Journal: PLoS ONE

Article Title: Distinct Epigenetic Domains Separated by a CTCF Bound Insulator between the Tandem Genes, BLU and RASSF1A

doi: 10.1371/journal.pone.0012847

Figure Lengend Snippet: ( A ) Genomic map of the RASSF1A and BLU CpG sites detected by MSO were indicated by thin vertical bars. Putative E2F1 binding sites are marked with black boxes. Star symbols indicated the potential transcriptionally important CpG sites. The locations of MSO probe were shown in black horizontal lines. The numbered probes indicated the potential transcriptionally important CpG sites. Blue horizontal lines indicated the regions amplified in the ChIP-PCR assay to detect E2F binding. MSP primers for RASSF1A (MSP1 and MSP2) and BLU were designed to analyze the methylation status of E2F1 binding sites. ( B ) MSO results are presented for 32 primary lung tumors (left column) in the RASSF1A potential transcriptionally important CpG sites (shown in the first row). The patients were then ranked by average methylation percentage determined by MSO method. The average methylation percentage of the potential transcriptionally important CpGs for each patient was listed in the second to the last (right) column. The relative mRNA expression level was calculated for each tumor (T) and matched normal (N) samples and was indicated by RT (T/N) in the right column. The extent of methylation of each CpG was indicated by blue color shown in the lower right corner. The darker shades of blue indicated increasing methylation. ( C ) MSO results were presented for 32 primary lung tumors in BLU potential transcriptionally important CpG sites. The heavily methylated and lightly methylated samples were separated by a cut-off line for the RASSF1A and BLU genes. The cut-off value is determined by the mean methylation percentage of potential transcriptionally important CpG sites.

Article Snippet: The gel-supershift assay included incubation of the nuclear extracts (12 μg) with the hot probe for 30 min, addition of 2 μg of CTCF polyclonal antibody (Upstate Biotechnology), or the negative control of 2 μg of E2F1 polyclonal antibody (Active Motif) or 2 μg of IgG polyclonal antibody (Upstate Biotechnology) in the binding reaction, and 30 min incubation at RT.

Techniques: Binding Assay, Amplification, Methylation, Expressing

Journal: PLoS ONE

Article Title: Distinct Epigenetic Domains Separated by a CTCF Bound Insulator between the Tandem Genes, BLU and RASSF1A

doi: 10.1371/journal.pone.0012847

Figure Lengend Snippet: ( A ) RT-PCR analysis for RASSF1A and BLU gene expressions in MRC5 and IMR90 normal cells and three cancer cell lines (A549, CL1-0, and H1299). GAPDH was amplified as an internal control. ( B ) ChIP-PCR analysis for E2F1 binding to RASSF1A and BLU sequences in MRC5 normal cell and A549 cancer cell line. The binding of E2F1 to RASSF1A and BLU promoters was observed in MRC5 cell line. “In”, total input DNA; “E2F”, DNA-protein complex precipitated by anti-E2F1; “IgG”, DNA-protein complex precipitated by rabbit IgG; and “No”, no antibody. ( C ) ChIP assay with anti-acetylated histone H3 for RASSF1A , BLU and GAPDH loci. “K9Ac”, DNA-protein complex precipitated by anti-acetylated histone H3. ( D ) Methylation status of E2F1 binding sites in RASSF1A gene was assessed by MSP in MRC5 normal cell and four cancer cell lines. U: unmethylated gene; M: methylated gene. Sss I methyltransferase-treated MRC5 DNA was used as methylation positive control. ( E ) Relative luciferase activities of different E2F1 mutated constructs are shown on the right side as bars in the bar chart, with the structure of each construct shown on the left side. Luc: luciferase gene sequence. E2F1-expression vector was cotransfected with different E2F1 binding sites mutated constructs (mutation sites shown as black circles).

Article Snippet: The gel-supershift assay included incubation of the nuclear extracts (12 μg) with the hot probe for 30 min, addition of 2 μg of CTCF polyclonal antibody (Upstate Biotechnology), or the negative control of 2 μg of E2F1 polyclonal antibody (Active Motif) or 2 μg of IgG polyclonal antibody (Upstate Biotechnology) in the binding reaction, and 30 min incubation at RT.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Binding Assay, Methylation, Positive Control, Luciferase, Construct, Sequencing, Expressing, Plasmid Preparation, Mutagenesis

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Preparation and characterization of the cell lines used in the present study. (A) Detection of SMS1, SMS2, and GAPDH in JAR, JAR4, JEG3, and HeLa-mCAT#8 cell lines by immunoblotting. (B) Nucleotide sequences around the target region for the SGMS1 gene-specific sgRNA in exon 9 of the SGMS1 gene of the SMS1KO22 clone (clone 22) were aligned with those of the parental JAR4 cells (parent). Target sequences for the sgRNA and following protospacer adjacent motifs are indicated by underlined and boxed sequences of the parental JAR4 cells, respectively. The SMS1KO22 clone has a homozygous 7-nucleotide deletion in the SGMS1 gene. (C) Detection of SMS1, SMS2, and GAPDH in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2 by immunoblotting. (D) Detection of clustered SM on the surface of JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Cells were treated with EGFP-NT-lysenin and analyzed by flow cytometry. Histograms with magenta line and gray fill represent EGFP-NT-lysenin-treated and untreated cells, respectively. Cells in the M1 region (fluorescent intensity of 50 and above) were defined as positive for binding to EGFP-NT-lysenin. The means and standard deviations of triplicate samples are reported in panel E. (E) Significant differences as determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (F) Quantification of sphingolipids in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2 by LC-MS analysis. Lipids were extracted from cells and quantified by LC-MS. The graphs indicate the means and standard deviations of triplicate samples. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. CMH, ceramide monohexoside (glucosylceramide and galactosylceramide); CDH, ceramide dihexoside (lactosylceramide and galabiosylceramide); Gb3, trisaccharide globo-series sphingolipid; GM3, monosialodihexosylganglioside.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Binding Assay, Comparison, Liquid Chromatography with Mass Spectroscopy

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of SGMS1 gene knockout on RuV growth. (A) Growth kinetics of RuV in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Supernatants of each cell line inoculated with the RuV TO-336WT strain at an MOI of 10 were harvested at 0, 1, 2, 3, or 4 days after inoculation. Infectious titers in the supernatants are represented as means and standard deviations of triplicate samples. (B) Fluorescent microscopy images of each cell line, NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, or SMS1KO22/SMS2, inoculated with the RuV TO-336WT strain at 3 days after inoculation. Green signals indicate the expression of the p150-AG1 protein. Nuclei were stained by DAPI (blue). (C) The rate of p150-AG1-expressing cells inoculated with the RuV TO-336WT strain under the same conditions as for panel B. Cells detached with trypsin-EDTA and fixed with 4% paraformaldehyde were analyzed by flow cytometry. The graph indicates the means and standard deviations of triplicate samples. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: *, P < 0.05; ****, P < 0.0001; ns, not significant. (D and E) Growth kinetics of SINV (D) and MeV (E) in NT1 or SMS1KO22 cells. Infectious titers of progeny viruses are represented as means and standard deviations of triplicate samples.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Gene Knockout, Derivative Assay, Microscopy, Expressing, Staining, Flow Cytometry, Comparison

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of knockout of the SGMS1 or SGMS2 gene on infectivity and entry of RuV in HeLa cells. (A) Detection of SMS1, SMS2, and GAPDH in HeLa-mCAT#8 cell line (Parent) and its gene-edited clones, SGMS1 or SGMS2 single-knockout (ΔSMS1 or ΔSMS2) and double-knockout (DKO) cells, by immunoblotting. (B) Growth kinetics of RuV in HeLa-derived cell lines. Each cell line was inoculated with the TO-336WT-AG1 RuV strain at an MOI of 10, and the supernatants were harvested at the indicated days after incubation. The infectious titers of RuV in the supernatants are represented as means and standard deviations of triplicate samples. (C) Infectivity of pseudotyped VSV in each cell line. Each cell line was inoculated with firefly luciferase gene-coding pseudotyped VSVs VSVFLuc-ΔG (ΔG), VSVFLuc-RV/CE2E1 (RuV-CE2E1), or VSVFLuc-G (VSV-G). The firefly luciferase activity was measured at 24 h postinoculation. The graph indicates the means and standard deviations of three independent assays. Significant differences as determined by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Knock-Out, Infection, Clone Assay, Double Knockout, Western Blot, Derivative Assay, Incubation, Luciferase, Activity Assay, Comparison

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of SGMS1 gene knockout on genome replication, entry, and binding of RuV. (A) Reporter assay of the RuV-subgenomic replicon. NT1 or SMS1KO22 cell line was transfected with the subgenomic replicon RNA HS-Rep-P2R (SGR) or replication-defective mutant HS-Rep-GND-P2R (GND), which expressed Rluc as a reporter, together with mRNAs encoding the RuV-C protein and firefly luciferase (Fluc). After 72 h of transfection, RLuc activity was determined and normalized by Fluc activity. The graph indicates the means and standard deviations of three independent assays. Significant differences as determined by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (B) Infectivity of pseudotyped VSVs in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Each cell line was inoculated with Fluc gene-encoding pseudotyped VSVs, VSVFLuc-ΔG (ΔG), VSVFLuc-RV/CE2E1 (RuV-CE2E1), or VSVFLuc-G (VSV-G). The Fluc activity was measured at 24 h postinoculation. The graph indicates means and standard deviations of three independent assays. Significant differences by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (C) Binding of RuV to JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Each cell line was incubated with RuV at an MOI of 4 on ice for 1 h and then washed to remove unbound viruses. Total RNA was extracted from the cells, and the amount of RuV genomic RNA was determined by quantitative RT-PCR and normalized by the amount of total RNA. The graph indicates the means and standard deviations of three independent assays. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Gene Knockout, Binding Assay, Reporter Assay, Transfection, Mutagenesis, Luciferase, Activity Assay, Comparison, Infection, Derivative Assay, Incubation, Quantitative RT-PCR

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Penetration of the RuV genome into the cytoplasm. NT1 or SMS1KO22 cells (A and B), or SMS1KO22cells, SMS1KO22/SMS1-WT clone 1, or SMS1KO22/SMS2 clone 1 (C and D) were inoculated with RuV. After incubation at 37°C for 3 h, cells were fixed with 4% paraformaldehyde. For the control experiment in panel A, NT1 cells were incubated with a medium containing BAPTA-AM (final concentration, 50 μM) before inoculation of RuV at 1 h. The RuV genome (pseudocolored in magenta) and the E1 protein (green) were stained by in situ hybridization and indirect immunofluorescence assay, respectively. Nuclei were stained by DAPI (blue). In panels A and C, representative z -stack images are shown. The three columns on the right are enlarged images of the areas enclosed by dashed boxes in the left column. Bars for original and enlarged images indicate 20 μm and 5 μm, respectively. In panels B and D, percentages of puncta in which RuV genomic RNA is present and colocalized with E1 protein are indicated. The graphs indicate the means and standard deviations of three independent assays. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: **, P < 0.01; *, P < 0.05.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Incubation, Control, Concentration Assay, Staining, In Situ Hybridization, Immunofluorescence, Comparison

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

Article Title: Modulation of apoptotic response by LAR family phosphatases–cIAP1 signaling during urinary tract morphogenesis

doi: 10.1073/pnas.1707229114

Figure Lengend Snippet: LAR family phosphatases regulate caspase 3-dependent apoptosis through cIAPs. (A) Ptprs; Ptprf; Ptprd triple knockout (TKO) MEF lines (n = 4 independent cell lines, with a minimum of 500 cells per condition) are resistant to intrinsic (cisplatin, etoposide) apoptotic triggers as revealed by TUNEL and DAPI stainings. (B) TKO MEFs fail to activate caspase 3 in response to cisplatin treatment. The amount of caspase activity was tracked temporally with a fluorogenic caspase 3,7-substrate by live cell imaging. A representative analysis at 18 h is shown. (C) Immunoblots of WT and TKO MEFs show that LAR-RPTP–deficient MEFs cleave upstream caspase 9 normally but fail to accumulate cleaved caspase 3 in response to 6-h stimulation with cisplatin. (D) Live cell imaging of MEFs transfected with a mitochondrially localized Smac-RFP construct (mito-Smac) in the presence of a fluorogenic caspase 3,7-substrate reveals normal release of Smac after 4 h of cisplatin stimulation (cyto-Smac), which is accompanied by an increase in caspase 3,7 activity (Casp3+) in WT but not TKO MEFs (200–500 cells per condition). (E) Mitochondrial outer membrane permeabilization triggered by 4-h treatment with the anti-apoptotic Bcl2-family inhibitors obatoclax (OBX) and ABT-737 (ABT) reveals that TKO MEFs are blocked in caspase 3,7 activation downstream of Bax/Bak-mediated MOMP. (F) Inactivation of IAPs by the Smac-mimetic BV6 (5 µM) restores caspase 3,7 activity in TKO MEFs upon cisplatin stimulation. (G) BV6-mediated rescue of TKO MEF apoptosis occurs downstream of mitochondrial Smac release. Imaging of MEFs transfected with a Smac-RFP construct in the presence of a fluorogenic caspase 3,7-substrate reveals normal release of Smac into the cytosol in response to cotreatment with BV6 and cisplatin (200–500 cells per condition). All experiments shown are representative of four independent experiments, performed in technical triplicates. All P values are calculated using one-way ANOVA. All error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Article Snippet: Antibodies used were mouse anti-GST (Abgent); rabbit anti-caspase 3 (Millipore); rabbit anti-cleaved caspase 3 (D175), rabbit anti-cleaved caspase 8 (D387), rabbit anti-cleaved caspase 9 (D353), and mouse anti-caspase 9 (all Cell Signaling Technology); rabbit anti-tubulin, rabbit anti-cIAP1, and rabbit anti-truncated Bid (all Abcam); and mouse anti-Smac (BD Biosciences).

Techniques: Triple Knockout, TUNEL Assay, Activity Assay, Live Cell Imaging, Western Blot, Transfection, Construct, Activation Assay, Imaging

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

Article Title: Modulation of apoptotic response by LAR family phosphatases–cIAP1 signaling during urinary tract morphogenesis

doi: 10.1073/pnas.1707229114

Figure Lengend Snippet: cIAP1 modulates the rate of apoptotic cell death during common nephric duct (CND) elimination. (A) Representative section immunofluorescence of WT or Birc2−/− caudal CND stained for TUNEL, cleaved caspase 3 (cC3), cleaved caspase 8 (cC8), or cleaved caspase 9 (cC9) and costained for DAPI and E-cadherin. (B) Quantification of apoptotic rates across the CND from WT or Birc2 mutant stainings (n = 20 CNDs per genotype) reveals an increase in caspase 3 and TUNEL positive cells but unaffected rates of cleaved caspase 8 and 9 upon loss of cIAP1. (C) Representative section immunofluorescence from the caudal CND (adjacent to the cloaca) from WT (n = 8 CNDs) and Birc4 mutant (n = 10 CNDs) E11.5 embryos costained for TUNEL, E-cadherin, and DAPI. (D) Quantification of apoptotic rates across the length of the CND fails to show any effect for Birc4. (E) Representative section immunofluorescence from the caudal CND (adjacent to the cloaca) from Smac heterozygous (n = 6 CNDs) and mutant (n = 6 CNDs) E11.5 embryos costained for TUNEL or cleaved caspase 3, E-cadherin, and DAPI. (F) Quantification of apoptotic rates across the length of the CND by TUNEL or cleaved caspase 3 shows a reduction in apoptosis upon loss of Smac. (Scale bars in A, C, and E, 5 µm.) All P values are calculated using one-way ANOVA. All error bars indicate SEM. *P < 0.05, ***P < 0.005.

Article Snippet: Antibodies used were mouse anti-GST (Abgent); rabbit anti-caspase 3 (Millipore); rabbit anti-cleaved caspase 3 (D175), rabbit anti-cleaved caspase 8 (D387), rabbit anti-cleaved caspase 9 (D353), and mouse anti-caspase 9 (all Cell Signaling Technology); rabbit anti-tubulin, rabbit anti-cIAP1, and rabbit anti-truncated Bid (all Abcam); and mouse anti-Smac (BD Biosciences).

Techniques: Immunofluorescence, Staining, TUNEL Assay, Mutagenesis

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

Article Title: Modulation of apoptotic response by LAR family phosphatases–cIAP1 signaling during urinary tract morphogenesis

doi: 10.1073/pnas.1707229114

Figure Lengend Snippet: Apoptotic defects in cIAP1 mutant mice are associated with vesicoureteral reflux. (A) Representative ex vivo urogenital cultures of E11.5 Pax2BACGFP transgenic animals either WT or mutant for Birc2 followed by time-lapse imaging for 20 h. (B) Quantification of CND elimination in WT and Birc2−/− ex vivo cultures treated with inhibitors for caspase 3/7 (DEVD), caspase 8 (IETD), or caspase 9 (LEHD) suggests a role for both extrinsic and intrinsic pathways. (C) Representative optical tomography 3D images of WT and Birc2-mutant urogenital systems at postnatal day 0 in front view (Left) and from inside bladder (Right). Ureters are cyan; arrow indicates urethral opening; and lines A and B represent distance to ureter insertion points. (D) Quantification of ureter insertion point ratios (lines A and B in A) of control and Birc2−/− pups based on optical tomography imaging show an increased variability in ureter positioning in mutants. (E) Dilation of Birc2-null postnatal day 0 ureters compared with control pups measured from dissected urogenital systems and normalized to body weight. (F and G) Ink injection assay measuring the frequency (F) and laterality (G) of vesicoureteral reflux on postnatal day 0 control, Birc2−/−, and Birc4−/− pups show a Birc2-specific reflux phenotype. (H) Model of apoptotic regulation by the LAR-RPTPs–cIAP1 axis to control CND elimination. (Scale bars, 100 µm in A.) P values are calculated using one-way ANOVA in B and using Mann–Whitney u tests in D–G. *P < 0.05, ***P < 0.005.

Article Snippet: Antibodies used were mouse anti-GST (Abgent); rabbit anti-caspase 3 (Millipore); rabbit anti-cleaved caspase 3 (D175), rabbit anti-cleaved caspase 8 (D387), rabbit anti-cleaved caspase 9 (D353), and mouse anti-caspase 9 (all Cell Signaling Technology); rabbit anti-tubulin, rabbit anti-cIAP1, and rabbit anti-truncated Bid (all Abcam); and mouse anti-Smac (BD Biosciences).

Techniques: Mutagenesis, Reflux, Ex Vivo, Transgenic Assay, Imaging, Tomography, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) Confocal imaging of ITGα2, Vimentin, and K8 in an IDC patient tissue section. Green contour: cluster of cancer cells with protrusive morphology; green arrowheads: basal-like cells (K8-low) at the tumor–stroma interface with high ITGα2 expression. White contour: cluster of cancer cells lacking basal-like cells (K8-high), with low ITGα2 expression; magenta arrowheads. White arrowheads: fibroblast-like cells (elongated, spindle-shaped). (B) Quantification of mean gray values for ITGα2 and Vimentin in basal-like (n = 33), luminal-like (n = 32), and fibroblast-like (n = 32) cells from one IDC patient tissue section. (C) Representative brightfield images of MMTV-PyMT organoids (ITGα2-WT or ITGα2-KO, gRNA1 and gRNA2) cultured in 3D Collagen I. Black arrowheads: invasive strands. (D) Percentage of organoids exhibiting one or more invasive strands in ITGα2-WT and ITGα2-KO (clones 1 and 2 from gRNA1) MMTV-PyMT organoids. (E) qPCR analysis of classical TGF-β and EMT target genes in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Values represent mean normalized mRNA expression (relative to housekeeping genes), shown for KO organoids relative to WT controls (dashed line). Data are presented as mean ± SD from three independent experiments. (F) Confocal imaging of Col ¾ and F-actin in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids after one day in 3D Collagen I. (G, H) qPCR analysis of Vimentin and Slug mRNA expression in ITGα2-KO versus ITGα2-WT MMTV-PyMT organoids treated with Activin A (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graphs represent mean normalized expression values ± SD from four independent experiments. (I) Kaplan–Meier analysis correlating high vs. low mRNA expression of INHBA, ITGA2, ITGB1, and their combinations (ITGA2 + ITGB1, or INHBA + ITGA2 + ITGB1) with distant metastasis-free survival (DMFS) in patients with grade 3 breast cancer. Scale bars: 100 μm (A, C), 50 μm (A, zoom-in), 50 μm (F), 10 μm (F, zoom-in). P values: two-sided unpaired Mann–Whitney test (E), two-sided Kruskal-Wallis test with Dunn’s multiple comparisons (G, H), Log-rank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Imaging, Expressing, Cell Culture, Clone Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) DNA sequence of the Itgα2 gene to confirm gene knockout by Crispr-Cas9 gene editing. Red regions indicate the insertion of one base pair compared to the wildtype Itgα2 sequence. (B) Western blot analysis showing Itgα2 and GAPDH expression from whole cell lysates of MMTV-PyMT organoids WT or KO (gRNA1 or gRNA2). (C) Confocal imaging of Itgα2 and K8 in MMTV-PyMT organoids with Itgα2 WT or knockout (KO) grown in Collagen I for 3 days. White arrowheads: invading strands in Itgα2 WT organoids led by basal-like cells (low K8, high Itgα2, insets), White arrows: non-invading basal-like cells at the ECM interface in Itgα2 KO organoids (low K8, low Itgα2, insets). (D) Percentage of invasive organoids in MMTV-PyMT organoids Itgα2-WT versus Itgα2-KO (gRNA2). (E) Mean gray value of Col ¾ relative to Collagen I reflection in MMTV-PyMT organoids with Itgα2-WT and Itgα2-KO. Median: red lines, from n = 11 organoids per group from two independent experiments. (F) Single confocal slice showing INHBA and K8 expression in Itgα2-WT and Itgα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Insets show basal-like cells (K8 low) guiding invasive strands (Itgα2-WT) or remaining at the organoid rim (Itgα2 KO, non-invasive). (G) Mean-gray value of Inhba in basal-like cells (K8-low) located at the rim of Itgα2 WT vs. KO MMTV-PyMT organoids (3D Collagen I, day 1). Median: red lines, n =11 cells from 6 Itgα2-WT organoids, and n = 12 cells from 7 Itgα2-KO organoids from one experiment. (H) qPCR analysis showing relative mRNA expression of CTGF in MMTV-PyMT Itgα2-KO organoids compared to Itgα2-WT organoids treated with Activin A ligand (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graph represents mean normalized expression values (relative to housekeeping genes) ± SD from four independent experiments. (I) Kaplan-Meier plot correlating ITGB1 gene expression with DMFS in grade 3 breast cancer patients. Scale bars: 50 μm (C, F), 25 μm (C, F, Zoom in). P values, two-sided unpaired Mann–Whitney test (E, G), two-sided Kruskal-Wallis test (Dunn’s multiple comparison) (H), Logrank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Sequencing, Gene Knockout, CRISPR, Western Blot, Expressing, Imaging, Knock-Out, Cell Culture, Control, Gene Expression, MANN-WHITNEY, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 3. REDD1 overexpression by neutrophils in colonic tissue of active UC or by control neutrophils stimulated in vitro with colonic tissue culture CM from active UC is paralleled with autophagy induction. (A) Colonic biopsy sections derived from patients with active UC stained with (A) REDD1 and NE (confocal microscopy; NE [green], REDD1 [red]) or (B) Beclin-1 and NE (confocal microscopy; Beclin-1 [green], NE [red]) compared with controls or active CD. (C) REDD1 mRNA levels and (D) REDD1, mTOR p2481, or p62/SQSTM1 immunoblotting in control neutrophils stimulated with CM from active UC patients in comparison with CM from healthy subjects or active CD. ***p , 0.001. (E) Results after measurement of the relative IOD of bands presented in (D), expressed as mTOR p2481/mTOR or p62/GAPDH. *,#p , 0.001. For (C) and (E), data presented as mean 6 SD. For (A) and (B), n = 4, (C)–(E) n = 6.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Over Expression, Control, In Vitro, Derivative Assay, Staining, Confocal Microscopy, Western Blot, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 4. In mucosal tissue of patients with active UC, neutrophil REDD1, Beclin-1 expression, and NETosis are diminished according to the distance from the inflamed area. Colonic biopsy sections derived from distinct mucosa areas of patients with active UC stained with (A) REDD1 and NE (confocal microscopy; NE [green], REDD1 [red]) or (B) Beclin-1 and NE (confocal microscopy; Beclin-1 [green], NE [red]). (C) REDD1 mRNA, (D) REDD1 immunoblotting, (E) percentage of NET-releasing neutrophils, (F) MPO–DNA complex, and (G) IL-1b expression in isolated NET structures (NET ELISA) of control neutrophils stimulated with colonic tissue culture CM from inflamed, marginal, or noninflamed mucosal area of patients with active UC. For (A) and (B), n = 4. For (C)–(G), data presented as mean 6 SD, n = 6. ***p , 0.001.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Expressing, Derivative Assay, Staining, Confocal Microscopy, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 5. Colonic tissue culture CM from inflamed area of patients with active UC constitute a more potent inducer of REDD1 expression than the respective sera. (A) REDD1 mRNA and (B) REDD1 immunoblotting in control neutrophils stimulated either with CM from the inflamed mucosal area of patients with active UC or with the respective sera. For (A), data presented as mean 6 SD. For (A) and (B), n = 6. ***p , 0.001.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Expressing, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 6. REDD1/neutrophils/IL-1b axis offers candidate targets for differential diagnosis and treatment of active UC. (A and B) Representative histograms and (C and D) data from flow cytometry analysis of CitH3+ or IL-1b–positive ex vivo isolated neutrophils from patients with active UC (n = 15) compared with controls (n = 25), active CD (n = 11), or IC (n = 15). (E) Heat map analysis of colonic tissue neutrophils positive for REDD1 expression (n = 6). (F) Visual analog scale (VAS) pain during sacroiliitis attacks of two active UC patients treated with anakinra (100 mg/daily s.c.). (G) Intracellular ex- pression of IL-1b in neutrophils or (H) CitH3-positive neutrophils ex vivo isolated from patients with active UC under mesalazine/5-ASA monotherapy (n = 8) compared with naive ones (n = 15), as assessed by flow cytometry analysis. For (C), (D), (G), and (H), data presented as mean 6 SD. ***p , 0.001.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Biomarker Discovery, Cytometry, Ex Vivo, Isolation, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 8. Schematic illustration of the proposed mechanism for active UC. Intestinal inflammatory environment activates colonic mucosa neutrophils to release inflammatory NETs through local upregulation of REDD1, leading to autophagy induction. Targeting the REDD1/autophagy/NETs/IL-1b pathway promises novel therapeutic approaches for intestinal and systemic inflammation in active UC.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Electroacupuncture Reduces Fibromyalgia Pain by Attenuating the HMGB1, S100B, and TRPV1 Signalling Pathways in the Mouse Brain

doi: 10.1155/2022/2242074

Figure Lengend Snippet: Comparative graph showing the hind paw withdrawal threshold and latency. Black: control group, red: ICS group, blue: ICS + electroacupuncture (EA) group, green: transient receptor vanilloid member one deletion ( Trpv1 −/− ) group. ∗ indicates statistical significance with p < 0.05 when compared with the control group. # indicates statistical significance with p < 0.05 when compared with the ICS group. (a) Mechanical hyperalgesia measured by the von Frey test in the four groups. (b) Thermal hyperalgesia assessed by the Hargreaves test in the four groups. (c) Schematic illustration showing the ICS procedure. Control mice stayed in a 24°C environment day and night for five days. Mice subjected to ICS were kept in a 4°C environment for 17.5 hours (from 4:30 p.m. to 10:00 a.m.) from Day 0 to 3. Between 10:00 a.m. and 4:30 p.m. (total duration: 6.5 hours), they were placed in a room at 24°C. In this 6.5-hour period, the mice were subjected to intermittent environment temperature change (24°C and 4°C) for 30 min each time. The procedure was terminated on Day 3 at 10:00 a.m., and fibromyalgia-like pain was assessed.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated with a primary antibody against TRPV1 (∼95 kDa, 1 : 1000; Alomone, Israel), HMGB1 (∼28 kDa, 1 : 1000; Alomone, Israel), S100B (∼10 kDa, 1 : 1000; Millipore, USA), TLR4 (∼35 kDa, 1 : 1000; Millipore, USA), RAGE (∼42 kDa, 1 : 1000; Millipore, USA), pPI3K (∼125 kDa, 1 : 1000; Millipore, USA), pAkt (∼60 kDa, 1 : 1000; Millipore, USA), pmTOR (∼60 kDa, 1 : 500; Millipore, USA), pERK1/2 (∼42–44 kDa, 1 : 1000; Millipore, USA), pp38 (∼42 kDa, 1 : 1000; Millipore, USA), pJNK (∼42 kDa, 1 : 1000; Millipore, USA), and pNF- κ B (∼65 kDa, 1 : 1000; Millipore, USA) in TBS-T with 1% bovine serum albumin.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Electroacupuncture Reduces Fibromyalgia Pain by Attenuating the HMGB1, S100B, and TRPV1 Signalling Pathways in the Mouse Brain

doi: 10.1155/2022/2242074

Figure Lengend Snippet: (a) Expression of extracellular neurotransmitters ((A) HMGB1, (B) S100B), receptors ((C) RAGE, (D) TLR2, (E) TLR4), and cytoplasmic inflammation signal molecules ((F) pPI3K, (G) pAkt, (H) pmTOR, (I) pERK, (J) pp38, (K) pJNK, (L) pNF- κ B) in the mouse PFC. The four lanes on the immunoblots correspond to the protein bands of, in order, control (Con), ICS-induced fibromyalgia (ICS), electroacupuncture (EA), and Trpv1 −/− groups. ∗ indicates statistical significance with P < 0.05 when compared with the Con group. # indicates statistical significance with P < 0.05 when compared with the ICS group. (b) Immunofluorescence staining of TLR2 and pNF- κ B in the mouse PFC. Immunopositive signals (light green, indicated by a red arrow) were detected for TLR2 and pNF- κ B. Scale bar: 100 μ m.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated with a primary antibody against TRPV1 (∼95 kDa, 1 : 1000; Alomone, Israel), HMGB1 (∼28 kDa, 1 : 1000; Alomone, Israel), S100B (∼10 kDa, 1 : 1000; Millipore, USA), TLR4 (∼35 kDa, 1 : 1000; Millipore, USA), RAGE (∼42 kDa, 1 : 1000; Millipore, USA), pPI3K (∼125 kDa, 1 : 1000; Millipore, USA), pAkt (∼60 kDa, 1 : 1000; Millipore, USA), pmTOR (∼60 kDa, 1 : 500; Millipore, USA), pERK1/2 (∼42–44 kDa, 1 : 1000; Millipore, USA), pp38 (∼42 kDa, 1 : 1000; Millipore, USA), pJNK (∼42 kDa, 1 : 1000; Millipore, USA), and pNF- κ B (∼65 kDa, 1 : 1000; Millipore, USA) in TBS-T with 1% bovine serum albumin.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Electroacupuncture Reduces Fibromyalgia Pain by Attenuating the HMGB1, S100B, and TRPV1 Signalling Pathways in the Mouse Brain

doi: 10.1155/2022/2242074

Figure Lengend Snippet: (a) Expression of extracellular neurotransmitters ((A) HMGB1, (B) S100B), receptors ((C) RAGE, (D) TLR2, (E) TLR4), and cytoplasmic inflammation signal molecules ((F) pPI3K, (G) pAkt, (H) pmTOR, (I) pERK, (J) pp38, (K) pJNK, (L) pNF- κ B) in the mouse SSC. The four lanes on the immunoblots correspond to the protein bands of, in order, control (Con), ICS-induced fibromyalgia (ICS), electroacupuncture (EA), and Trpv1 −/− groups. ∗ indicates statistical significance with P < 0.05 when compared with the Con group. # indicates P < 0.05 statistical significance when compared with the ICS group. (b) Immunofluorescence staining of TLR2 and pNF- κ B in the mouse SSC. Immunopositive signals (light green, indicated by a red arrow) were detected for TLR2 and pNF- κ B. Scale bar: 100 μ m.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated with a primary antibody against TRPV1 (∼95 kDa, 1 : 1000; Alomone, Israel), HMGB1 (∼28 kDa, 1 : 1000; Alomone, Israel), S100B (∼10 kDa, 1 : 1000; Millipore, USA), TLR4 (∼35 kDa, 1 : 1000; Millipore, USA), RAGE (∼42 kDa, 1 : 1000; Millipore, USA), pPI3K (∼125 kDa, 1 : 1000; Millipore, USA), pAkt (∼60 kDa, 1 : 1000; Millipore, USA), pmTOR (∼60 kDa, 1 : 500; Millipore, USA), pERK1/2 (∼42–44 kDa, 1 : 1000; Millipore, USA), pp38 (∼42 kDa, 1 : 1000; Millipore, USA), pJNK (∼42 kDa, 1 : 1000; Millipore, USA), and pNF- κ B (∼65 kDa, 1 : 1000; Millipore, USA) in TBS-T with 1% bovine serum albumin.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Electroacupuncture Reduces Fibromyalgia Pain by Attenuating the HMGB1, S100B, and TRPV1 Signalling Pathways in the Mouse Brain

doi: 10.1155/2022/2242074

Figure Lengend Snippet: (a) Expression of extracellular neurotransmitters (A) HMGB1, (B) S100B), receptors (C) RAGE, (D) TLR2, (E) TLR4) and cytoplasmic inflammation signal molecules (F) pPI3K, (G) pAkt, (H) pmTOR, (I) pERK, (J) pp38, (K) pJNK, (L) pNF- κ B) in the mouse thalamus. The four lanes on the immunoblots correspond to the protein bands of, in order, control (Con), ICS-induced fibromyalgia (ICS), electroacupuncture (EA) and Trpv1 −/− groups. ∗ indicates statistical significance with P < 0.05 when compared with the Con group. # indicates statistical significance with P < 0.05 when compared to the ICS group. (b) Immunofluorescence staining of TLR2 and pNF- κ B in the mouse thalamus. Immunopositive signals (light green, indicated by a red arrow) were detected for TLR2 and pNF- κ B. Scale bar: 100 μ m.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated with a primary antibody against TRPV1 (∼95 kDa, 1 : 1000; Alomone, Israel), HMGB1 (∼28 kDa, 1 : 1000; Alomone, Israel), S100B (∼10 kDa, 1 : 1000; Millipore, USA), TLR4 (∼35 kDa, 1 : 1000; Millipore, USA), RAGE (∼42 kDa, 1 : 1000; Millipore, USA), pPI3K (∼125 kDa, 1 : 1000; Millipore, USA), pAkt (∼60 kDa, 1 : 1000; Millipore, USA), pmTOR (∼60 kDa, 1 : 500; Millipore, USA), pERK1/2 (∼42–44 kDa, 1 : 1000; Millipore, USA), pp38 (∼42 kDa, 1 : 1000; Millipore, USA), pJNK (∼42 kDa, 1 : 1000; Millipore, USA), and pNF- κ B (∼65 kDa, 1 : 1000; Millipore, USA) in TBS-T with 1% bovine serum albumin.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Electroacupuncture Reduces Fibromyalgia Pain by Attenuating the HMGB1, S100B, and TRPV1 Signalling Pathways in the Mouse Brain

doi: 10.1155/2022/2242074

Figure Lengend Snippet: (a) The expression of extracellular neurotransmitters ((A) HMGB1, (B) S100B), receptors ((C) RAGE, (D) TLR2, (E) TLR4), and cytoplasmic inflammation signal molecules ((F) pPI3K, (G) pAkt, (H) pmTOR, (I) pERK, (J) pp38, (K) pJNK, (L) pNF- κ B) in the mouse amygdala. The four lanes on the immunoblots correspond to the protein bands of, in order, control (Con), ICS-induced fibromyalgia (ICS), electroacupuncture (EA), and Trpv1 −/− groups. ∗ indicates statistical significance with P < 0.05 when compared with the Con group. # indicates statistical significance with P < 0.05 when compared with the ICS group. (b) Immunofluorescence staining of TLR2 and pNF- κ B in the mouse amygdala. Immunopositive signals (light green, indicated by a red arrow) were detected for TLR2 and pNF- κ B. Scale bar: 100 μ m.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated with a primary antibody against TRPV1 (∼95 kDa, 1 : 1000; Alomone, Israel), HMGB1 (∼28 kDa, 1 : 1000; Alomone, Israel), S100B (∼10 kDa, 1 : 1000; Millipore, USA), TLR4 (∼35 kDa, 1 : 1000; Millipore, USA), RAGE (∼42 kDa, 1 : 1000; Millipore, USA), pPI3K (∼125 kDa, 1 : 1000; Millipore, USA), pAkt (∼60 kDa, 1 : 1000; Millipore, USA), pmTOR (∼60 kDa, 1 : 500; Millipore, USA), pERK1/2 (∼42–44 kDa, 1 : 1000; Millipore, USA), pp38 (∼42 kDa, 1 : 1000; Millipore, USA), pJNK (∼42 kDa, 1 : 1000; Millipore, USA), and pNF- κ B (∼65 kDa, 1 : 1000; Millipore, USA) in TBS-T with 1% bovine serum albumin.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Electroacupuncture Reduces Fibromyalgia Pain by Attenuating the HMGB1, S100B, and TRPV1 Signalling Pathways in the Mouse Brain

doi: 10.1155/2022/2242074

Figure Lengend Snippet: Schematic illustration of neuronal and non-neuronal mechanisms underlying EA-mediated analgesic effect on ICS-induced fibromyalgia pain. The summary diagram shows the importance of and mechanisms involving glial cells (astrocytes and microglia) and TRPV1 in fibromyalgia pain. EA inhibits HMGB1 and S100B release from non-neuronal cells or directly inhibits TRPV1 on the plasma membrane. Mice with a TRPV1 gene deletion ( Trpv1 −/− ) have the same phenotype than mice treated with EA.

Article Snippet: The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated with a primary antibody against TRPV1 (∼95 kDa, 1 : 1000; Alomone, Israel), HMGB1 (∼28 kDa, 1 : 1000; Alomone, Israel), S100B (∼10 kDa, 1 : 1000; Millipore, USA), TLR4 (∼35 kDa, 1 : 1000; Millipore, USA), RAGE (∼42 kDa, 1 : 1000; Millipore, USA), pPI3K (∼125 kDa, 1 : 1000; Millipore, USA), pAkt (∼60 kDa, 1 : 1000; Millipore, USA), pmTOR (∼60 kDa, 1 : 500; Millipore, USA), pERK1/2 (∼42–44 kDa, 1 : 1000; Millipore, USA), pp38 (∼42 kDa, 1 : 1000; Millipore, USA), pJNK (∼42 kDa, 1 : 1000; Millipore, USA), and pNF- κ B (∼65 kDa, 1 : 1000; Millipore, USA) in TBS-T with 1% bovine serum albumin.

Techniques:

Journal: Nature immunology

Article Title: Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway

doi: 10.1038/ni.1645

Figure Lengend Snippet: Expression of rapamycin-sensitive mTOR pathway components in pDCs. (a) Immunoblot analysis of phosphorylated (phospho-) and total mTOR in lysates of RAW cells (4 × 106) transiently transfected for 40 h with cyan fluorescent protein–tagged MyD88 and IRF7-YFP, treated with vehicle (−) or rapamycin (+) and then stimulated for 0–60 min with CpG-A–DOTAP. Results are representative of two independent experiments. (b) Immunoblot analysis of phosphorylated and total mTOR, p70S6K, 4E-BP1 and Akt in lysates of purified pDCs (1 × 106) stimulated for 15 min with CpG-A in the presence (Rap) or absence (Medium) of rapamycin. Results are representative of three independent experiments.

Article Snippet: The following primary antibodies were from Cell Signaling (all at a dilution of 1:1,000): anti-mTOR (2972); antibody to mTOR phosphorylated at Ser2448 (2971); anti-Akt (9272); antibody to Akt phosphorylated at Thr308 (9275); anti-4E-BP1 (9452); antibody to 4E-BP1 phosphorylated at Thr37 and Thr46 (9459), Ser65 (9451) or Thr70 (9455); anti-p70S6K (9202); and antibody to p70S6K phosphorylated at Thr389 (9205).

Techniques: Expressing, Western Blot, Transfection, Purification

Journal: Nature immunology

Article Title: Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway

doi: 10.1038/ni.1645

Figure Lengend Snippet: TLR-mediated induction of IFN-α in pDCs depends on the mTOR signaling pathway. (a) ELISA of IFN-α in supernatants (above) and immunoblot analysis of mTOR in lysates (below) of purified mouse pDCs (5 × 105) transfected for 5 h with control or mTOR-specific siRNA and then stimulated for 24 h with CpG-A. Below, β-actin serves as a loading control. (b) ELISA of IFN-α in supernatants of pDCs treated with rapamycin or the PI(3)K inhibitor LY294002 (1–25 μM), then stimulated with CpG-A and assessed 24 h later. (c) ELISA of IFN-α in supernatants (above) and immunoblot analysis of p70S6K in lysates (below) of mouse pDCs transfected for 5 h with siRNA pools specific for p70S6K1 (S6K1), p70S6K2 (S6K2) or both (S6K1,2), then stimulated for 24 h with CpG-A. *, P < 0.05. (d) ELISA of IFN-α in supernatants of pDCs isolated from wild-type (WT) and Rps6k1−/−Rps6k2−/− double-knockout (S6K1,2-KO) mouse spleens and stimulated in vitro with CpG-A. Data are representative of three independent experiments (a–c) or two independent experiments with at least two mice per group per experiment (d); error bars, s.e.m. of replicate wells.

Article Snippet: The following primary antibodies were from Cell Signaling (all at a dilution of 1:1,000): anti-mTOR (2972); antibody to mTOR phosphorylated at Ser2448 (2971); anti-Akt (9272); antibody to Akt phosphorylated at Thr308 (9275); anti-4E-BP1 (9452); antibody to 4E-BP1 phosphorylated at Thr37 and Thr46 (9459), Ser65 (9451) or Thr70 (9455); anti-p70S6K (9202); and antibody to p70S6K phosphorylated at Thr389 (9205).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Purification, Transfection, Isolation, Double Knockout, In Vitro

Journal:

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability †

doi: 10.1128/MCB.25.20.8874-8886.2005

Figure Lengend Snippet: Gene trap event in the NPM locus leads to a null NPM mutation and early embryonic lethality in mice. (A) Schematic representation of the targeting retroviral construct (middle) and the wild-type (upper) or mutated (lower) NPM alleles. Positions of oligonucleotides used for the PCR-based genotyping (see panel B) are indicated by arrows (a, b, and c). LTR: long terminal repeats; SA: splicing acceptor site; SD: splicing donor sites; β-Geo: neomycin resistance gene. (B) Left panel: PCR analysis of embryos with the indicated NPM genotypes (primer pairs used are indicated on the right). Right panel: expression of NPM protein as determined by Western blotting of lysates prepared from whole embryos. A polyclonal antibody anti-NPM (Santa Cruz) was used. (C) Wild-type (WT) and knockout (KO) NPM embryos at 10.5 dpc of embryonic development (upper panels) and immunohistochemistry analysis of NPM expression in neuroepithelial (NE) sections from the same embryos, using NPMc antibodies (lower panels). (D and E) immunohistochemistry analysis of apoptosis (D; anti-caspase-3 staining) and p53 expression levels (E; anti-p53 staining) in the developing neuroepithelium and dorsal root ganglia (DRG) of wild-type NPM and knockout 10.5 dpc embryos. (F and G) Expression of p53 target genes analyzed by quantitative PCR (p21, Pig8, Mdm2, and Bax) using mRNA from wild-type NPM (black bars), heterozygous (white bars), and knockout (gray bars) embryos. (G) Western blotting analysis of p21 protein expression in total embryo lysates.

Article Snippet: The following antisera were used: anti-BrdU (1/200; Boehringer Mannheim), anti-cleaved caspase 3 (1/200; Cell Signaling), anti-NPM (NPMc) ( 10 ), anti-H2AX (1:100; Upstate), and anti-p53 (monoclonal antibody raised against mouse p53 protein, clone AI25, kindly provided by Kristian Helin) An appropriate biotinylated secondary antibody was then applied to the slides.

Techniques: Mutagenesis, Construct, Expressing, Western Blot, Knock-Out, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal:

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability †

doi: 10.1128/MCB.25.20.8874-8886.2005

Figure Lengend Snippet: Loss of p53 gene rescues apoptosis in NPM knockout (KO) embryos. (A) p53−/− and double-knockout (p53−/− NPM−/−) embryos at different stages of development (10.5 and 12.5 dpc). (B) Analysis of apoptosis in the developing neuroepithelium (NE) of wild-type (WT), NPM−/−, p53−/− and double-knockout embryos. Immunohistochemistry analysis was performed using anti-activated caspase-3 antibody and counterstained with hematoxylin end eosin. (C) Down-regulation of NPM expression in wild-type MEFs by short interfering RNA. Cells were infected with a control (CTRL) lentivirus or a lentivirus expressing short interfering RNA for NPM (siNPM), as indicated, and analyzed by immunofluorescence using an anti-NPM antibody (NPMa) and by Western blot using anti-NPM and anti-p21 antibodies. For the growth curves, infected cells were seeded on six-well plates at a density of 104 cells per well. Cultures were harvested every day, and the number of cells was determined. The numbers refer to mean values of triplicate determinations. At day 3 of the growth curve, levels of BrdU incorporation were determined by FACS analysis (results refers to mean values of triplicate determinations). (D) Left panel: 2,000 cells derived from the yolk sac of knockout and wild-type embryos and knockout embryos reconstituted with GFP-NPM were seeded in semisolid medium supplemented with interleukin-3, interleukin-6, and stem cell factor and scored after 10 days for number of colonies. Middle panel: expression of phosphorylated p53 (anti-Ser18) and its target p21 in yolk sac-derived cells. Right panel: yolk sac-derived cells were cultured in suspension for 24 h, with (control) or without cytokines, and counted after 10 days (results are expressed as a percentage of the colonies obtained with control cells).

Article Snippet: The following antisera were used: anti-BrdU (1/200; Boehringer Mannheim), anti-cleaved caspase 3 (1/200; Cell Signaling), anti-NPM (NPMc) ( 10 ), anti-H2AX (1:100; Upstate), and anti-p53 (monoclonal antibody raised against mouse p53 protein, clone AI25, kindly provided by Kristian Helin) An appropriate biotinylated secondary antibody was then applied to the slides.

Techniques: Knock-Out, Double Knockout, Immunohistochemistry, Expressing, Small Interfering RNA, Infection, Immunofluorescence, Western Blot, BrdU Incorporation Assay, Derivative Assay, Cell Culture

Journal: Molecular and Cellular Biology

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability

doi: 10.1128/mcb.25.20.8874-8886.2005

Figure Lengend Snippet: FIG. 1. Gene trap event in the NPM locus leads to a null NPM mutation and early embryonic lethality in mice. (A) Schematic representation of the targeting retroviral construct (middle) and the wild-type (upper) or mutated (lower) NPM alleles. Positions of oligonucleotides used for the PCR-based genotyping (see panel B) are indicated by arrows (a, b, and c). LTR: long terminal repeats; SA: splicing acceptor site; SD: splicing donor sites; -Geo: neomycin resistance gene. (B) Left panel: PCR analysis of embryos with the indicated NPM genotypes (primer pairs used are indicated on the right). Right panel: expression of NPM protein as determined by Western blotting of lysates prepared from whole embryos. A polyclonal antibody anti-NPM (Santa Cruz) was used. (C) Wild-type (WT) and knockout (KO) NPM embryos at 10.5 dpc of embryonic development (upper panels) and immunohistochemistry analysis of NPM expression in neuroepithelial (NE) sections from the same embryos, using NPMc antibodies (lower panels). (D and E) immunohistochemistry analysis of apoptosis (D; anti-caspase-3 staining) and p53 expression levels (E; anti-p53 staining) in the developing neuroepithelium and dorsal root ganglia (DRG) of wild-type NPM and knockout 10.5 dpc embryos. (F and G) Expression of p53 target genes analyzed by quantitative PCR (p21, Pig8, Mdm2, and Bax) using mRNA from wild-type NPM (black bars), heterozygous (white bars), and knockout (gray bars) embryos. (G) Western blotting analysis of p21 protein expression in total embryo lysates.

Article Snippet: The primary antibodies used were: sheep polyclonal anti-p53 (1:1000; Ab7; Oncogene); rabbit polyclonal anti-p53 phospho-Ser15 (1/1,000; Cell Signaling), monoclonal anti-p21 (1/300; F5; Santa Cruz); polyclonal anti-p19Arf (1:1,000; Abcam); monoclonal anti-NPM (NPMc; NPMa) (10); polyclonal anti- -tubulin (1:1,000, H-235, Santa Cruz); monoclonal antivinculin (1:1,000; hVIN-1; Sigma), monoclonal anti-Flag (1:1,000; Sigma), polyclonal anti-NPM (B19) (9), and anti-H2AX (1:1,000; Upstate) Immunofluorescence.

Techniques: Mutagenesis, Retroviral, Construct, Expressing, Western Blot, Knock-Out, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability

doi: 10.1128/mcb.25.20.8874-8886.2005

Figure Lengend Snippet: FIG. 2. Loss of p53 gene rescues apoptosis in NPM knockout (KO) embryos. (A) p53/ and double-knockout (p53/ NPM/) embryos at different stages of development (10.5 and 12.5 dpc). (B) Analysis of apoptosis in the developing neuroepithelium (NE) of wild-type (WT), NPM/, p53/ and double-knockout embryos. Immunohistochemistry analysis was performed using anti-activated caspase-3 antibody and counterstained with hematoxylin end eosin. (C) Down-regulation of NPM expression in wild-type MEFs by short interfering RNA. Cells were infected with a control (CTRL) lentivirus or a lentivirus expressing short interfering RNA for NPM (siNPM), as indicated, and analyzed by immunofluorescence using an anti-NPM antibody (NPMa) and by Western blot using anti-NPM and anti-p21 antibodies. For the growth curves, infected cells were seeded on six-well plates at a density of 104 cells per well. Cultures were harvested every day, and the number of cells was determined. The numbers refer to mean values of triplicate determinations. At day 3 of the growth curve, levels of BrdU incorporation were determined by FACS analysis (results refers to mean values of triplicate determinations). (D) Left panel: 2,000 cells derived from the yolk sac of knockout and wild-type embryos and knockout embryos reconstituted with GFP-NPM were seeded in semisolid medium supplemented with interleukin-3, interleukin-6, and stem cell factor and scored after 10 days for number of colonies. Middle panel: expression of phosphorylated p53 (anti-Ser18) and its target p21 in yolk sac-derived cells. Right panel: yolk sac-derived cells were cultured in suspension for 24 h, with (control) or without cytokines, and counted after 10 days (results are expressed as a percentage of the colonies obtained with control cells).

Article Snippet: The primary antibodies used were: sheep polyclonal anti-p53 (1:1000; Ab7; Oncogene); rabbit polyclonal anti-p53 phospho-Ser15 (1/1,000; Cell Signaling), monoclonal anti-p21 (1/300; F5; Santa Cruz); polyclonal anti-p19Arf (1:1,000; Abcam); monoclonal anti-NPM (NPMc; NPMa) (10); polyclonal anti- -tubulin (1:1,000, H-235, Santa Cruz); monoclonal antivinculin (1:1,000; hVIN-1; Sigma), monoclonal anti-Flag (1:1,000; Sigma), polyclonal anti-NPM (B19) (9), and anti-H2AX (1:1,000; Upstate) Immunofluorescence.

Techniques: Knock-Out, Double Knockout, Immunohistochemistry, Expressing, Small Interfering RNA, Infection, Control, Western Blot, BrdU Incorporation Assay, Derivative Assay, Cell Culture, Suspension

Journal: Molecular and Cellular Biology

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability

doi: 10.1128/mcb.25.20.8874-8886.2005

Figure Lengend Snippet: FIG. 3. p53 activation in NPM null cells is part of a DNA damage response. (A) Western blots of lysates from the same cells as in panel B analyzed with antibodies against NPM, p21, and p53. (B) Growth curves of Arf/ MEFs infected with a control lentivirus or a lentivirus expressing short interfering RNA for NPM (siNPM); 2 104 cells were plated in six-well plates. Cultures were harvested every day, and the number of cells was determined. The numbers refer to mean values of triplicate determinations. (C) Western blotting analysis of H2AX expression in lysates of total embryo and yolk sac cells. (D) Analysis of H2AX expression in the developing brain of wild-type (WT) NPM and knockout (KO) 10.5 dpc embryos. Embryo sections were stained with a monoclonal antibody against the phosphorylated form of H2AX and counterstained with hematoxylin and eosin. (E and F) Immunofluorescence analysis of H2AX and phosphorylated ATM (p-ATM) expression in wild-type and knockout yolk sac cells and in knockout cells reconstituted with GFP-NPM.

Article Snippet: The primary antibodies used were: sheep polyclonal anti-p53 (1:1000; Ab7; Oncogene); rabbit polyclonal anti-p53 phospho-Ser15 (1/1,000; Cell Signaling), monoclonal anti-p21 (1/300; F5; Santa Cruz); polyclonal anti-p19Arf (1:1,000; Abcam); monoclonal anti-NPM (NPMc; NPMa) (10); polyclonal anti- -tubulin (1:1,000, H-235, Santa Cruz); monoclonal antivinculin (1:1,000; hVIN-1; Sigma), monoclonal anti-Flag (1:1,000; Sigma), polyclonal anti-NPM (B19) (9), and anti-H2AX (1:1,000; Upstate) Immunofluorescence.

Techniques: Activation Assay, Western Blot, Infection, Control, Expressing, Small Interfering RNA, Knock-Out, Staining

Journal: Molecular and Cellular Biology

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability

doi: 10.1128/mcb.25.20.8874-8886.2005

Figure Lengend Snippet: FIG. 4. Loss of NPM leads to increased proliferation. (A) Left panel: immunohistochemistry analysis of proliferation (anti-BrdU staining) in the developing neuroepithelium and dorsal root ganglia (DRG) of wild-type (WT) NPM and knockout (KO) 10.5 dpc embryos. Right panel: evaluation of BrdU-positive cells in wild-type and knockout embryos through FACS analysis of cells derived directly from embryos after treatment with 0.1% collagenase. (B) Right panel: growth curves of p53/ and double-knockout MEFs at passage 4 (2 104 cells were plated into six-well plates). Cultures were harvested every day, and the total number of cells was determined. The numbers represent the mean values of triplicate determinations. The experiment was repeated twice, using two independently derived MEF cultures, and gave comparable results. Left panels: evaluation of BrdU-positive cells in growing p53/ and double-knockout MEFs. Bars represent mean values of triplicate determinations. The experiments were repeated three times, giving comparable results.

Article Snippet: The primary antibodies used were: sheep polyclonal anti-p53 (1:1000; Ab7; Oncogene); rabbit polyclonal anti-p53 phospho-Ser15 (1/1,000; Cell Signaling), monoclonal anti-p21 (1/300; F5; Santa Cruz); polyclonal anti-p19Arf (1:1,000; Abcam); monoclonal anti-NPM (NPMc; NPMa) (10); polyclonal anti- -tubulin (1:1,000, H-235, Santa Cruz); monoclonal antivinculin (1:1,000; hVIN-1; Sigma), monoclonal anti-Flag (1:1,000; Sigma), polyclonal anti-NPM (B19) (9), and anti-H2AX (1:1,000; Upstate) Immunofluorescence.

Techniques: Immunohistochemistry, BrdU Staining, Knock-Out, Derivative Assay, Double Knockout

Journal: Molecular and Cellular Biology

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability

doi: 10.1128/mcb.25.20.8874-8886.2005

Figure Lengend Snippet: FIG. 5. Regulation of Arf stability and function by NPM. (A) Immunofluorescence analysis of p19Arf localization (red) in p53/ and double-knockout (KO) MEFs. Nucleolin staining (green) was used as a marker for nucleoli. (B) Left panels: Western blotting analysis of p19Arf

Article Snippet: The primary antibodies used were: sheep polyclonal anti-p53 (1:1000; Ab7; Oncogene); rabbit polyclonal anti-p53 phospho-Ser15 (1/1,000; Cell Signaling), monoclonal anti-p21 (1/300; F5; Santa Cruz); polyclonal anti-p19Arf (1:1,000; Abcam); monoclonal anti-NPM (NPMc; NPMa) (10); polyclonal anti- -tubulin (1:1,000, H-235, Santa Cruz); monoclonal antivinculin (1:1,000; hVIN-1; Sigma), monoclonal anti-Flag (1:1,000; Sigma), polyclonal anti-NPM (B19) (9), and anti-H2AX (1:1,000; Upstate) Immunofluorescence.

Techniques: Double Knockout, Staining, Marker, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability

doi: 10.1128/mcb.25.20.8874-8886.2005

Figure Lengend Snippet: FIG. 6. Effects of NPM expression on transformation by Myc and Ras. (A) p53/ and double-knockout (KO) MEFs were infected with a control retrovirus or a retrovirus expressing the c-myc oncogene. At the end of the selection, 2 104 cells were plated into six-well plates and cells were counted at daily intervals. The numbers refer to the mean values of triplicate determinations. The same cells were analyzed by Western blotting using antibodies against tubulin, p19Arf and Myc (right upper panel) and plated in semisolid medium (in triplicate). The number of colonies (right lower panel) and representative plates and colonies (left lower panels) are shown. (C) Same experiment as described for panel A, using a retrovirus expressing the RasV12 oncogene. We evaluated tubulin and p19Arf protein levels (right upper panel) and numbers of colonies formed after plating in semisolid medium (left panel). Representative colonies are also shown (right lower panel).

Article Snippet: The primary antibodies used were: sheep polyclonal anti-p53 (1:1000; Ab7; Oncogene); rabbit polyclonal anti-p53 phospho-Ser15 (1/1,000; Cell Signaling), monoclonal anti-p21 (1/300; F5; Santa Cruz); polyclonal anti-p19Arf (1:1,000; Abcam); monoclonal anti-NPM (NPMc; NPMa) (10); polyclonal anti- -tubulin (1:1,000, H-235, Santa Cruz); monoclonal antivinculin (1:1,000; hVIN-1; Sigma), monoclonal anti-Flag (1:1,000; Sigma), polyclonal anti-NPM (B19) (9), and anti-H2AX (1:1,000; Upstate) Immunofluorescence.

Techniques: Expressing, Transformation Assay, Double Knockout, Infection, Control, Selection, Western Blot