Journal: Neurogenetics

Article Title: Dysregulated expression of lipid storage and membrane dynamics factors in Tia1 knockout mouse nervous tissue

doi: 10.1007/s10048-014-0397-x

Figure Lengend Snippet: Transcriptome profiling in four Tia1 KO mice tissues at two ages identifies consistent expression dysregulations. Tia1 KO and WT mice (3 vs. 3 at age 12 and 24 weeks) were compared, the significance of expression changes was determined, and consistently dysregulated transcript levels were shown with average fold changes. Negative values represent reduced expression (with green color highlighting its significance), while positive values represent induced expression (with red color highlighting its significance). Bold values illustrates transcripts with established induction by fasting conditions. The transcripts were grouped to reflect the convergent functions of the corresponding gene products in three pathways and were shown in alphabetical order

Article Snippet: Subsequently, expression levels were measured with the StepOnePlus Real-Time PCR System (Applied Biosystems) using 25 ng cDNA, 10 μl of FastStart Universal Probe Master (Rox) Mix (04914058001, Roche), and 1 μl of one of the following TaqMan Assays (Applied Biosystems): Atxn2 (Mm01199894_m1), Bid (Mm00432073_m1), Ccnf (Mm00432385_m1), Cdkn1a (Mm00432448_m1), Cntn4 (Mm00476065_m1), Dcp1b (Mm01183995_m1), Inca1 (Mm01243670_m1), Pabpc1 (Mm00849569_s1), Plin4 (Mm01272159_m1), Pnpla2 (Mm00503046_g1), Tbc1d24 (Mm00557451_m1), Tardbp (Mm00523870_g1), Tia1 Exon 3–4 (Mm01183616_m1), Tial1 (Mm00437049_m1), Tprkb (Mm00616325_m1), Tsen2 (Mm01184390_m1), Wdfy1 (Mm00840455_m1), and Tbp (Mm00446973_m1) as endogenous control.

Techniques: Expressing, Control, Binding Assay, Membrane

Journal: Neurogenetics

Article Title: Dysregulated expression of lipid storage and membrane dynamics factors in Tia1 knockout mouse nervous tissue

doi: 10.1007/s10048-014-0397-x

Figure Lengend Snippet: Quantitative immunoblots demonstrate significantly increased levels of perilipin-4 and WDFY1, but decreased levels of CNTN4 in Tia1 KO tissue. In cerebellum of 12-week-old mice ( a ), the PLIN4 levels were elevated in the RIPA-soluble protein fraction, whereas ( b ) the WDFY1 levels were elevated in the SDS fraction and ( c ) the CNTN4 levels were decreased in the SDS fraction ( n = 4 WT vs. 5 KO mice)

Article Snippet: Subsequently, expression levels were measured with the StepOnePlus Real-Time PCR System (Applied Biosystems) using 25 ng cDNA, 10 μl of FastStart Universal Probe Master (Rox) Mix (04914058001, Roche), and 1 μl of one of the following TaqMan Assays (Applied Biosystems): Atxn2 (Mm01199894_m1), Bid (Mm00432073_m1), Ccnf (Mm00432385_m1), Cdkn1a (Mm00432448_m1), Cntn4 (Mm00476065_m1), Dcp1b (Mm01183995_m1), Inca1 (Mm01243670_m1), Pabpc1 (Mm00849569_s1), Plin4 (Mm01272159_m1), Pnpla2 (Mm00503046_g1), Tbc1d24 (Mm00557451_m1), Tardbp (Mm00523870_g1), Tia1 Exon 3–4 (Mm01183616_m1), Tial1 (Mm00437049_m1), Tprkb (Mm00616325_m1), Tsen2 (Mm01184390_m1), Wdfy1 (Mm00840455_m1), and Tbp (Mm00446973_m1) as endogenous control.

Techniques: Western Blot

Journal: BMC Cancer

Article Title: Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells

doi: 10.1186/1471-2407-11-81

Figure Lengend Snippet: Primers used for DNA methylation analysis.

Article Snippet: Total RNA extraction, cDNA synthesis and real-time PCR were carried out on the cells prepared above, by the same methods as described previously [ ]. mRNA levels of the CYP3A4 (exon 3-4, Hs01546612_m1), PXR (exon 5-6, Hs00243666_m1) and vitamin D receptor (VDR) (exon 10-11, Hs01045840_m1) were evaluated by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA).

Techniques: DNA Methylation Assay, Sequencing, Combined Bisulfite Restriction Analysis Assay

Journal: BMC Cancer

Article Title: Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells

doi: 10.1186/1471-2407-11-81

Figure Lengend Snippet: Basal expression profile of colon cancer cell lines . Basal levels of (a), PXR exon 5-6; (b), PXR exon 1a-2; (c), CYP3A4; and (d), VDR transcripts in LS180, LoVo, Caco-2, HT29, HCT116 and SW48 cells. The vertical axis indicates a relative transcript level (ratio to LS180 cells). The transcripts indicated by * (PXR exon 5-6 transcript in SW48 cells and CYP3A4 transcript in HCT116 cells) were not detected and we estimated the minimum detectable levels among gradually diluted calibration samples (0.00257 and 0.0399, respectively).

Article Snippet: Total RNA extraction, cDNA synthesis and real-time PCR were carried out on the cells prepared above, by the same methods as described previously [ ]. mRNA levels of the CYP3A4 (exon 3-4, Hs01546612_m1), PXR (exon 5-6, Hs00243666_m1) and vitamin D receptor (VDR) (exon 10-11, Hs01045840_m1) were evaluated by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells

doi: 10.1186/1471-2407-11-81

Figure Lengend Snippet: Expression profile of colon cancer cell lines after 5-aza-dC treatment . Levels of PXR, VDR and CYP3A4 transcripts in (a), LS180; (b), Caco-2; (c), HCT116; (d), LoVo; (e), HT29; and (f), SW48 cells. Cells were treated with 5-aza-2'-deoxycytidine (0, 0.5 or 5 μM). The vertical axis indicates a relative transcript level (ratio to cells without 5-aza-dC treatment). Numbers in italics indicate relative transcript levels that were over the maximum scale on the vertical axis. The transcripts indicated by * (PXR exon 5-6 transcript in SW48 cells and CYP3A4 transcript in HCT116 cells) were not detected and we estimated the minimum detectable levels among gradually diluted calibration samples.

Article Snippet: Total RNA extraction, cDNA synthesis and real-time PCR were carried out on the cells prepared above, by the same methods as described previously [ ]. mRNA levels of the CYP3A4 (exon 3-4, Hs01546612_m1), PXR (exon 5-6, Hs00243666_m1) and vitamin D receptor (VDR) (exon 10-11, Hs01045840_m1) were evaluated by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA).

Techniques: Expressing

Journal: Oncotarget

Article Title: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

doi: 10.18632/oncotarget.7857

Figure Lengend Snippet: A. IP-Western analysis of the binding of WTp53 and mutp53 to various markers distinguishing between WT and mutp53 conformations. 50ng of each purified protein was subjected for immunoprecipitation with a subset of different antibodies and proteins shown to bind differentially WTp53 or mutp53: PAb421 (binds both WT and mutant), PAb1620 (WT specific), PAb240 (mutant specific), PAb419+LTag (WT specific), biotinilated-p53RE DNA oligo (WT specific), and a control oligo mutated at two bases. Immunoprecipitated material was subjected to Western blotting using αp53-HRP as second antibody. B. Schematic diagram representing the protocol for identification, screening and selection of mutp53 reactivating peptides. The protocol consists of various selection strategies, at increasing stringencies, for screening and identifying mutp53 reactivating peptides, by utilizing phage display. Strategy A (left): Conformation-based selection: selection of peptides presented by a phage, which can bind a mutp53 protein bound to immobilized WTp53 conformation-specific antibody (PAb1620), thereby enabling selection of a bound phage capable of stabilizing WTp53 conformation. Strategy B (middle): selection according to binding: selection of peptides, which can bind a mutp53 protein bound to immobilized LT-antigen. Strategy C (right): selection according to function: selection of peptides binding to immobilized WTp53-p53RE DNA complex. C. Western blot analysis of IP with PAb1620 antibody of purified either p53 R175H (upper panel) or p53 R249S -DBD (lower panel) in the presence of selected phage pools. Non-selected phage (ns) and no phage (nt) were used as controls. Incubation was for 3 hours at 4°C. Bound p53 in the immunoprecipitate (IP) was analyzed by Western blot using antibody against p53 (αp53). “In” stands for 10% of the IP input material, loaded directly on the gel. D. Western blot analysis of IP experiments of streptavidin-coated beads bound either to p53RE-DNA or control-RE-DNA oligonucleotides labeled with biotin were incubated with purified WTp53-DBD or mutant p53 R249S -DBD in the presence of phage selected by phage display. Non selected phage (ns) were used as control. Incubation was for 3 hours at 4°C. E. A schematic illustration of several consensus peptide motifs identified as described.

Article Snippet: Plasmid #42230, containing a TP53 exon 3 single guide RNA (sgRNA), was from Addgene.

Techniques: Western Blot, Binding Assay, Purification, Immunoprecipitation, Mutagenesis, Control, Selection, Incubation, Labeling

Journal: Oncotarget

Article Title: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

doi: 10.18632/oncotarget.7857

Figure Lengend Snippet: A. Bar graph demonstrating representative ELISA experiments for determining the effect of selected peptides on the conformation of mutp53 in H1299-p53 R175H cell extract, as determined by immunoassay. Cell extracts were added to ELISA plates coated with the indicated antibodies to allow mutp53 to react with the peptides and antibodies. αp53-HRP Ab was used for assessment of p53 levels. Numbers represent ratio of absorbance between the PAb1620 and PAb240 samples. All reads were normalized to the control PAb241 reading of each extract. MCF7 (WTp53) and H1299-mutp53 A135V (tsp53) cells were used as positive controls for the WTp53 conformation (1620/240 ratio equals or exceeds 5:1). B. - Bar graph demonstrating representative ELISA experiments of determining the effect of selected peptides on the DNA binding activity of mutp53 in H1299-p53 R175H cell extract. 96 well plates were coated with anti-p53 antibody, cell extracts containing p53 were reacted with oligonucleotides that contain a p53RE consensus binding site, labeled with biotin, in the presence or absence (NT) of test peptides. Streptavidin-HRP is used to quantify the amount of oligos in the well. TMB assay was performed to determine bound mutp53 levels (450nm). MCF7 and the H1299-mutp53 A135V (TS) cells serve as positive controls for WTp53. C. and D. - Bar graphs illustrating the effect of various selected peptides on mut-p53 dependent expression on cell viability. C represents H1299 p53-null (light blue bars) and H1299 stably overexpressing mutp53 R175H (blue bars). D, ES2 cells expressing endogenous mutp53 (blue bars) and ES2 cells knocked out for p53 by CRISPR construct (light blue bars). Cell lines were treated with selected peptides, Cis-platinum was used as positive control for cell death. 48 hours after treatment, cells were washed with PBS, and the remaining attached cells were stained with Crystal violet and washed 4 times with PBS. Stained cells were dissolved in 10% acetic acid and plates were taken for optical density measurement at 595nM.

Article Snippet: Plasmid #42230, containing a TP53 exon 3 single guide RNA (sgRNA), was from Addgene.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Activity Assay, Labeling, Expressing, Stable Transfection, CRISPR, Construct, Positive Control, Staining

Journal: Oncotarget

Article Title: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

doi: 10.18632/oncotarget.7857

Figure Lengend Snippet: A. - Bar graph illustrating the effect of selected peptides on activation of mutp53 by measuring transactivation of p53 target genes as determined by qRT-PCR. H1299 cells stably transfected with mutp53 (ts) A135V were used. The indicated peptides were added directly to the medium at a concentration of 5ug/ml and cells were then either moved to 32°C or returned to 37°C. 18 hours later cells were harvested, extracted for RNA, cDNA was synthesis subjected to qrt time PCR analysis. The expression level of 3 representative p53 target genes, p21, PUMA and Mdm2, were examined. The figure illustrates the relative fold induction of transcription of the cells treated with the selected peptides as compared to non-treated cells. B. - Bar graph illustrating the effect of selected peptides on activation of mutp53 by measuring transactivation of p53 target genes as determined by qRT-PCR. ES2and ES2-p53KO cells were treated with indicated peptides 5ug/ml, for 18 hours. Cells were harvested, and cDNA was subjected to qRT-PCR analysis. Expression of 4 representative p53 target genes, p21, PUMA, Mdm2 and CD95, was examined. The figure illustrates the relative fold induction of transcription in cells treated with the selected peptides compared to non-treated cells. C. - Binding of mutp53 to promoters of representative p53 target genes in live cells, assessed by chromatin immunoprecipitation. BT-549 breast cancer cells endogenously expressing mutant p53 R249S were treated for 5 hours with a mix of 3 pCAPs - 250, 242 and 325. Cells treated with a mix of inert peptides served as a negative control. DNA cross-linked p53 was immunoprecipitated, and binding to the p53 responsive elements of the PUMA , p21, CD95 and MDM2 gene promoters was quantified by qPCR. Results were normalized to input total DNA. Cell extracts immunoprecipitated with beads without antibody (beads) served as negative controls. A genomic segment containing no p53RE served as negative control (white and gray bars). D. - ELISA analysis of the binding of WTp53 and mutp53 to lead peptides. Peptides were conjugated to the bottoms of the wells of 96 well plates, employing a commercial conjugation kit (TAKARA). Control wells were coated with the αp53 monoclonal antibodies PAb1801, PAb1620 and PAb240. Recombinant WTp53 or mutp53R175H was added to the wells and incubated with the bound peptides or antibodies. Where indicated, soluble peptides were added as competitors (+ comp), to confirm the specificity of the binding to p53. pCAP-76 served as a negative control peptide. After removal of recombinant protein, plates were washed and incubated with αp53-HRP followed by TMB and optical density determination. Results are presented as relative absorbance at 450nm. E. - Microscale thermophoresis (MST) analysis of the binding of fluorescently labeled WTp53 DBD and pCAP-250. See Materials and Methods for details.

Article Snippet: Plasmid #42230, containing a TP53 exon 3 single guide RNA (sgRNA), was from Addgene.

Techniques: Activation Assay, Quantitative RT-PCR, Stable Transfection, Transfection, Concentration Assay, Expressing, Binding Assay, Chromatin Immunoprecipitation, Mutagenesis, Negative Control, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Control, Bioprocessing, Recombinant, Incubation, Microscale Thermophoresis, Labeling

Journal: Oncotarget

Article Title: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

doi: 10.18632/oncotarget.7857

Figure Lengend Snippet: A. , C. - Apoptosis of ES2 cells treated with peptides. At the indicated time points, peptides were added directly to the medium of growing ES2 cells at a concentration of 12ug/ml. Cells were harvested and stained with Annexin FITC to detect apoptotic cells, and PI (propidium iodide) staining for dead cells. Stained cells were then analyzed by flow cytometry. A total of 10,000 cells were counted for each sample. B. , D. - Expression of p53 target genes p21, PUMA , BTG2 and CD95 in ES2 cells following peptide treatment. Either pCAP-242 (figure ) or pCAP-250 (Figure ) was added directly to the medium at a concentration of 12μg/ml. At the indicated time points cells were harvested and RNA subjected to qRT-PCR analysis.

Article Snippet: Plasmid #42230, containing a TP53 exon 3 single guide RNA (sgRNA), was from Addgene.

Techniques: Concentration Assay, Staining, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

doi: 10.18632/oncotarget.7857

Figure Lengend Snippet: A. - D. - Effect of indicated peptides in a mouse breast cancer xenograft model.10 6 MDA-MB-231 breast cancer cells, expressing endogenous mutp53 and stably expressing luciferase, were injected into the hips of nude mice. When tumors reached visible size, mice were treated by intra-tumoral injection, three times a week, with either a mixture of 3 control peptides that showed no phenotype in vitro (pCAPs 76, 77 and 12; 2μg of each peptide) or a mixture of 3 test peptides that exhibited mutp53-reactivating ability (pCAPs D60R, 24R and 174; 2μg of each peptide). A. - Live imaging of control group mice at the beginning of treatment (day 18) and at termination of the experiment (day 30). B. - Live imaging of mice treated with effective peptide mix at the beginning of treatment (day 18) and at termination of the experiment (day 30). C. - (control group mice) and D. (effective pCAP mix group): Logarithmic scale box-plot showing the luciferase readings in tumors as a function of time; average (horizontal line), standard deviation (box), highest and lowest reads (error bars) are shown, before (until day 18) and after initiation of treatment. The background threshold detection level of the IVIS system in this experiment was about 5×10 6 photons. E. - Box-plot of excised tumor weights at termination of the experiment. Average weight (horizontal line), standard deviation (box), highest and lowest reads are shown. F. - Western blot analysis of two p53 target gene products, p21 and MDM2. Part of the excised tumors of mice #7 and #8 treated with control peptides and mouse #3 treated with effective peptide mix were homogenized and lysed. Protein concentration was determined using Bio-Rad reagent. 50μg protein of each sample was loaded and subjected to Western blot analysis with antibodies against p21 and MDM2. G. - K. - In-vivo effect of indicated peptides in a mouse xenograft model. 5*10 5 ES2 cells expressing luciferase were injected into the hips of nude mice. Bioluminescence was measured. 10 days after injection, mice were randomly divided to 2 groups and injected intratumorally, three times a week, with either a mixture of 2 control peptides (pCAPs 76 and 12; 5μg of each peptide) or pCAP-250 (10μg). G. , H. - Live imaging of control group mice and pCAP-250 treated mice, respectively, at the beginning of treatment (day 0) and at termination of experiment (day 18). I. (control mice) and J. (effective pCAP-250 group): box-plot showing the luciferase readings in tumors as a function of time; average (horizontal line), standard deviation (box), highest and lowest reads are shown, before (until day 0) and after initiation of treatment. The background threshold detection level of the IVIS system was about 5×10 6 photons. K. - Box-plot of excised tumor weights at termination of the experiment. Average weight (horizontal line), standard deviation (box), highest and lowest reads are shown. L. - Live imaging of a single mouse treated with pCAP-250 over the indicated time points.

Article Snippet: Plasmid #42230, containing a TP53 exon 3 single guide RNA (sgRNA), was from Addgene.

Techniques: Expressing, Stable Transfection, Luciferase, Injection, Control, In Vitro, Imaging, Standard Deviation, Western Blot, Protein Concentration, In Vivo

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Protein expression of NOX2 in different tissues. ( A ) Western blot analysis with different tissues from wt (+/+) and NOX2ko (−/−) mice (protein/lane: 25 µg RAW cell (as the NOX2 positive control) and 25 µg aorta, 40 µg heart and lung). Signals for NOX2 were observed at 58 kDa in wildtype mice, and additionally as smaller sized bands in NOX2ko mice, especially with stronger intensity noticed in the heart. Additionally, the ≈ 30 kDa NOX2β isoform, as described by Harrison et al. , was probably detected in lung tissues. Loading and transfer of proteins was assessed by Ponceau S staining of the nitrocellulose membrane. ( B ) Immunohistochemical staining for pulmonary NOX2 expression. Signals for NOX2 were observed in lungs of wildtype and NOX2ko mice (brown color). The nuclei were stained by a blue color. Representative microscopy images are shown for two control (wildtype) and three NOX2ko mice. Magnification is 20× and 40×.

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Expressing, Western Blot, Positive Control, Staining, Membrane, Immunohistochemical staining, Microscopy, Control

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Analysis of the NOX2 mRNA expression by qRT-PCR ( A , B ) or NGS-analyses ( C ). ( A ) Shown is the statistical analysis of qRT-PCR experiments performed with RNA isolated from the brain of NOX2 wt (NOX2wt) or NOX2ko (NOX2ko) animals using total brain homogenates and the primer-probe-mixes purchased from Applied Biosystems. The relative NOX2 mRNA expression in NOX2wt animals was set to 100%. Shown are the mean ± SEM of n = 6–8 experiments (*** p < 0.001 vs. NOX2wt; two tailed unpaired t -test). ( B ) Shown is the statistical analysis of qRT-PCR experiments performed with RNA isolated from the brain of NOX2 wt (NOX2wt) or NOX2ko (NOX2ko) animals using total liver homogenates and the primers qRT-PCR-5P2/3P2 shown in the Materials and Methods section. The relative NOX2 mRNA expression in NOX2wt animals was set to 100%. Shown are the mean ± SEM of n = 8 experiments (* p < 0.05 vs. NOX2wt; two tailed unpaired t -test). ( C ) Shown is the statistical analysis of the mRNA expression analyzed using the NGS data published by Adane et al. (, GEO: GSE117657). The RNAs were isolated from leukemia-initiating stem cells. The rpkm (reads per kilo base per million mapped reads) data of the NOX2 wt (NOX2wt) animals were set to 100%. Shown are the mean ± SEM of n = 2 experiments (* p < 0.05 vs. NOX2wt; two tailed unpaired t -test).

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Two Tailed Test

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Structure of the murine wildtype NOX2 gene ( A ) and the inactivated NOX2 gene ( B ) in NOX2ko mice. ( A ) Shown is a schematic picture of the murine wildtype NOX2 (gp91phox) gene (mouse Chromosome X, NC_000086.7 (9,435,252.9487766, complement)). ( B ) Shown is a schematic picture of the inactivated NOX2 gene (as describe by ). ATG = translational start codon; Cybb = cybb/NOX2/gp91phox gene sequence; ex = exon; ko = inactivated gene, knockout; neoR = neomycin resistance gene; PGK1polyA = mouse phosphoglycerate kinase 1 gene polyadenylation signal; PGK1 promoter = mouse phosphoglycerate kinase 1 gene promoter sequence; wt = wildtype.

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Sequencing, Gene Knockout

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Structure of the mRNAs encoded by the murine wt NOX2 gene ( A ) and the inactivated NOX2 gene ( B ) in NOX2ko mice. ( A ) Shown is a schematic picture of the mRNA encoded by the murine wildtype NOX2 (gp91phox) gene (NM_007807). ( B ) Shown is a schematic picture of the mRNA encoded by the inactivated NOX2 gene. 5′-end wt-prot = sequence of the putative small 5′-part of the wt NOX2 protein; ATG = translational start codon; ex = exon; Mm00432775_m1 = location of the PCRF generated in the qRT-PCR assays using RNA isolated from brain tissues; neoR = neomycin resistance gene; NOX2ko_f and NOX2ko_r = primer used for qualitative RT-PCR analyses detecting the mutated NOX2 mRNA only; NOX2ko_prot = sequence of the fusion protein encoded by the mutated NOX2 mRNA; NOX2wt_f and NOX2wt_r = primer used for qualitative qRT-PCR analyses, detecting both the wt and mutated NOX2 mRNA; PGK1 promoter = mouse phosphoglycerate kinase gene promoter sequence; qRT-PCR-5P2 and -3P2 = primers used for quantitative RT-PCR with RNA isolated from liver tissues; seq = sequenced part of the mRNA; UTR = untranslated region; wt = wildtype.

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Sequencing, Generated, Quantitative RT-PCR, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Alignment of sequences of the proteins encoded by the NOX2wt and NOX2ko mRNA. Additionally, the consensus amino acid sequence is shown. Shown is a picture of the alignment of the wt and mutated NOX2 mRNAs. The wt NOX2 mRNA encodes a protein with 571 amino acids, whereas the NOX2ko mRNA codes for a 507 amino acid protein (uppermost sequence = NOX2ko; middle sequence = wt; lowest sequence = consensus).

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Sequencing

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Qualitative RT-PCR reaction using RNAs isolated from liver from NOX2wt and NOX2ko animals. ( A ) Shown is a representative picture of an agarose gel analyzing the RT-PCR products using RNAs isolated from the liver of NOX2 wildtype (wt) and NOX2ko (ko) animals. The RT reaction was performed with oligo-dT primers. The PCR reaction was performed with the primers NOX2wt_f and NOX2wt_r (see B for the location of the primers). This primer pair resulted in a 395 bp DNA fragment only in PCR reactions using RNA from wt animals. ( B ) Shown is a representative picture of an agarose gel analyzing the RT-PCR products using RNAs isolated from the liver of NOX2 wildtype (wt) and NOX2ko (ko) animals. The RT reaction was performed with oligo-dT primers. The PCR reaction was performed with the primers NOX2ko_f and NOX2ko_r (see B for the location of the primers). This primer pair resulted in a 306 bp DNA fragment only in PCR reactions using RNA from ko animals. c = control reaction without RNA; ko = RNA from NOX2 ko animals; M = DNA marker; wt = wildtype.

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Control, Marker

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Alignment of NGS reads to the mRNA expressed in NOX2ko animals. ( A ) Shown is a schematic picture of the mRNA encoded by the inactivated NOX2 gene (as described by , which is the same as B). ( B ) Shown is a picture of the alignment of NGS reads, obtained from RNA, isolated from leukemia-initiating stem cells, obtained from NOX2ko animals (, GEO: GSE117657) to the mRNA sequence of the mutated NOX2 gene in NOX2ko animals. The NGS sequence reads published by Adane et al. (GEO: GSE117657) were analyzed with a CLC genomics workbench (QIAGEN, version 20.0.4), as described by the manufacturer (using the preinstalled parameters). The reads were aligned to the NOX2ko mRNA sequence using the “Map reads to reference” algorithm. Shown are reads with >99% identity. The black rectangle are reads of the upper strand, whereas the gray rectangles are reverse reads (lower strand). 5′-end wt-prot = sequence of the putative small 5′-part of the wt NOX2 protein; ATG = translational start codon; ex = exon; Mm00432775_m1 = location of the PCR fragment generated in the qRT-PCR assays using RNA isolated from brain tissues; neoR = neomycin resistance gene; NOX2ko_f and NOX2ko_r = primer used for qualitative RT-PCR analyses detecting the mutated NOX2 mRNA only; NOX2ko_prot = sequence of the fusion protein encoded by the mutated NOX2 mRNA; NOX2wt_f and NOX2wt_r = primer used for qualitative qRT-PCR analyses, detecting both the wt and mutated NOX2 mRNA; PGK1 promoter = mouse phosphoglycerate kinase gene promoter sequence; qRT-PCR-5P2 and -3P2 = primers used for quantitative RT-PCR with RNA isolated from liver tissues; seq = sequenced part of the mRNA; UTR = untranslated region; wt = wildtype.

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Isolation, Sequencing, Generated, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants

Article Title: NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

doi: 10.3390/antiox9111043

Figure Lengend Snippet: Analysis of the NOX2 mRNA expression in mice with the inactivated p47phox gene by qRT-PCR. ( A ) Shown is the statistical analysis of qRT-PCR experiments performed with RNA isolated from the liver of NOX2 wt or animals with the inactivated p47phox gene (p47phox ko) using total liver homogenates and the primers qRT-PCR-5P2/3P2 shown in the Materials and Methods section. The relative NOX2 mRNA expression in NOX2wt animals was set to 100%. Shown are the mean ± SEM of n = 6–8 experiments (** p < 0.01 vs. NOX2wt; two-sided unpaired t -test). ( B ) Shown is the statistical analysis of qRT-PCR experiments performed with RNA isolated from the spleen of NOX2 wt (NOX2wt) or animals with inactivated p47phox gene (p47phox ko), using total spleen homogenates and the primers qRT-PCR-5P2/3P2 shown in the Materials and Methods section. The relative NOX2 mRNA expression in NOX2wt animals was set to 100%. Shown are the mean ± SEM of n = 6 experiments (* p < 0.05 vs. NOX2wt; two tailed unpaired t -test).

Article Snippet: Primer-Probe-Mixes purchased from Applied Biosystems (Foster City, CA) were used to analyze the mRNA expression patterns of NADPH oxidase (NOX-2 Mm00432775_m1, binds to exon 3 and 4 of the mRNA, see figures below) and normalized on the TATA box binding protein (TBP, Mm_00446973_m1) as an internal control.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Two Tailed Test

Journal: iScience

Article Title: Mice deficient in TWIK-1 are more susceptible to kainic acid-induced seizures

doi: 10.1016/j.isci.2024.111587

Figure Lengend Snippet:

Article Snippet: Exon 3 targeting Twik-1 shRNA (5′-GGAAGATGTTCTACGTG-AAGA-3′) was obtained through oligonucleotide-directed mutagenesis (Enzynomics).

Techniques: Sequencing