AZD6244 (Ei) for 16 h. Three experiments with duplicate samples per treatment and condition (temperature) were performed; ( B , D ) Clonal growth analyses were performed as in Figure 1 by exposing the cells for 24 h to mock treatment or 5 μM TWS119 (GSKi); ( E ) Representative Western blot analyses with total cell lysates of HCT-116 cells exposed for 16 h to 42 °C and 5 or 10 μM TWS119 (GSKi) and 20 mM lithium chloride.
Figure Legend Snippet: Increase of WNT/beta-catenin activity in HCT-116 cells results in higher levels of ERK1/2 activation and decreased ability for clonal growth at 42 °C. ( A , C ) WNT/beta-catenin transcriptional activity was measured as in Figure 5 after exposure of the cells to mock treatment (M), 5 μM of the GSK-beta kinase inhibitor (GSKi) TWS119, or 0.5 μM AZD6244 (Ei) for 16 h. Three experiments with duplicate samples per treatment and condition (temperature) were performed; ( B , D ) Clonal growth analyses were performed as in Figure 1 by exposing the cells for 24 h to mock treatment or 5 μM TWS119 (GSKi); ( E ) Representative Western blot analyses with total cell lysates of HCT-116 cells exposed for 16 h to 42 °C and 5 or 10 μM TWS119 (GSKi) and 20 mM lithium chloride.

Techniques Used: Activity Assay, Activation Assay, Western Blot

Combined effect of hyperthermia and propolis on CRC cells. ( A ) Representative Western blot analyses with total cell lysates after exposure of HCT-116 and HKH2 cells to 37 °C or 42 °C, for 8, 16, or 24 h in absence or presence of 100 μg/ml propolis (P); ( B ) Clonal growth assays of HCT-116 (H), HKH2 (K) and CDC841CoN (C) cells were performed as described in the legend of Figure 1 . Cells were exposed simultaneously for 24 h to 37 °C or 42 °C and to mock (m) treatment or propolis (p) at 100 μg/mL. Results are the mean from minimum of four clonal growth assays with triplicate samples per treatment. Only statistically significant different values are indicated; ( C ) WNT/beta-catenin transcriptional activity was measured with the luciferase reporter pair TOP-Flash and FOP-Flash as described in Materials and Methods. Mock and propolis treatment were performed for 16 h as in ( B ).
Figure Legend Snippet: Combined effect of hyperthermia and propolis on CRC cells. ( A ) Representative Western blot analyses with total cell lysates after exposure of HCT-116 and HKH2 cells to 37 °C or 42 °C, for 8, 16, or 24 h in absence or presence of 100 μg/ml propolis (P); ( B ) Clonal growth assays of HCT-116 (H), HKH2 (K) and CDC841CoN (C) cells were performed as described in the legend of Figure 1 . Cells were exposed simultaneously for 24 h to 37 °C or 42 °C and to mock (m) treatment or propolis (p) at 100 μg/mL. Results are the mean from minimum of four clonal growth assays with triplicate samples per treatment. Only statistically significant different values are indicated; ( C ) WNT/beta-catenin transcriptional activity was measured with the luciferase reporter pair TOP-Flash and FOP-Flash as described in Materials and Methods. Mock and propolis treatment were performed for 16 h as in ( B ).

Techniques Used: Western Blot, Activity Assay, Luciferase

34) Product Images from "Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9"

Article Title: Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

Journal: BMB Reports

doi: 10.5483/BMBRep.2013.46.11.053

Effects of BVT948 on the viability of MCF-7 cells and TPA-induced MMP-9 expression. Cells were cultured in 96-well plates until 90% confluence, and various concentrations of BVT948 were then added to cells for 24 h. An established MTT assay was used to detect the viability of the cells (A). MCF-7 cells were treated with the indicated BVT948 concentrations in the presence of TPA for 24 h. MMP-9 mRNA levels were analyzed by real-time PCR, and GAPDH was used as an internal control (B). Cell lysates were analyzed by Western blot with an anti-MMP-9 antibody. The blot was retaken with anti β-actin to confirm equal loading (C). Conditioned medium was prepared and used for gelatin zymography (D). Each value represents the mean ± SEM of three independent experiments. *P < 0.01 vs. TPA.
Figure Legend Snippet: Effects of BVT948 on the viability of MCF-7 cells and TPA-induced MMP-9 expression. Cells were cultured in 96-well plates until 90% confluence, and various concentrations of BVT948 were then added to cells for 24 h. An established MTT assay was used to detect the viability of the cells (A). MCF-7 cells were treated with the indicated BVT948 concentrations in the presence of TPA for 24 h. MMP-9 mRNA levels were analyzed by real-time PCR, and GAPDH was used as an internal control (B). Cell lysates were analyzed by Western blot with an anti-MMP-9 antibody. The blot was retaken with anti β-actin to confirm equal loading (C). Conditioned medium was prepared and used for gelatin zymography (D). Each value represents the mean ± SEM of three independent experiments. *P < 0.01 vs. TPA.

Techniques Used: Expressing, Cell Culture, MTT Assay, Real-time Polymerase Chain Reaction, Western Blot, Zymography

35) Product Images from "Celecoxib and acetylbritannilactone interact synergistically to suppress breast cancer cell growth via COX-2-dependent and -independent mechanisms"

Article Title: Celecoxib and acetylbritannilactone interact synergistically to suppress breast cancer cell growth via COX-2-dependent and -independent mechanisms

Journal: Cell Death & Disease

doi: 10.1038/cddis.2011.64

Effects of the combination of celecoxib and ABL on COX-2 expression and activity in MDA-MD-231 cells. ( a ) MDA-MD-231 cells were treated with celecoxib, ABL or their combination for 24 h (left) or 48 h (right). COX-2 mRNA and protein expression was examined using RT-PCR (left) and western blot analysis (right). Quantification of COX-2 mRNA and protein expression normalized to actin levels, were provided at the bottom. ( b ) MDA-MB-231 cells were treated with the combination for 48 h, and PGE 2 levels in the culture medium were measured using ELISA. Results were expressed as mean±S.E.M. from at least three independent experiments
Figure Legend Snippet: Effects of the combination of celecoxib and ABL on COX-2 expression and activity in MDA-MD-231 cells. ( a ) MDA-MD-231 cells were treated with celecoxib, ABL or their combination for 24 h (left) or 48 h (right). COX-2 mRNA and protein expression was examined using RT-PCR (left) and western blot analysis (right). Quantification of COX-2 mRNA and protein expression normalized to actin levels, were provided at the bottom. ( b ) MDA-MB-231 cells were treated with the combination for 48 h, and PGE 2 levels in the culture medium were measured using ELISA. Results were expressed as mean±S.E.M. from at least three independent experiments

Techniques Used: Expressing, Activity Assay, Multiple Displacement Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

36) Product Images from "Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster"

Article Title: Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025087

Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear phosphorylated Jun-Kinase (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) Anti-BRDU (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.
Figure Legend Snippet: Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear phosphorylated Jun-Kinase (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) Anti-BRDU (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.

Techniques Used: Mutagenesis

37) Product Images from "AP-1 Is a Component of the Transcriptional Network Regulated by GSK-3 in Quiescent Cells"

Article Title: AP-1 Is a Component of the Transcriptional Network Regulated by GSK-3 in Quiescent Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020150

Analysis of c-Jun binding and recruitment to predicted AP-1 sites by chromatin immunoprecipitation. A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1 ) relative to the transcription start site. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1 . Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).
Figure Legend Snippet: Analysis of c-Jun binding and recruitment to predicted AP-1 sites by chromatin immunoprecipitation. A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1 ) relative to the transcription start site. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1 . Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Autoradiography, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Negative Control, Inhibition

38) Product Images from "Anti-melanogenic activity of the novel herbal medicine, MA128, through inhibition of tyrosinase activity mediated by the p38 mitogen-activated protein kinases and protein kinase signaling pathway in B16F10 cells"

Article Title: Anti-melanogenic activity of the novel herbal medicine, MA128, through inhibition of tyrosinase activity mediated by the p38 mitogen-activated protein kinases and protein kinase signaling pathway in B16F10 cells

Journal: Pharmacognosy Magazine

doi: 10.4103/0973-1296.139774

Effects of MA128 on the levels of melanogenic proteins, p-c-AMP-dependent protein kinase (PKA), and p-c-AMP-related element binding protein (CREB) in alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. (a) B16F10 cells preincubated with or without 100 and 250 μg/mL MA128 for 12 h were simulated with 1 μM α-MSH for additional 36 h. After harvest of cells, lysates were subjected to western blotting for the detection of tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor levels. (b) B16F10 cells were preincubated with or without 100 and 250 μg/mL MA128 for 12 h, and then further stimulated with 1 μM α-MSH for 30 min. Changes in the level of p-PKA and p-CREB were examined by western blotting followed by quantitation with ImageJ. Relative ratios were determined after normalization to α-tubulin expression. Data are expressed as mean ± standard deviation of two independent experiments. * P
Figure Legend Snippet: Effects of MA128 on the levels of melanogenic proteins, p-c-AMP-dependent protein kinase (PKA), and p-c-AMP-related element binding protein (CREB) in alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. (a) B16F10 cells preincubated with or without 100 and 250 μg/mL MA128 for 12 h were simulated with 1 μM α-MSH for additional 36 h. After harvest of cells, lysates were subjected to western blotting for the detection of tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor levels. (b) B16F10 cells were preincubated with or without 100 and 250 μg/mL MA128 for 12 h, and then further stimulated with 1 μM α-MSH for 30 min. Changes in the level of p-PKA and p-CREB were examined by western blotting followed by quantitation with ImageJ. Relative ratios were determined after normalization to α-tubulin expression. Data are expressed as mean ± standard deviation of two independent experiments. * P

Techniques Used: Binding Assay, Western Blot, Quantitation Assay, Expressing, Standard Deviation

39) Product Images from "Inactivation of SAG E3 Ubiquitin Ligase Blocks Embryonic Stem Cell Differentiation and Sensitizes Leukemia Cells to Retinoid Acid"

Article Title: Inactivation of SAG E3 Ubiquitin Ligase Blocks Embryonic Stem Cell Differentiation and Sensitizes Leukemia Cells to Retinoid Acid

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027726

Induction of SAG-SCF E3 ligase substrates by RA and/or MLN4924. Cells were treated with indicated concentrations of MLN4924 for 24 hrs, followed by immunoblotting using cullin-1 antibody with β-actin as the loading control ( A ). Cells were treated with RA (1.5 µM), MLN4924 (0.1 µM for HL-60, 1.0 µM for KG-1) alone or in combination for 24 hrs, followed by immunoblotting using indicated antiobodies ( B C ). mES cells were treated with RA at indicated concentrations for 24 hrs, followed by immunoblotting using NOXA antibody with β-actin as the loading control ( D ).
Figure Legend Snippet: Induction of SAG-SCF E3 ligase substrates by RA and/or MLN4924. Cells were treated with indicated concentrations of MLN4924 for 24 hrs, followed by immunoblotting using cullin-1 antibody with β-actin as the loading control ( A ). Cells were treated with RA (1.5 µM), MLN4924 (0.1 µM for HL-60, 1.0 µM for KG-1) alone or in combination for 24 hrs, followed by immunoblotting using indicated antiobodies ( B C ). mES cells were treated with RA at indicated concentrations for 24 hrs, followed by immunoblotting using NOXA antibody with β-actin as the loading control ( D ).

Techniques Used:

Sag deletion sensitizes mES cells to RA-induced apoptosis. mES cells with genotype of Sag +/+ and Sag −/− were treated with DMSO control or 1 µM RA for indicated time periods, followed by trypan blue staining ( A ), TUNEL staining at 36 hrs ( B , left panel), with quantification of TUNEL positive cells graphed ( B , right panel), DNA fragmentation assay at 36 hrs ( C ), and caspase-3 activity assay at the indicated time point ( D ). *, p
Figure Legend Snippet: Sag deletion sensitizes mES cells to RA-induced apoptosis. mES cells with genotype of Sag +/+ and Sag −/− were treated with DMSO control or 1 µM RA for indicated time periods, followed by trypan blue staining ( A ), TUNEL staining at 36 hrs ( B , left panel), with quantification of TUNEL positive cells graphed ( B , right panel), DNA fragmentation assay at 36 hrs ( C ), and caspase-3 activity assay at the indicated time point ( D ). *, p

Techniques Used: Staining, TUNEL Assay, DNA Fragmentation Assay, Caspase-3 Activity Assay

Correlation between SAG overexpression and RA resistance in AML cell lines and their sensitivity to RA and/or MLN4924. Proteins were extracted from 7 AML lines and subjected to immunoblotting using antibody against SAG with β-actin as the loading control ( A ). Cells were seeded in 96-well plate in triplicate and treated with various concentrations of RA ( B , left panel), MLN4924 ( B , right panel) or in combination at indicated concentrations of each drug for 48 hrs ( C , HL-60 cells) and ( D , KG-1 cells), followed by ATP-lite cell viability assay. Values were normalized to the untreated control. Shown is x ± SEM from three independent experiments with IC50 curve generated and IC50 value calculated using the Prism software.
Figure Legend Snippet: Correlation between SAG overexpression and RA resistance in AML cell lines and their sensitivity to RA and/or MLN4924. Proteins were extracted from 7 AML lines and subjected to immunoblotting using antibody against SAG with β-actin as the loading control ( A ). Cells were seeded in 96-well plate in triplicate and treated with various concentrations of RA ( B , left panel), MLN4924 ( B , right panel) or in combination at indicated concentrations of each drug for 48 hrs ( C , HL-60 cells) and ( D , KG-1 cells), followed by ATP-lite cell viability assay. Values were normalized to the untreated control. Shown is x ± SEM from three independent experiments with IC50 curve generated and IC50 value calculated using the Prism software.

Techniques Used: Over Expression, Viability Assay, Generated, Software

40) Product Images from "CP690,550 inhibits oncostatin M-induced JAK/STAT signaling pathway in rheumatoid synoviocytes"

Article Title: CP690,550 inhibits oncostatin M-induced JAK/STAT signaling pathway in rheumatoid synoviocytes

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3333

CP690,550 suppresses OSM-induced JAKs/STATs activation in RA-FLS; CP690,550 inhibits OSM-induced MAPKs activation RA-FLS . (a) Quiescent RA-FLS were pretreated with various concentrations of CP690,550 for 2 hours, then stimulated with OSM (20 ng/ml) for 20 minutes. Cellular lysates were subjected to Western blotting using phospho-specific antibodies against JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5. Three experiments were performed using different RA-FLS and a representative result is shown. (b) Quiescent RA-FLS were pretreated with various concentrations of CP690,550 for 2 hours, then stimulated with OSM (20 ng/ml) for 20 minutes. Cellular lysates were subjected to Western blotting using phospho-specific antibodies against ERK1/2, p38 and JNK1/2. Three experiments were performed using different RA-FLS and a representative result is shown.
Figure Legend Snippet: CP690,550 suppresses OSM-induced JAKs/STATs activation in RA-FLS; CP690,550 inhibits OSM-induced MAPKs activation RA-FLS . (a) Quiescent RA-FLS were pretreated with various concentrations of CP690,550 for 2 hours, then stimulated with OSM (20 ng/ml) for 20 minutes. Cellular lysates were subjected to Western blotting using phospho-specific antibodies against JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5. Three experiments were performed using different RA-FLS and a representative result is shown. (b) Quiescent RA-FLS were pretreated with various concentrations of CP690,550 for 2 hours, then stimulated with OSM (20 ng/ml) for 20 minutes. Cellular lysates were subjected to Western blotting using phospho-specific antibodies against ERK1/2, p38 and JNK1/2. Three experiments were performed using different RA-FLS and a representative result is shown.

Techniques Used: Activation Assay, Western Blot

Pyridone 6 suppresses OSM-induced JAKs/STATs activation in RA-FLS; Pyridone 6 suppresses OSM-induced IL-6 synthesis in RA-FLS . (a) Quiescent RA-FLS were pretreated with vehicle (DMSO -) or pyridone 6 for 2 hours, then stimulated with OSM (20 ng/ml) for 20 minutes. Cellular lysates were subjected to Western blotting using phospho-specific antibodies against JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5. Two experiments were performed using different RA-FLS and a representative result is shown. (b) Quiescent RA-FLS were pretreated with vehicle (DMSO, -) or pyridone 6 for two hours, then stimulated with OSM (20 ng/ml) for 24 hours. IL-6 protein in the conditioned media was determined by ELISA. The data were expressed as the mean ± SD of two independent experiments. * P
Figure Legend Snippet: Pyridone 6 suppresses OSM-induced JAKs/STATs activation in RA-FLS; Pyridone 6 suppresses OSM-induced IL-6 synthesis in RA-FLS . (a) Quiescent RA-FLS were pretreated with vehicle (DMSO -) or pyridone 6 for 2 hours, then stimulated with OSM (20 ng/ml) for 20 minutes. Cellular lysates were subjected to Western blotting using phospho-specific antibodies against JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5. Two experiments were performed using different RA-FLS and a representative result is shown. (b) Quiescent RA-FLS were pretreated with vehicle (DMSO, -) or pyridone 6 for two hours, then stimulated with OSM (20 ng/ml) for 24 hours. IL-6 protein in the conditioned media was determined by ELISA. The data were expressed as the mean ± SD of two independent experiments. * P

Techniques Used: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Phosphorylation of JAKs/STATs in OSM-treated RA-FLS . Quiescent RA-FLS were stimulated with OSM (20 ng/ml) for indicated times. Phosphorylation of JAKs (A; JAK1, JAK2, JAK3) and STATs (B; STAT1, STAT3, STAT5) were determined by Western blotting using phospho-specific or pan antibodies against JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5. Three experiments were performed using different RA-FLS and a representative result is shown.
Figure Legend Snippet: Phosphorylation of JAKs/STATs in OSM-treated RA-FLS . Quiescent RA-FLS were stimulated with OSM (20 ng/ml) for indicated times. Phosphorylation of JAKs (A; JAK1, JAK2, JAK3) and STATs (B; STAT1, STAT3, STAT5) were determined by Western blotting using phospho-specific or pan antibodies against JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5. Three experiments were performed using different RA-FLS and a representative result is shown.

Techniques Used: Western Blot

Related Articles

Pyrolysis Gas Chromatography:

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway
Article Snippet: .. Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. .. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature.

Blocking Assay:

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway
Article Snippet: .. Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. .. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature.

Incubation:

Article Title: Protective Effect of Hesperidin Against Sepsis-Induced Lung Injury by Inducing the Heat-Stable Protein 70 (Hsp70)/Toll-Like Receptor 4 (TLR4)/ Myeloid Differentiation Primary Response 88 (MyD88) Pathway
Article Snippet: .. Proteins were transferred onto nitrocellulose membranes and incubated overnight with caspase-3 (1: 500), Bcl-2 (1: 1000), TLR4 (1: 1000), Hsp70 (1: 1000), and β-Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, USA). ..

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway
Article Snippet: .. Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. .. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature.

other:

Article Title: Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway
Article Snippet: Antibodies The primary antibodies described in this article include anti-supervillin (H340 [ ]), anti-ERK1/2 (#4695; Cell Signaling Technology; MA, USA), anti-p-ERK1/2 (#4370; Cell Signaling Technology), anti-p38 (#8690; Cell Signaling Technology), anti-p-p38 (#4511; Cell Signaling Technology), anti-c-Jun N-terminal kinase (JNK)1/2 (#9252; Cell Signaling Technology), anti-p-JNK1/2 (#4668; Cell Signaling Technology), anti-E-cadherin (#sc7870; Santa Cruz Biotechnology, Inc.; CA, USA), anti-Vimentin (#sc73258; Santa Cruz Biotechnology, Inc.; CA, USA), anti-Snail1 (#sc393172; Santa Cruz Biotechnology, Inc), anti-β-actin (#3700; Cell Signaling Technology), and anti-β-tubulin (#TA506805; Origene; China).

Article Title: DeSUMOylation of MKK7 kinase by the SUMO2/3 protease SENP3 potentiates lipopolysaccharide-induced inflammatory signaling in macrophages
Article Snippet: The antibody against IκBα (sc-371) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy
Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

Expressing:

Article Title: TRPM7 overexpression enhances the cancer stem cell-like and metastatic phenotypes of lung cancer through modulation of the Hsp90α/uPA/MMP2 signaling pathway
Article Snippet: .. Our results demonstrate that 48 h treatment of the 95D cells with 5 μM or 10 μM Waixenicin A significantly and dose-dependently inhibited the expression level of TRPM7, Vimentin, Survivin, STAT3, HSP90α, uPA, and MMP2 proteins, compared to the untreated control cells (Fig. b). .. In parallel experiments using the tumorsphere formation assay, we demonstrated that like the inhibitory effect of shTRPM7 on tumorsphere formation, Waixenicin A not only more efficiently suppressed tumorsphere formation by the lung cancer cells, but also enhanced the anti-CSCs effect of shTRPM7 (Fig. c and d).

Western Blot:

Article Title: MCL-1 is a prognostic indicator and drug target in breast cancer
Article Snippet: .. Western blotting Standard western blot procedures were used on whole cell lysates and probed with antibodies specific to MCL-1 (Proteintech, UK), ACTIN (Sigma, UK), PARP (Cell Signaling, UK), BAK (Cell Signaling, UK), BAX (Santa Cruz, CA, USA), HSP70 (Cell Signaling, UK), Active Caspase 3 (Cell Signaling, UK). .. Xenograft experiments For assessment of UMI-77 anti-tumour activity in vivo, 3 million MDA-MB-468 breast cancer cells were injected bilaterally into the inguinal mammary fat pads in 1:1 PBS:matrigel mix into 8-week BALB/c-Nu female mice (Charles River, UK).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Santa Cruz Biotechnology mouse anti phosphorylated jun kinase
    Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear <t>phosphorylated</t> <t>Jun-Kinase</t> (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) <t>Anti-BRDU</t> (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.
    Mouse Anti Phosphorylated Jun Kinase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phosphorylated jun kinase/product/Santa Cruz Biotechnology
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti phosphorylated jun kinase - by Bioz Stars, 2020-10
    85/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology anti c jun
    AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in <t>anti-c-Jun</t> antibody-immunoprecipited candidates, but not in <t>anti-IgG</t> antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P
    Anti C Jun, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti c jun/product/Santa Cruz Biotechnology
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    anti c jun - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    Image Search Results


    Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear phosphorylated Jun-Kinase (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) Anti-BRDU (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.

    Journal: PLoS ONE

    Article Title: Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster

    doi: 10.1371/journal.pone.0025087

    Figure Lengend Snippet: Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear phosphorylated Jun-Kinase (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) Anti-BRDU (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.

    Article Snippet: Goat anti-Vasa (1∶1000) and mouse anti-phosphorylated Jun-Kinase (1∶50) were obtained from Santa Cruz Biotechnology.

    Techniques: Mutagenesis

    AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in anti-c-Jun antibody-immunoprecipited candidates, but not in anti-IgG antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P

    Journal: Oncotarget

    Article Title: The histidine-rich calcium binding protein (HRC) promotes tumor metastasis in hepatocellular carcinoma and is upregulated by SATB1

    doi:

    Figure Lengend Snippet: AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in anti-c-Jun antibody-immunoprecipited candidates, but not in anti-IgG antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P

    Article Snippet: The purified chromatin was immunoprecipitated using 2 μg of anti-c-Jun, or irrelevant antibody anti-IgG (Santa Cruz).

    Techniques: Expressing, Luciferase, Activity Assay, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Mutagenesis, Polymerase Chain Reaction, Activation Assay

    Effects of Cf-GP on the expression levels of Tcf, LEF-1, ICAM-1, c-jun, c-myc and cyclin D. Cells were treated with Cf-GP (5, 10 or 20 μ g/ml) for 24 h. Gene expression was determined by western blot analysis and RT-PCR. (A) Western blot analysis using anti-Tcf, anti-LEF-1, anti-ICAM-1, anti-c-jun, anti-c-myc, anti-cyclin D and anti-β-actin antibodies. SDS-PAGE was performed on acrylamide gel. (B) cDNA and primers were synthesized. PCR was then performed at the indicated annealing temperatures. Reaction products were electrophoresed on a 1% agarose gel and visualized with RedSafe reagent.

    Journal: International Journal of Oncology

    Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling

    doi: 10.3892/ijo.2013.2079

    Figure Lengend Snippet: Effects of Cf-GP on the expression levels of Tcf, LEF-1, ICAM-1, c-jun, c-myc and cyclin D. Cells were treated with Cf-GP (5, 10 or 20 μ g/ml) for 24 h. Gene expression was determined by western blot analysis and RT-PCR. (A) Western blot analysis using anti-Tcf, anti-LEF-1, anti-ICAM-1, anti-c-jun, anti-c-myc, anti-cyclin D and anti-β-actin antibodies. SDS-PAGE was performed on acrylamide gel. (B) cDNA and primers were synthesized. PCR was then performed at the indicated annealing temperatures. Reaction products were electrophoresed on a 1% agarose gel and visualized with RedSafe reagent.

    Article Snippet: The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, SDS Page, Acrylamide Gel Assay, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Effect of TBH on electrophoretic mobility shift and supershift assays for AP-1 binding. Nuclear protein extracts (10 μg) were obtained from WT, F1, and F2 cells treated with TBH (60 μM for 0, 4, or 8 h), and EMSA was done as described in Materials and Methods using a consensus AP-1 probe. Panel A shows supershift analysis using anti-c-Jun antibodies, and panel B shows supershift analysis using anti-c-Fos antibodies. The arrows to the right point to complexes that were supershifted in the presence of specific antibodies. Representative EMSAs are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

    doi: 10.1128/MCB.25.14.5933-5946.2005

    Figure Lengend Snippet: Effect of TBH on electrophoretic mobility shift and supershift assays for AP-1 binding. Nuclear protein extracts (10 μg) were obtained from WT, F1, and F2 cells treated with TBH (60 μM for 0, 4, or 8 h), and EMSA was done as described in Materials and Methods using a consensus AP-1 probe. Panel A shows supershift analysis using anti-c-Jun antibodies, and panel B shows supershift analysis using anti-c-Fos antibodies. The arrows to the right point to complexes that were supershifted in the presence of specific antibodies. Representative EMSAs are shown.

    Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay

    EMSA and supershift analysis of the rat GCLC AP-1 and NF-κB sites. WT cells were treated with TBH (60 μM for 8 h) or vehicle control and subjected to EMSA with supershift analysis for the AP-1 site at −356 or the NF-κB site at −378. Supershift analysis was performed using antibodies directed against c-Jun, Nrf1, and Nrf2 for the AP-1 site (A) and p50, Nrf1, and Nrf2 for the NF-κB site (B). Note that supershift occurred only with anti-c-Jun antibodies for the AP-1 site and anti-p50 antibodies for the NF-κB site. As a positive control, TBH treatment induced Nrf1 and Nrf2 binding to the ARE site of the mouse GCLM (C). Arrows in panel C point to the Nrf1 and Nrf2 supershifts.

    Journal: Molecular and Cellular Biology

    Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

    doi: 10.1128/MCB.25.14.5933-5946.2005

    Figure Lengend Snippet: EMSA and supershift analysis of the rat GCLC AP-1 and NF-κB sites. WT cells were treated with TBH (60 μM for 8 h) or vehicle control and subjected to EMSA with supershift analysis for the AP-1 site at −356 or the NF-κB site at −378. Supershift analysis was performed using antibodies directed against c-Jun, Nrf1, and Nrf2 for the AP-1 site (A) and p50, Nrf1, and Nrf2 for the NF-κB site (B). Note that supershift occurred only with anti-c-Jun antibodies for the AP-1 site and anti-p50 antibodies for the NF-κB site. As a positive control, TBH treatment induced Nrf1 and Nrf2 binding to the ARE site of the mouse GCLM (C). Arrows in panel C point to the Nrf1 and Nrf2 supershifts.

    Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

    Techniques: Positive Control, Binding Assay

    Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

    doi: 10.1128/MCB.25.14.5933-5946.2005

    Figure Lengend Snippet: Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P

    Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

    Techniques: Western Blot