Journal: American Journal of Human Genetics

Article Title: Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

doi: 10.1016/j.ajhg.2017.07.010

Figure Lengend Snippet: Mutations in TBC1D23 Cause PCH

Article Snippet: Three different 29-mer shRNA sequences targeting mouse Tbc1d23 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_026254","term_id":"27754078","term_text":"NM_026254"}} NM_026254 ) commercially designed and provided by Origene were cloned in the psiStrike vector under the control of a U6 promoter: shRNA-1, targeting the sequence 5′-AGAATCCATCTGAGTTTGCACAGTCAGT-3′, specific for both mouse and human TBC1D23 ; shRNA-2, targeting the sequence 5′-TTACATCCAGTCTCGGCAAGCACTAAATT-3′ and specific for mouse Tbc1d23 ; and shRNA-3, targeting the sequence 5′-TGTTAGCATTGCCAGTGGAGGATTTATGG-3′, also specific for mouse Tbc1d23 .

Techniques:

Journal: American Journal of Human Genetics

Article Title: Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

doi: 10.1016/j.ajhg.2017.07.010

Figure Lengend Snippet: Representation of the TBC1D23 Polypeptide

Article Snippet: Three different 29-mer shRNA sequences targeting mouse Tbc1d23 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_026254","term_id":"27754078","term_text":"NM_026254"}} NM_026254 ) commercially designed and provided by Origene were cloned in the psiStrike vector under the control of a U6 promoter: shRNA-1, targeting the sequence 5′-AGAATCCATCTGAGTTTGCACAGTCAGT-3′, specific for both mouse and human TBC1D23 ; shRNA-2, targeting the sequence 5′-TTACATCCAGTCTCGGCAAGCACTAAATT-3′ and specific for mouse Tbc1d23 ; and shRNA-3, targeting the sequence 5′-TGTTAGCATTGCCAGTGGAGGATTTATGG-3′, also specific for mouse Tbc1d23 .

Techniques:

Journal: American Journal of Human Genetics

Article Title: Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

doi: 10.1016/j.ajhg.2017.07.010

Figure Lengend Snippet: Clinical and Radiological Features of Individuals with TBC1D23 Mutations

Article Snippet: Three different 29-mer shRNA sequences targeting mouse Tbc1d23 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_026254","term_id":"27754078","term_text":"NM_026254"}} NM_026254 ) commercially designed and provided by Origene were cloned in the psiStrike vector under the control of a U6 promoter: shRNA-1, targeting the sequence 5′-AGAATCCATCTGAGTTTGCACAGTCAGT-3′, specific for both mouse and human TBC1D23 ; shRNA-2, targeting the sequence 5′-TTACATCCAGTCTCGGCAAGCACTAAATT-3′ and specific for mouse Tbc1d23 ; and shRNA-3, targeting the sequence 5′-TGTTAGCATTGCCAGTGGAGGATTTATGG-3′, also specific for mouse Tbc1d23 .

Techniques: Expressing

Journal: American Journal of Human Genetics

Article Title: Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

doi: 10.1016/j.ajhg.2017.07.010

Figure Lengend Snippet: Effects of TBC1D23 Downregulation on Neuronal Migration and Positioning

Article Snippet: Three different 29-mer shRNA sequences targeting mouse Tbc1d23 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_026254","term_id":"27754078","term_text":"NM_026254"}} NM_026254 ) commercially designed and provided by Origene were cloned in the psiStrike vector under the control of a U6 promoter: shRNA-1, targeting the sequence 5′-AGAATCCATCTGAGTTTGCACAGTCAGT-3′, specific for both mouse and human TBC1D23 ; shRNA-2, targeting the sequence 5′-TTACATCCAGTCTCGGCAAGCACTAAATT-3′ and specific for mouse Tbc1d23 ; and shRNA-3, targeting the sequence 5′-TGTTAGCATTGCCAGTGGAGGATTTATGG-3′, also specific for mouse Tbc1d23 .

Techniques: Migration

Journal: American Journal of Human Genetics

Article Title: Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

doi: 10.1016/j.ajhg.2017.07.010

Figure Lengend Snippet: TBC1D23 Truncating Mutation Alters Intracellular Transport

Article Snippet: Three different 29-mer shRNA sequences targeting mouse Tbc1d23 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_026254","term_id":"27754078","term_text":"NM_026254"}} NM_026254 ) commercially designed and provided by Origene were cloned in the psiStrike vector under the control of a U6 promoter: shRNA-1, targeting the sequence 5′-AGAATCCATCTGAGTTTGCACAGTCAGT-3′, specific for both mouse and human TBC1D23 ; shRNA-2, targeting the sequence 5′-TTACATCCAGTCTCGGCAAGCACTAAATT-3′ and specific for mouse Tbc1d23 ; and shRNA-3, targeting the sequence 5′-TGTTAGCATTGCCAGTGGAGGATTTATGG-3′, also specific for mouse Tbc1d23 .

Techniques: Mutagenesis

Journal: American Journal of Human Genetics

Article Title: Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development

doi: 10.1016/j.ajhg.2017.07.010

Figure Lengend Snippet: TBC1D23 Downregulation Alters Neurite Outgrowth

Article Snippet: Three different 29-mer shRNA sequences targeting mouse Tbc1d23 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_026254","term_id":"27754078","term_text":"NM_026254"}} NM_026254 ) commercially designed and provided by Origene were cloned in the psiStrike vector under the control of a U6 promoter: shRNA-1, targeting the sequence 5′-AGAATCCATCTGAGTTTGCACAGTCAGT-3′, specific for both mouse and human TBC1D23 ; shRNA-2, targeting the sequence 5′-TTACATCCAGTCTCGGCAAGCACTAAATT-3′ and specific for mouse Tbc1d23 ; and shRNA-3, targeting the sequence 5′-TGTTAGCATTGCCAGTGGAGGATTTATGG-3′, also specific for mouse Tbc1d23 .

Techniques:

Journal: Frontiers in Oncology

Article Title: Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation

doi: 10.3389/fonc.2022.958155

Figure Lengend Snippet: TRIAP1 overexpression supports HCT116 cancer cell proliferation and tumorigenesis. (A–E) HCT116 cells were transfected with the control (pCtrl) or TRIAP1-overexpressing (pTRIAP1) plasmids. (A) Control (pCtrl) and TRIAP1-overexpressing (pTRIAP1) cells were seeded (10 4 cells) in standard culture medium, and their proliferation rate was determined every day by manual counting. Data are represented as mean ± SEM of triplicates. (B) Similar cells were analyzed (crystal violet staining) for their capacity to form colonies at low density (clonogenic assay). (C) Monitoring of the proliferation rate of control (pCtrl) and TRIAP1-overexpressing (pTRIAP1) HCT116 cells using flow cytometry and the vital lipophilic fluorescent dye PKH67, which is lost upon cell proliferation. One day after staining with PKH67, cells were analyzed by flow cytometry on the indicated days. At day 0 (16 h after staining), all the cells have a high intensity of fluorescence (PKH67 High). The decrease in fluorescence (PKH67 Low), which is observed at the indicated time points, correlates with the proliferation of the labeled cells. The separation of the two populations (PKH67 High and PKH67 Low) is indicated with a red bar. (D) Quantification of data in (C) . Data are represented as mean ± SEM of triplicates. (E) Growth curves of HCT116 tumors developed as xenografts in athymic mice. Nude mice were subcutaneously injected with 5 × 10 6 HCT116 cells transfected with the control (pCtrl) or TRIAP1-overexpressing (pTRIAP1) plasmid. On the indicated days post injection, tumor volumes were measured as described in the Material and Methods section. Data are represented as the mean ± SEM. For statistical comparison, Student’s t -tests were performed *p < 0.05; ***p < 0.001.

Article Snippet: The following primer couples are used for qPCR analysis: for TRIAP1 mRNA (OriGene): 5′-CGCTGGTTCGCCGAGAAATTTC-3′ (forward) and 5′-TGAAGGACTGGAGTTCATGGGC-3′ (reverse).

Techniques: Over Expression, Transfection, Staining, Clonogenic Assay, Flow Cytometry, Fluorescence, Labeling, Injection, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation

doi: 10.3389/fonc.2022.958155

Figure Lengend Snippet: TRIAP1 depletion impairs HCT116 cancer cell proliferation and tumorigenesis. (A) Proliferation rate of HCT116 cells transduced with lentiviral control (Ctrl) and TRIAP1 (TRIAP1 #1, #2, and #3) shRNAs was monitored using flow cytometry and the vital lipophilic fluorescent dye PKH67 (similar to 1C), which is lost upon cell proliferation. On the indicated day, triplicate samples of PKH67-labeled HCT116 cells were analyzed and the % of “PKH67 Low” population was calculated compared with day 0. Data are represented as mean ± SEM of triplicate. (B) Representative images of colony-forming assay for HCT116 cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, and #3) shRNAs. Cells were seeded at the indicated densities, and 11 days post plating, colonies were stained with crystal violet. Note the amelioration of the colony-forming capacity of TRIAP1 knocked down cells at higher cell density. (C) Effect of TRIAP1 depletion on cell proliferation. HCT116 cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, #3) shRNAs were seeded in 48-well plates (7 × 10 3 cells/well; left panel or 25 × 10 3 cells/panel; right panel). Cell proliferation was monitored in real time, for the indicated number of days, using the label-free Incucyte Live-Cell Analysis system. Changes in cell confluence are used as an indicator of cell proliferation. Data are represented as the mean ± SD. Note the amelioration of the proliferative capacity of TRIAP1 knocked-down cells at higher cell density. (D) Growth curves of HCT116 tumors developed as xenografts in athymic mice. Nude mice were subcutaneously injected with 5 × 10 6 HCT116 cells transduced with the control (Ctrl) or TRIAP1 (TRIAP1 #1 and #3) shRNAs. On the indicated days post injection, tumor volumes were determined as described in the Material and Methods section. Data are represented as the mean ± SEM. For statistical comparison, ANOVA tests were performed, *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: The following primer couples are used for qPCR analysis: for TRIAP1 mRNA (OriGene): 5′-CGCTGGTTCGCCGAGAAATTTC-3′ (forward) and 5′-TGAAGGACTGGAGTTCATGGGC-3′ (reverse).

Techniques: Transduction, Flow Cytometry, Labeling, Staining, Injection

Journal: Frontiers in Oncology

Article Title: Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation

doi: 10.3389/fonc.2022.958155

Figure Lengend Snippet: Impact of TRIAP1 depletion on mitochondrial structure and function. (A) Mitochondrial structure analysis of HCT116 cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, #3) shRNAs. Transmission electron microscopy (TEM) images were analyzed for mitochondrial ultrastructure. N = nucleus. Scale bar = 1 µm. Examples of elongated mitochondria are shown with a red arrow. (B) TEM images were used for the calculation of mitochondria number/µm 2 of cytoplasm surface area and aspect factor (major axis/minor axis; reflecting length/width of mitochondria). Statistical analyses were performed using Mann–Whitney non-parametric tests, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Monitoring of the activity of specific segments of the respiratory chain in HCT116 cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, #3) shRNAs and cultured in standard medium. Succinate cytochrome c reductase (SCCR; complexes CII and CIII); malonate-sensitive succinate quinone DCPIP reductase (SQDR; complex CII); decylubiquinol cytochrome c reductase (QCCR; complex CIII); glycerol-3-phosphate quinone DCPIP reductase (GQDR; G3PDH); cyanide-sensitive cytochrome c oxydase (complex CIV); rotenone-sensitive NADH quinone reductase (complex CI); oligomycin-sensitive ATPase (complex CV). (D) Measurement of ATP levels. Average ATP levels were quantified in HCT116 cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, #3) shRNAs and cultured in standard medium. For statistical comparison, Kruskal–Wallis tests were performed. *p < 0.05 is considered significant (ns, non-significant).

Article Snippet: The following primer couples are used for qPCR analysis: for TRIAP1 mRNA (OriGene): 5′-CGCTGGTTCGCCGAGAAATTTC-3′ (forward) and 5′-TGAAGGACTGGAGTTCATGGGC-3′ (reverse).

Techniques: Transduction, Transmission Assay, Electron Microscopy, MANN-WHITNEY, Activity Assay, Cell Culture

Journal: Frontiers in Oncology

Article Title: Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation

doi: 10.3389/fonc.2022.958155

Figure Lengend Snippet: Impact of TRIAP1 depletion on cellular lipidome and metabolome. (A) Lipidomics analysis of HCT116 cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, #3) shRNAs. Relative abundance of indicated lipid classes is expressed as the fold change in combined TRIAP1 shRNA (#1, #2, and #3) treated cells relative to control cells. Data are represented as the mean ± SEM (Ctrl n = 6 vs. TRIAP1 n = 18). Mann–Whitney tests are used for statistical comparisons *p < 0.05. Cer, ceramides; Chol.Ester, cholesteryl esters; CL, cardiolipins; DG, diacylglycerols; DiHexCer, dihexosylceramides; FA, free fatty acids; FA.Carn, fatty acyl carnitines; HexCer, hexosylceramides; LPC, lyso-glycerophosphocholines; LPE, lyso-glycerophosphoethanolamine; LPG, lyso-glycerophosphoinositols; LPI, lyso-glycerophosphoinositols; LPS, lyso-glycerophosphoserines; PC, glycerophosphocholine; PE, glycerophosphoethanolamines; PFAA, primary amides; PG, glycerophosphoglycerols; PI, glycerophosphoinositols; PS, glycerophosphoserines; SGL, gangliosides; SM, sphingomyelins; Sphing. Base, sphingoid bases; TG, triacylglycerols; Su, sulfoglycosphingolipids; Sterol, sterols. (B) Untargeted metabolomic profiling of HCT116 cells transduced with lentiviral control (Ctrl) and TRIAP1 (TRIAP1#1, #2, #3) shRNAs and grown in standard growth conditions. Heatmap showing significantly different intracellular metabolites (Adj p-value <0.1; univariate non-parametric Kruskal–Wallis test, Benjamini–Hochberg correction). Data are represented as –log 2 (Fold Change) related to control cells. (C) Metabolite Set Enrichment Analysis (MSEA) of the significantly altered metabolites (Adj p-value <0.1) in TRIAP1-depleted HCT116 cells was performed using MetaboAnalyst 5.0 and KEGG open database . Significantly enriched pathways (p-value <0.05) are represented.

Article Snippet: The following primer couples are used for qPCR analysis: for TRIAP1 mRNA (OriGene): 5′-CGCTGGTTCGCCGAGAAATTTC-3′ (forward) and 5′-TGAAGGACTGGAGTTCATGGGC-3′ (reverse).

Techniques: Transduction, shRNA, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation

doi: 10.3389/fonc.2022.958155

Figure Lengend Snippet: TRIAP1 depletion activates a p53-related stress response. (A) Transcriptomics analysis of HCT116 cells transduced with control (ctrl#1, #2) and TRIAP1 (TRIAP1 #1, #2, #3) shRNAs. Heat map and hierarchical clustering based on genes that are differentially expressed between TRIAP1-depleted (shRNA TRIAP1 #1, #2, #3) and control (shRNA Ctrl #1, #2) cells analyzed by RNA-seq, 6 days post transduction (adjusted p value < 0.05). Genes are listed by row and analyzed samples by column. The color intensity represents the Z score, where green corresponds to upregulated and red downregulated expression. (B) Top enriched biological processes (top panel). The differentially expressed genes were analyzed for Gene Ontology (GO) using the “ToppGene Suite” . The top GO terms for biological processes are represented with a bar plot with most significant p-values (Adj. p value <0.07). Top enriched signaling and functional pathways (bottom panel). Significantly enriched pathway term analysis for differentially expressed genes in relation to TRIAP1 depletion was carried out using the online PANTHER website . (C) mRNA expression analyses of several P53 target genes identified as differentially expressed during transcriptomics studies performed in (A) qPCR studies were realized for the indicated mRNA. Relative expression fold variation was calculated for TRIAP1-depleted cells (shRNA TRIAP1 #1, #2, #3) compared with control (shRNA Ctrl) cells, using the comparative Ct method (after normalization against actin mRNA). Data represent mean ± SEM of three experiments. Kruskal–Wallis tests were used for statistical comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ns, non significant. (D) Immunoblot analysis of extracts of HCT116 cells transduced with lentiviral control (Ctrl) and TRIAP1 (TRIAP1 #1, #2, #3) shRNAs and grown in standard growth conditions. Actin was used as a loading control.

Article Snippet: The following primer couples are used for qPCR analysis: for TRIAP1 mRNA (OriGene): 5′-CGCTGGTTCGCCGAGAAATTTC-3′ (forward) and 5′-TGAAGGACTGGAGTTCATGGGC-3′ (reverse).

Techniques: Transduction, shRNA, RNA Sequencing Assay, Expressing, Functional Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation

doi: 10.3389/fonc.2022.958155

Figure Lengend Snippet: TRIAP1 depletion exacerbates a p53-dependent adaptation to glutamine deprivation. (A) Clonogenic assay of HCT116 p53 +/+ or p53 -/- transduced with lentiviral control (shRNA Ctrl) or TRIAP1 (shRNA TRIAP1#1, #2, #3) shRNAs. Cells were seeded at a density of 500 cells/well in six-well plates. Seven days after, cells were stained with crystal violet and the number of clones was counted. Bar charts show the percentage of counted colonies relative to control cells. Data are represented as mean ± SEM (n = 3). Wilcoxon tests were used for statistical comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. (B) HCT116 cells p53 +/+ (left panel) or p53 -/- (right panel) were transduced with lentiviral control (Ctrl) and TRIAP1 (TRIAP1 #1, #2, and #3) shRNAs. Five days after transduction, cells were seeded in complete medium. After 24 h (day 0), the medium was replaced by the starvation one (10 mM glucose, 1 mM pyruvate, no glutamine). The cell proliferation rate was determined every 2 days by manual counting and represented as percentage of day 0. Data are shown as mean ± SEM (n = 6). Kruskal–Wallis tests were used for statistical comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Immunoblot analysis of extracts of HCT116 P53 +/+ cells transduced with lentiviral control (Ctrl) and TRIAP1 (TRIAP1 #1, #2, #3) shRNAs and grown in glutamine starvation conditions for the indicated times. (D) Intracellular glutamate levels were quantified in HCT116 P53 +/+ cells transduced with lentiviral control (Ctrl) or TRIAP1 (TRIAP1 #1, #2, #3) shRNAs and cultured in glutamine starvation medium for 72 h. Data are represented as mean ± SEM (n = 3) of the fold change related to control cells. Kruskal–Wallis tests were used for statistical comparisons. **p < 0.01, ***p < 0.001.

Article Snippet: The following primer couples are used for qPCR analysis: for TRIAP1 mRNA (OriGene): 5′-CGCTGGTTCGCCGAGAAATTTC-3′ (forward) and 5′-TGAAGGACTGGAGTTCATGGGC-3′ (reverse).

Techniques: Clonogenic Assay, Transduction, shRNA, Staining, Clone Assay, Western Blot, Cell Culture

Journal:

Article Title: A mutation in a mitochondrial transmembrane protein is responsible for the pleiotropic hematological and skeletal phenotype of flexed-tail ( f/f ) mice

doi: 10.1101/gad.873001

Figure Lengend Snippet: Genetic and physical maps of the flexed-tail region. (A) Genetic map of the f locus on mouse chromosome 13 determined from 1000 CAST/Ei backcross animals. PCR polymorphism markers are listed above, and recombination distances in centimorgans (cM) are below. (B) Bacterial artificial chromosome (BAC) contig spanning the f critical genetic interval. Individual sequence tagged sites (STSs) present on each BAC are indicated by black diamonds. STSs mapped in the backcross are indicated by a dashed line. (C) The intron/exon structure of the Sfxn1 gene. The gene spans approximately 35 kb and is divided into 11 exons (gray boxes). Exon 2, which was nonrecombinant with the phenotype in the backcrosses and contains the f mutation, is indicated by an asterisk. The position on the physical map of the centromeric flanking markers J2T7 and 506Hd28T7 are indicated by a single arrow and a double arrow, respectively.

Article Snippet: Northern blotting was performed on an adult mouse multitissue Northern blot (Origene Technologies) by sequential hybridization with radiolabeled probes of the entire open reading frames of mouse Sfxn1, Sfxn2, Sfxn3 , Sfxn4 , and β-actin.

Techniques: Sequencing, Mutagenesis

Journal:

Article Title: A mutation in a mitochondrial transmembrane protein is responsible for the pleiotropic hematological and skeletal phenotype of flexed-tail ( f/f ) mice

doi: 10.1101/gad.873001

Figure Lengend Snippet: Western blotting and subcellular localization of Sfxn1. (A) Western blots from HEK293T cells transfected with epitope-tagged (FLAG) wild-type Sfxn1 cloned in a sense and antisense (α-sense) orientation, as well as the f frameshift mutation (f mut), and on kidney tissue extracts from wild-type (+/+) and f/f animals on a C57BL/6J background. The FLAG-tagged protein runs as a doublet with an apparent molecular mass 4–6 kD greater than the endogenous human or mouse proteins. (B) HEK293T cells were transfected with FLAG-tagged Sfxn1 and pulsed with MitoTracker Red CMXRos (MTR). Colocalization of the epitope-tagged protein and mitochondria is seen as yellow fluorescence in the red and green merged image (MTR + FLAG). (C) Western blots of whole cell extracts (c) and purified mitochondria (m) from transfected HEK293T cells using antibodies against the cytoplasmic enzyme GAPDH, and the mitochondrial proteins porin and cytochrome C (Cyt C).

Article Snippet: Northern blotting was performed on an adult mouse multitissue Northern blot (Origene Technologies) by sequential hybridization with radiolabeled probes of the entire open reading frames of mouse Sfxn1, Sfxn2, Sfxn3 , Sfxn4 , and β-actin.

Techniques: Western Blot, Transfection, Clone Assay, Mutagenesis, Fluorescence, Purification

Journal: BMC Biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: The identification of the 8F1 cross-reactive protein with protein microarray chip and Western blot confirmation. A . Protein microarray hybridization with 8F1. The OriGene overexpression protein microarray chip was immunostained with the most commonly used 8F1 monoclonal anti-ERCC1 antibody. The positive reactive proteins are highlighted with red arrows. These data show that 8F1 recognizes not only its specific target (two ERCC1 transcript variants), but also another unrelated nuclear membrane protein PCYT1A. A number of internal controls were also labeled on the panel. B . Western Blot analysis. Seven OriGene VERIFY™ overexpression lysate antigen standards (Lane 1 to 7) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (Upper panel). The recombinant protein expression levels within the lysates were analyzed with anti-DDK antibody (Lower panel). Lanes 1 to 7 are loaded with samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and PCYT1A (NM_005017).

Article Snippet: TissueScan 24 lung cancer panels (HLRT104, OriGene Technologies) were used to examine ERCC1 and PCYT1A mRNA expression profiles.

Techniques: Microarray, Western Blot, Hybridization, Over Expression, Labeling, SDS Page, Recombinant, Expressing

Journal: BMC Biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: 8F1 immunoblot analyses with purified proteins and antigen absorption test. A . Coomassie staining of purified ERCC1 and PCYT1A proteins. 1ug of affinity purified recombinant ERCC1 and PCYT1A proteins were fractionated on the SDS-PAGE gel and then commassie-stained. B . Immunoblot analysis with 8F1 anti-ERCC1 mAb.0.5ug of purified ERCC1 (Lane 2) and PCYT1A (Lane 4) were loaded on an SDS-PAGE gel and then immunobloted with 8F1 antibody. Empty vector transfected HEK293T cell lysates (Lanes 1 and 3) were used as a negative control. C . Antigen absorption test. Overexpression lysates for PCYT1A (Lane1), ERCC1 (Lane 3), and empty vector transfected control (Lanes 2 and 4) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (upper panel) or 8F1 pre-depleted with purified PCYT1A protein (lower panels).

Article Snippet: TissueScan 24 lung cancer panels (HLRT104, OriGene Technologies) were used to examine ERCC1 and PCYT1A mRNA expression profiles.

Techniques: Western Blot, Purification, Staining, Affinity Purification, SDS Page, Plasmid Preparation, Transfection, Negative Control, Over Expression

Journal: BMC Biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: The immunohistochemistry staining on NSCLC tissue sections with rabbit monoclonal anti-PCYT1A antibody or 8F1 mouse monoclonal anti-ERCC1 antibody. Pathologist-validated NSCLC FFPE tissue blocks were cut into thin sections, and then further deparaffinized, re-hydrated before IHC testing. The primary antibodies (Left: rabbit anti-PCYT1A mAb, Right: 8F1 anti-ERCC1 mAb) used for these experiments were diluted at 1:150 dilution with blocking buffer.

Article Snippet: TissueScan 24 lung cancer panels (HLRT104, OriGene Technologies) were used to examine ERCC1 and PCYT1A mRNA expression profiles.

Techniques: Immunohistochemistry, Staining, Blocking Assay

Journal: BMC Biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: qPCR analysis of mRNA expression profile for ERCC1 and PCYT1A genes on 24 lung cancer patient tissues. A . qPCR standard. The experimental data using OriGene’s ERCC1 and PCYT1A qPCR standards. A formula for copy number calculation is shown on the chart. B . qPCR lung cancer tissue expression analysis. qPCRdata on OriGene’s TissueScan lung cancer cDNA array panel (HLRT104). The data were grouped based on cancer patient stages.

Article Snippet: TissueScan 24 lung cancer panels (HLRT104, OriGene Technologies) were used to examine ERCC1 and PCYT1A mRNA expression profiles.

Techniques: Expressing

Journal: BMC Biotechnology

Article Title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

doi: 10.1186/1472-6750-12-88

Figure Lengend Snippet: To develop the most highly specific anti-ERCC1 monoclonal antibody with a protein microarray chip. A . Specificity evaluation with a protein microarray chip. The overexpression protein microarray chip was immunostained with the 4F9 clone. This data shows that 4F9 is highly specific to ERCC1 (indicated with red arrows). No cross-reactivity was observed with any other test protein. B . Western Blot confirmation analysis. Seven OriGene VERIFY TM overexpression lysate antigen standards (Lane 1 to 7) were fractionated on SDS-PAGE, and then immunoblotted with 4F9. The recombinant protein expression levels for the each of the different protein overexpression lysates were confirmed with the anti-DDK antibody (Figure B). Lanes 1 to 7 are loaded with samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and PCYT1A (NM_005017).

Article Snippet: TissueScan 24 lung cancer panels (HLRT104, OriGene Technologies) were used to examine ERCC1 and PCYT1A mRNA expression profiles.

Techniques: Microarray, Over Expression, Western Blot, SDS Page, Recombinant, Expressing

Journal: PLoS ONE

Article Title: Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2

doi: 10.1371/journal.pone.0155771

Figure Lengend Snippet: A-C) YFP Fluorescence measurement showing inhibition of mouse ANO2 activity by Ani9, T16A inh -A01 and MONNA in FRT cells expressing ANO2 and a halide sensor YFP. The indicated concentrations of the inhibitors were applied 20 min prior to ANO2 activation by 100 μM ATP. D) Apical membrane current was measured in FRT-ANO1cells. 1 μM Ani9, 10 μM T16A inh -A01 and 10 μM MONNA were applied 20 min prior to ANO1 activation. E) Apical membrane current was measured in FRT-ANO2 cells. 1 μM Ani9, 10 μM T16A inh -A01 and 10 μM MONNA were applied 20 min prior to ANO2 activation. F) Summary of peak currents of ANO1 and ANO2 (mean ± S.E., n = 4). G) Representative current traces showing Ani9-induced ANO2 inhibition at the indicated concentration. H) Summary of dose responses of ANO1 and ANO2 (mean ± S.E., n = 4–6).

Article Snippet: ANO2 expressing FRT cells were obtained by stable transfection of FRT cells with a pCMV6-ANO2 (Origene Technologies Inc), plasmid which expresses the mouse ANO2 gene and a pcDNA3.1-YFP-F46L/H148Q/I152L plasmid which expresses halide sensor YFP gene.

Techniques: Fluorescence, Inhibition, Activity Assay, Expressing, Activation Assay, Concentration Assay

Journal: Cancer Science

Article Title: SIX 1 maintains tumor basal cells via transforming growth factor‐β pathway and associates with poor prognosis in esophageal cancer

doi: 10.1111/cas.13135

Figure Lengend Snippet: Selective expression of SIX 1 in esophageal squamous cell carcinoma ( ESCC ). (a) Semi‐quantitative RT ‐ PCR analyses of SIX 1 and PDPN in eight ESCC cell lines (30 PCR cycles for SIX 1 and PDPN ) and three layers (Differentiated, Epibasal, and Basal cell layer) of normal esophageal mucosa (50 PCR cycles for SIX 1 and 30 PCR cycles for PDPN ). (b) Quantitative RT ‐ PCR of SIX 1 in 60 pairs of primary ESCC s and the matched normal tissues. (c) Quantitative RT ‐ PCR of SIX 1 in 42 ESCC specimens classified with no lymph node metastasis (N0, n = 12) and high metastasis (N ≥ 5, n = 30). Data are mean ± SD from three independent experiments. * P < 0.05. (d) Overall survival of 42 ESCC patients stratified by SIX 1 expression level. P‐ values are calculated by log‐rank test.

Article Snippet: Cells were incubated with primary antibody for PDPN (1:50, Acris Antibodies GmbH), and then incubated with Alexa488‐conjugated anti‐mouse IgG antibody (1:800, Invitrogen) and stained with DAPI.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: SIX 1 maintains tumor basal cells via transforming growth factor‐β pathway and associates with poor prognosis in esophageal cancer

doi: 10.1111/cas.13135

Figure Lengend Snippet: Continues expression of SIX 1 alter esophageal squamous cell carcinoma ( ESCC ) cell to tumor basal phenotype. (a) Semi‐quantitative RT ‐ PCR of SIX 1 and normal basal cell or stem cell markers ( NGFR , TP 63 , PDPN , PTPRZ 1 , LGR 6, and ALDH 1A1 ) in three layers (differentiated, epibasal, and basal cell layer) of normal esophageal mucosa. A size marker lane is cropped from a photograph. (b) Semi‐quantitative RT ‐ PCR of SIX 1 and the six above stem cell markers in two SIX 1 ‐transfectants ( TE 6‐ SIX 1‐13 and TE 6‐ SIX ‐17) and mock‐transfectants ( TE 6‐Mock). Lanes of a size marker, positive control (left of TE 6‐Mock), and another unstable clone (right) are cropped from a photograph. Photographs of four full uncut gels were shown in Fig. . (c) Matrigel invasion assay of SIX 1 ‐transfectants. The migrated cells counted at 24 h after transfer into wells. Data are mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01. (d) Flow cytometry analysis of SIX 1 ‐transfectants for PDPN expression. Cells are double‐stained by anti‐ PDPN antibody and PI . Only viable cells are analyzed. (e) Immunohistochemical study of xenografted tumors of TE 6‐Mock and TE 6‐ SIX 1‐17. HE (left), anti SIX 1 antibody (middle) anti‐ PDPN antibody (right). Scale bars represent 20 μm.

Article Snippet: Cells were incubated with primary antibody for PDPN (1:50, Acris Antibodies GmbH), and then incubated with Alexa488‐conjugated anti‐mouse IgG antibody (1:800, Invitrogen) and stained with DAPI.

Techniques: Expressing, Quantitative RT-PCR, Marker, Positive Control, Invasion Assay, Flow Cytometry, Staining, Immunohistochemical staining

Journal: BMC Neuroscience

Article Title: Expression patterns of Neil3 during embryonic brain development and neoplasia

doi: 10.1186/1471-2202-10-45

Figure Lengend Snippet: Expression analysis of Neil3 during embryonic development . (A) Expression profiles of Neil3 based on the EST profile viewer at the NCBI homepage. TPM, transcripts per million. (B) A multiple tissue northern blot containing poly(A) + RNA from mouse embryo tissues at four different developmental stages was hybridized with 32 P-labeled cDNA probes for Neil3 and β-actin .

Article Snippet: For measurement of NEIL3 mRNA expression in normal and tumor tissues, we used a Disease Tissue qPCR array (CSRT303; OriGene Technologies).

Techniques: Expressing, Northern Blot, Labeling

Journal: BMC Neuroscience

Article Title: Expression patterns of Neil3 during embryonic brain development and neoplasia

doi: 10.1186/1471-2202-10-45

Figure Lengend Snippet: Expression analysis of Neil3 in embryonic brain development . (A) Real-time quantitative PCR was performed on mRNA isolated from mouse brains at four different developmental stages. The data were normalized to Gapdh and expressed relative to the level of Neil3 at P0. (B) A detailed expression profile of Neil3 during brain development was obtained by using a mouse developmental tissue qPCR array. The array contains one concentration of cDNAs, normalized to Gapdh. Neil3 mRNA levels were calculated relative to the sample expressing the lowest level of Neil3 (Fron Cx, P7). The experiment was conducted twice and the standard deviations are indicated. Abbreviations: Tel, telencephalon; Die, diencephalon; Mes, mesencephalon (midbrain); Rhom, Rhombencephalon; Sp C, spinal cord; Mid B, midbrain; Med, medulla; Fron Cx, frontal cortex; Post Cx, posterior cortex; Ent Cx, entorhinal cortex; Olf B, olfactory bulb; Hipp, hippocampus; Str, Striatum; Tha, thalamus; Hyp, hypothalamus; Cer, cerebellum.

Article Snippet: For measurement of NEIL3 mRNA expression in normal and tumor tissues, we used a Disease Tissue qPCR array (CSRT303; OriGene Technologies).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Concentration Assay

Journal: BMC Neuroscience

Article Title: Expression patterns of Neil3 during embryonic brain development and neoplasia

doi: 10.1186/1471-2202-10-45

Figure Lengend Snippet: Expression of Neil3 in developing mouse forebrain . In situ hybridisation was performed on coronal sections of mouse brains at different embryonic stages. Magnifications of Neil3 -positive cells are shown to the right. Abbreviations: CA, hippocampal differentiation fields; Ctx, cortex; DG, dentate gyrus; Hy, hypothalamus; LV, lateral ventricle; SM, secondary matrix; SVZ, subventricle zone.

Article Snippet: For measurement of NEIL3 mRNA expression in normal and tumor tissues, we used a Disease Tissue qPCR array (CSRT303; OriGene Technologies).

Techniques: Expressing, In Situ, Hybridization

Journal: BMC Neuroscience

Article Title: Expression patterns of Neil3 during embryonic brain development and neoplasia

doi: 10.1186/1471-2202-10-45

Figure Lengend Snippet: Expression of NEIL3 in human cancers . Transcript profiling was performed by using a disease tissue qPCR array. cDNA concentrations are normalized to β-actin. Neil3 mRNA levels are calculated relative to the sample expressing the lowest level of Neil3 (one of the stomach cancer samples). Tumor and normal tissue samples were grouped according to their origin and the average and standard deviations were calculated for each group. The experiment was conducted twice and the results from one of the experiments are shown. Abbreviations: Adr G, adrenal gland; Bre, breast; Cer, cervix; Col, colon; End, endometrium; Eso, esophagus, Kid, kidney; Liv, liver; Lun, lung; Lym N, lymph node; Ova, ovary; Pan, pancreas; Pro, prostate; Sto, stomach; Tes, testis; Thy G, thyroid gland; Uri B, urinary bladder; Ute, uterus.

Article Snippet: For measurement of NEIL3 mRNA expression in normal and tumor tissues, we used a Disease Tissue qPCR array (CSRT303; OriGene Technologies).

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Mono-ADP-Ribosylhydrolase MACROD2 Is Dispensable for Murine Responses to Metabolic and Genotoxic Insults

doi: 10.3389/fgene.2018.00654

Figure Lengend Snippet: Please provide promoter the meaning of “ ∗ ” specified in Figure 1. (A) qPCR screening of MACROD2 mRNA expression levels normalized to GAPDH across 36 murine somatic and reproductive organs and tissues. (B) Generation of MACROD2 mice. Design diagram of VelociGene ® KOMP constitutive MACROD2 deletion. LacZ, β-galactosidase; TM-lacZ, transmembrane sequence fused to β-galactosidase; neo or hyg, coding sequences for neomycin or hygromycin phosphotransferases; hUbCpro, promoter from the human ubiquitin C gene; p(A), polyadenylation signal. (C) Immunoblot analysis. Protein extracts (10 μg/lane) of liver (L) and kidney (K) were prepared from wild-type (WT) and macroD2 KO (KO) mice and analyzed using anti-macroD2 and anti-β-actin. ∗ BacVec = BAC based targeting vector.

Article Snippet: MACROD2 mRNA expression screen was performed on a cDNA library (96 wells coated plates, normalized against GAPDH) from ORIGENE (MNRT101) – TissueScan Mouse Normal Tissue qPCR Array, according to manufacturer’s instructions.

Techniques: Expressing, Sequencing, Western Blot, Plasmid Preparation

Journal: Frontiers in Genetics

Article Title: Mono-ADP-Ribosylhydrolase MACROD2 Is Dispensable for Murine Responses to Metabolic and Genotoxic Insults

doi: 10.3389/fgene.2018.00654

Figure Lengend Snippet: MACROD2 deletion has no effect on γ-irradiation induced lethality and DNA damage. (A) A Kaplan–Meier survival curve of wild type (WT), MACROD2 HET (+/-) and MACROD2 KO (HOM; -/-) mice ( n = 25–30 mice/group). Survivals of mice were closely monitored several times per day. (B) Comet Assay. Images of comet assay showing DNA damage mouse fibroblasts in response to γ-irradiation. The cells were processed for comet assays immediately after the irradiation.

Article Snippet: MACROD2 mRNA expression screen was performed on a cDNA library (96 wells coated plates, normalized against GAPDH) from ORIGENE (MNRT101) – TissueScan Mouse Normal Tissue qPCR Array, according to manufacturer’s instructions.

Techniques: Irradiation, Single Cell Gel Electrophoresis

Journal: Frontiers in Genetics

Article Title: Mono-ADP-Ribosylhydrolase MACROD2 Is Dispensable for Murine Responses to Metabolic and Genotoxic Insults

doi: 10.3389/fgene.2018.00654

Figure Lengend Snippet: Figure MACROD2 deletion has no effect on high-fat diet induced dysmetabolism. (A) lean and fat masses were determined by CT scan in WT and MACROD2 KO mice fed a HF diet (HFD). (B) body weight in WT and MACROD2 KO mice fed a chow or a HF diet at the experimental end point. (C,D) GTT and ITT were performed in WT and Tg mice fed a chow or a HFD following a 6 h fast. Mice were injected with 2 g glucose/kg of body weight intraperitoneally, and blood glucose concentrations were measured at the indicated time points (minutes). Data are expressed as means ± S.E.M. ( n = 8–9 per group). ∗∗∗ p < 0.001 change versus WT fed a chow diet.

Article Snippet: MACROD2 mRNA expression screen was performed on a cDNA library (96 wells coated plates, normalized against GAPDH) from ORIGENE (MNRT101) – TissueScan Mouse Normal Tissue qPCR Array, according to manufacturer’s instructions.

Techniques: Computed Tomography, Injection

Journal: Frontiers in Genetics

Article Title: Mono-ADP-Ribosylhydrolase MACROD2 Is Dispensable for Murine Responses to Metabolic and Genotoxic Insults

doi: 10.3389/fgene.2018.00654

Figure Lengend Snippet: Pie-charts of frequencies of the interested genomic regions (A) and functional consequences (B) of COSMIC variants hitting the MACROD2 genes.

Article Snippet: MACROD2 mRNA expression screen was performed on a cDNA library (96 wells coated plates, normalized against GAPDH) from ORIGENE (MNRT101) – TissueScan Mouse Normal Tissue qPCR Array, according to manufacturer’s instructions.

Techniques: Functional Assay