Journal: PLoS ONE

Article Title: Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

doi: 10.1371/journal.pone.0076793

Figure Lengend Snippet: Immunofluorescent flow cytometry. HEK293T transfectants were labeled with AP3 (anti-β3), 7E3 (anti-β3), 10E5 (anti-αIIb), and LM609 (anti-αV). Thick and thin lines show labeling of the αIIbβ3 transfectant and the mock transfectant, respectively.

Article Snippet: The expression levels of αIIb and β3 were detected by flow cytometry staining with the following monoclonal antibodies: AP3 (nonfunctional anti-β3 mAb, American Type Culture Collection), 7E3 (anti-β3 mAb), 10E5 (anti-αIIb mAb, kindly provided by B.S.

Techniques: Flow Cytometry, Labeling, Transfection

Journal: Oncogene

Article Title: CRTC1-MAML2 fusion-induced lncRNA LINC00473 expression maintains the growth and survival of human mucoepidermoid carcinoma cells

doi: 10.1038/s41388-017-0104-0

Figure Lengend Snippet: (a,b) Heatmap and volcano plot show differentially expressed genes in shLnc473 knockdown in compared with shRNA control in fusion-positive H3118 cells. The cutoff criteria were absolute fold-change of ≥2 and p<0.05. This analysis led to the identification of a total of 1320 LINC00473-regulated candidate coding genes, with 675 up-regulated and 645 down-regulated genes. (c) Functional classification of LINC00473 down-regulated genes was performed using Ingenuity Pathway Analysis (IPA). The top 6 molecular and cell functions were ranked based on p-value and activation z-score. Negative z-score indicates inhibition. (d) The qRT-PCR analysis showed that LncRNA LINC00473 was significantly enriched in the NONO immunoprecipitates relative to the IgG control in H3118 MEC cells. ASNS was used as a negative control (n=3, ***p<0.0001). (e) Overexpression of LINC00473 in HEK293T cells significantly increased the interaction of CRTC1-MAML2 and NONO by a Gal4-NONO reporter assay (n=3, *p<0.05). (f) Knockdown of NONO or LINC00473 expression significantly reduced the pCRE-luc reporter activities in HEK293T cells with CRTC1-MAML2 overexpression (n=3, *p<0.05 and **p<0.001). (g) A model for the molecular basis of LINC00473 induction by CRTC1-MAML2 and function in fusion-positive MEC cells.

Article Snippet: Cell lysates were collected for immunoprecipitation overnight at 4°C using anti-NONO antibodies (A300-587A, Bethyl Laboratories) control and IgG control (SC-2027, Santa Cruz) and protein A/G beads.

Techniques: Knockdown, shRNA, Control, Functional Assay, Activation Assay, Inhibition, Quantitative RT-PCR, Negative Control, Over Expression, Reporter Assay, Expressing

Journal: Nature

Article Title: Evolution of a central neural circuit underlies Drosophila mate preferences

doi: 10.1038/s41586-018-0322-9

Figure Lengend Snippet: Mutant males and males lacking foreleg tarsi still court, but display altered courtship preferences. a , Courtship indices of males with foreleg tarsi intact (+) or surgically removed (−). Data is replotted from . b , c , Courtship indices of D. melanogaster ( b ) and D. simulans ( c ) males with either foreleg tarsi or rear leg tarsi ablated towards D. melanogaster or D. simulans females. d , Schematic of CRISPR/Cas9-induced mutations (top) in Gr32a (left) and ppk23 (right) gene loci. Cas9 was targeted by gRNA to the first exon (cut site) of Gr32a or ppk23 . Cleaved DNA was repaired by non-homologous end-joining resulting in a 36 bp insertion/2 bp deletion in the Gr32a coding sequence and 90 bp insertion into the ppk23 coding sequence. Both indels resulted in in-frame stop codons (bottom, * highlighted red in resulting amino acid sequence). Forward (F) and reverse (R) genotyping primers are marked with a line. e , Courtship indices towards females of different Drosophila species by wild-type (+/+), Gr32a −/− and ppk23 −/− D. simulans males. f , Courtship indices of D. simulans males towards D. melanogaster and D. simulans females in preference assays. Data is replotted from . g , Courtship indices of D. simulans males towards D. simulans female perfumed with 7,11-HD (7, green) or ethanol (E, EtOH, blue). Data is replotted from . e , Kruskal-Wallis test, different letters mark significant differences. Black bars and dots: mean and s.d. Lines connect courtship indices of the same male towards the different female targets in a preference assay. Since the male can only court one female at a time, the paired points are inherently interdependent on each other, thus inappropriate for statistical analysis. See for details of statistical analyses.

Article Snippet: Primary antibodies used were 1:20 Mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank), 1:1000 Sheep anti-GFP (sim fru GFP , mel fru Gal4 >UAS-GCaMP , ppk23-Gal4>UAS GCaMP and R25E04-Gal4 , Bio-Rad #4745-1051) and 1:100 rabbit anti-GABA antibody ( D. simulans fru GFP , Catalog #A2052; Sigma, St. Louis, MO).

Techniques: Mutagenesis, CRISPR, Non-Homologous End Joining, Sequencing

Journal: Nature

Article Title: Evolution of a central neural circuit underlies Drosophila mate preferences

doi: 10.1038/s41586-018-0322-9

Figure Lengend Snippet: a , Predominant cuticular hydrocarbons of related species. b , Courtship preferences of D. melanogaster and D. simulans males with foreleg tarsi intact (+) or removed (−). c , d , Courtship indices ( c ) or preference indices ( d ) of wild-type (+/+), Gr32a −/− , and ppk23 −/− D. simulans males offered D. simulans and/or D. melanogaster females. e , Preference indices of wild-type or ppk23 −/− D. simulans males offered D. simulans females perfumed with ethanol (EtOH) or 7,11-HD. b , d , e , one-sample t-test. c, Kruskal-Wallis test. Bars: mean and s.d. See for details of statistical analyses.

Article Snippet: Primary antibodies used were 1:20 Mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank), 1:1000 Sheep anti-GFP (sim fru GFP , mel fru Gal4 >UAS-GCaMP , ppk23-Gal4>UAS GCaMP and R25E04-Gal4 , Bio-Rad #4745-1051) and 1:100 rabbit anti-GABA antibody ( D. simulans fru GFP , Catalog #A2052; Sigma, St. Louis, MO).

Techniques:

Journal: Nature

Article Title: Evolution of a central neural circuit underlies Drosophila mate preferences

doi: 10.1038/s41586-018-0322-9

Figure Lengend Snippet: a , Schematic of ventral nerve cord preparation used for imaging with Fru+ foreleg sensory neurons expressing GCaMP. b , c , Functional responses of Fru+ foreleg afferents in D. melanogaster ( b ) or D. simulans ( c ) males evoked by the taste of a D. melanogaster (m) or D. simulans (s) female. Representative activity traces with time of taps indicated (left) and average ΔF/F in wild-type and ppk23 −/− males (right). d , Functional responses (average ΔF/F) of ppk23+ foreleg afferents evoked by the taste of a D. melanogaster (m) or D. simulans (s) female. e , Courtship indices (CI) towards conspecific females during optogenetic stimulation of ppk23+ neurons in D. melanogaster and D. simulans males. b - d , Wilcoxon matched-pairs test, d, Kruskal-Wallis test. Black bars and dots: mean and s.d. Scale bar: 10 μm. See for details of statistical analyses.

Article Snippet: Primary antibodies used were 1:20 Mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank), 1:1000 Sheep anti-GFP (sim fru GFP , mel fru Gal4 >UAS-GCaMP , ppk23-Gal4>UAS GCaMP and R25E04-Gal4 , Bio-Rad #4745-1051) and 1:100 rabbit anti-GABA antibody ( D. simulans fru GFP , Catalog #A2052; Sigma, St. Louis, MO).

Techniques: Imaging, Expressing, Functional Assay, Activity Assay

Journal: Nature

Article Title: Evolution of a central neural circuit underlies Drosophila mate preferences

doi: 10.1038/s41586-018-0322-9

Figure Lengend Snippet: a - c , ppk23 promoter expression in D. melanogaster and D. simulans males in forelegs ( a ), ventral nerve cord ( b , VNC) and brain ( c ). Green: GFP. Grey: DIC. Magenta: neuropil counterstain. a , Number of ppk23+ sensory neuron soma in the first three tarsal segments of the foreleg (middle right). d , ppk23 neuron innervation in the first thoracic ganglion of the VNC of D. simulans females. ppk23+ sensory neurons display a characteristic sexually dimorphic expression pattern in the ventral nerve cord where they do not cross the midline in females, but do in males. e , Schematic of VNC imaging preparation (left). Functional responses evoked by individual taps of a female abdomen in Fru+ neurons (middle) and ppk23+ neurons (right) in the VNC of wild-type (WT) and ppk23 −/− D. melanogaster and D. simulans males. Data replotted from . f , Schematic of paired ppk23+ somatic imaging preparation (left) and functional responses of the paired neurons (cell A and B, see ) within a sensory bristle stimulated with 7,11-HD or ethanol (middle). Comparison of 7,11-HD responses in ppk23+ soma across species (right). a , unpaired t-test, e , Kruskal-Wallis test, different letters mark significant differences, and f , paired and unpaired t-tests. Scale bars represent 10 μm. See for details of statistical analyses.

Article Snippet: Primary antibodies used were 1:20 Mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank), 1:1000 Sheep anti-GFP (sim fru GFP , mel fru Gal4 >UAS-GCaMP , ppk23-Gal4>UAS GCaMP and R25E04-Gal4 , Bio-Rad #4745-1051) and 1:100 rabbit anti-GABA antibody ( D. simulans fru GFP , Catalog #A2052; Sigma, St. Louis, MO).

Techniques: Expressing, Imaging, Functional Assay, Comparison

Journal: Nature

Article Title: Evolution of a central neural circuit underlies Drosophila mate preferences

doi: 10.1038/s41586-018-0322-9

Figure Lengend Snippet: a - f , Anatomy ( a ) and optogenetic behavioral manipulations ( b - f) of P1 neurons using 71G01-Gal4 to drive the expression of CsChrimson. b , Courtship indices (top, right) towards a rotating magnet by D. melanogaster males pre-, during, and post-P1 neuron stimulation. Fraction of male flies courting (bottom, grey boxes: bright light illumination, see ). c , Courtship indices towards a magnet moving at different speeds during optogenetic P1 neuron stimulation in D. simulans males. d - f , Comparison of courtship indices towards magnet ( d ), D. simulans female ( e ), or D. melanogaster female ( f ) by D. simulans males of denoted genotypes, fed, or not fed retinal. g , In vivo preparation used to measure pheromone responses in the P1 neurons (top left) and overlay of the Fru+ (green) neurons and fasciculated P1 neuron processes (magenta). White box indicates approximate ROI imaged to measure P1 responses. h - j , Functional responses evoked by individual taps of a female abdomen in P1 neurons ( h ) and Fru+ neurons in the LPC ( i , j ) of wild-type (WT) and ppk23 −/− D .melanogaster and D. simulans males (data replotted from e, f). b , d - f, h - j , Kruskal-Wallis test and c , One-way ANOVA). Black bars: mean and s.d. See for details of statistical analyses.

Article Snippet: Primary antibodies used were 1:20 Mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank), 1:1000 Sheep anti-GFP (sim fru GFP , mel fru Gal4 >UAS-GCaMP , ppk23-Gal4>UAS GCaMP and R25E04-Gal4 , Bio-Rad #4745-1051) and 1:100 rabbit anti-GABA antibody ( D. simulans fru GFP , Catalog #A2052; Sigma, St. Louis, MO).

Techniques: Expressing, Comparison, In Vivo, Functional Assay

Journal: Nature

Article Title: Evolution of a central neural circuit underlies Drosophila mate preferences

doi: 10.1038/s41586-018-0322-9

Figure Lengend Snippet: a , Schematic of in vivo preparation used to measure pheromone responses in vAB3 processes in the brain (top). Representative fluorescence increase of vAB3 responses in D. melanogaster male evoked by tapping a D. melanogaster female (bottom left). GCaMP was expressed in vAB3 neurons using the AbdB-Gal4 driver. Anatomy of fasciculated vAB3 processes co-labeled by fru Gal4 (green) and AbdB-Gal4 (magenta) in the same in vivo preparation used for imaging (right). White box indicates approximate ROI analyzed for functional imaging. b - d , Functional responses evoked by the taste of female pheromones in the vAB3 processes of wild-type (WT) and ppk23 −/− males. b , Functional responses evoked by individual taps in vAB3 neurons in D. melanogaster labeled using AbdB-Gal4. c, Average responses of vAB3 neurons in ppk23 −/− D. melanogaster and D. simulans males in response to the taste of D. melanogaster (m) and D. simulans (s) females. GCaMP was expressed in vAB3 neurons using fru Gal4 . d , Functional responses evoked by individual taps in vAB3 neurons in wild type and ppk23 −/− mutant males. Data replotted from and Extended Data Fig. 6c. e , Expression of 25E04-Gal4 > UAS-GCaMP (green) with neuropil counterstain (magenta) in the brains of D. melanogaster (left) and D. simulans (right) males. f , Courtship indices towards conspecific females during optogenetic activation of mAL neurons in D. simulans males with parental controls. g , Functional responses evoked by individual taps in mAL neurons. Data replotted from . h , Average ΔF/F responses in Fru+ neurons of the LPC evoked by the taste of a female before and after local injection of picrotoxin, a GABA receptor antagonist, into the LPC. In males of both species, application of picrotoxin increased responses only to D. melanogaster female stimuli. Lines connect average functional responses in the same male towards the different female targets. b , d , f , g , Kruskal-Wallis test, different letters mark significant differences, and c , h , paired t-test. Black bars: mean and s.d. See for details of statistical analyses.

Article Snippet: Primary antibodies used were 1:20 Mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank), 1:1000 Sheep anti-GFP (sim fru GFP , mel fru Gal4 >UAS-GCaMP , ppk23-Gal4>UAS GCaMP and R25E04-Gal4 , Bio-Rad #4745-1051) and 1:100 rabbit anti-GABA antibody ( D. simulans fru GFP , Catalog #A2052; Sigma, St. Louis, MO).

Techniques: In Vivo, Fluorescence, Labeling, Imaging, Functional Assay, Mutagenesis, Expressing, Activation Assay, Injection

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of HIF-1α overexpression on several functional proteins associated with β-catenin nuclear translocation in the prostate cancer cell lines, LNCaP and C4-2B. (A) Western blot analysis of the protein expression of HIF-1α, Glut-1, VEGF, t-β-catenin, p-β-catenin-Ser45, p-β-catenin-Y654, t-GSK-3β and p-GSK-3β Ser9 at 72 h in the negative control, HIF-1α overexpression, β-catenin silenced and scrambled shRNA groups. (B–I) Statistic analysis of the expression of these functional proteins. Bar 1 indicates untreated cells, bar 2 indicates PcDNA3.1(−) control vector-transfected cells, bar 3 indicates PcDNA3.1(−)/HIF-1α plasmid-transfected cells, bar 4 indicates HIF-1α-overexpressing cells transfected with PSuper-Scramble-β-catenin-ShRNA control vector, and bar 5 indicates HIF-1α-overexpressing cells transfected with PSuper-β-catenin-shRNA plasmid.

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Over Expression, Functional Assay, Translocation Assay, Western Blot, Expressing, Negative Control, shRNA, Control, Plasmid Preparation, Transfection

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: HIF-1α transfection led to β-catenin translocation to the nucleus in the prostate cancer cell lines, LNCaP and C4-2B. (A and B) Immunofluorescence showing the expression and intercellular location of β-catenin at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups. (C and D) Nuclear expression of β-catenin at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups.

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Transfection, Translocation Assay, Immunofluorescence, Expressing, Negative Control, Over Expression

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of activation of the HIF-1α/β-catenin signaling pathway on the biological behavior in the prostate cancer cell lines, LNCaP and C4-2B. (A) Western blot analysis of the protein expression of epithelial transmembrane proteins (E-cadherin and CK18) and mesenchymal cytoplasm proteins (vimentin, VEGF, fibronectin and MMP-2) at 72 h. (B) Images of invasion assays in the negative control, HIF-1α overexpression and β-catenin silenced groups at 72 h. (C and D) Cell count of invasion assays in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells at 24, 48 and 72 h. (E and F) MTT assays in the negative control, HIF-1α overexpression and β-catenin silenced groups at 24, 48 and 72 h.

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Activation Assay, Western Blot, Expressing, Negative Control, Over Expression, Cell Counting

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of β-catenin nuclear translocation on cell cycle distribution in the prostate cancer cell lines, LNCaP and C4-2B. (A) Western blot analysis of the protein expression of cell cycle regulatory proteins, including p21, CDK1, p-CDK1, Chk1, p-Chk1, Chk2, p-Chk2, Rb and p-Rb at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells. (B–F) Statistic analysis of the expression of cell cycle regulators. Bar 1 indicates untreated cells, bar 2 indicates PcDNA3.1(−)/HIF-1α plasmid transfected cells, and bar 3 indicates HIF-1α high expression cells transfected with PSuper-β-catenin-shRNA plasmid. ● P>0.05; ▲ P<0.05; * P<0.01; ** P<0.001.

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Translocation Assay, Western Blot, Expressing, Negative Control, Over Expression, Plasmid Preparation, Transfection, shRNA

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of β-catenin nuclear translocation on apoptosis and non-homologous end joining (NHEJ) repairing potential in the prostate cancer cell lines, LNCaP and C4-2B. (A and B) Flow cytometric analysis of the cell cycle distribution (G0/G1, S and G2/M phase) at 72 h in the negative control, HIF-1α over-expression and β-catenin silenced groups. (C) Western blot analysis of the protein expression of apoptotic regulators (including caspase-3, cleaved-caspase-3, caspase-7, cleaved-caspase-7, cleaved-PARP-1 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL), the DNA double strand breaks (DSB) marker, γH2AX, and NHEJ repair proteins (Ku70, Ku80 and DNA-PKcs) at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups.

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Translocation Assay, Non-Homologous End Joining, Negative Control, Over Expression, Western Blot, Expressing, Marker

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of β-catenin nuclear translocation on cell cycle distribution, apoptosis and non-homologous end joining (NHEJ) repair following irradiation. (A–D) Results of flow cytometry of the cell cycle distribution and sub-G1 cell ratio at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (E and F) Colony formation assay showing colony-forming capability at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (G and H) ELISA results at 72 h showing cell death (DNA fragmentation) in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (I) Western blot analysis of the protein expression of cell cycle regulators (p21, CDK1, p-CDK1, Chk1, p-Chk1, Chk2, p-Chk2, Rb and p-Rb), apoptotic proteins (including caspase-3, cleaved-caspase-3, caspase-7, cleaved-caspase-7, cleaved-PARP-1 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL), the DNA double strand breaks (DSB) marker, γH2AX, and non-homologous end joining (NHEJ) repair proteins (Ku70, Ku80 and DNA-PKCs) at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation.

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Translocation Assay, Non-Homologous End Joining, Irradiation, Flow Cytometry, Negative Control, Over Expression, In Vitro, Colony Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Marker

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of β-catenin nuclear translocation on tumor growth following in vivo irradiation in mice. (A and B) Luminescence signal intensity representing tumor volume measured at 1, 6, 11, 16 and 21 days in the negative control, HIF-1α overexpression, and β-catenin silenced groups of the LNCaP orthotopic model using BALB/c-nu mice following irradiation. (C) Tumor wet weight on day 21 in the negative control, HIF-1α overexpression and β-catenin silenced groups of the LNCaP orthotopic model using BALB/c-nu mice following irradiation. (D and E) Vital imaging of tumor and tumor volume at 1, 6, 11, 16 and 21 days in the negative control, HIF-1α overexpression and β-catenin silenced groups of the C4-2B subcutaneous model using SCID mice following irradiation. (F) Tumor wet weight on day 21 in the negative control, HIF-1α overexpression and β-catenin silenced groups of the C4-2B subcutaneous model using SCID mice following irradiation (n=15/group).

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Translocation Assay, In Vivo, Irradiation, Negative Control, Over Expression, Imaging

Journal: International Journal of Oncology

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

doi: 10.3892/ijo.2018.4368

Figure Lengend Snippet: Effect of β-catenin nuclear translocation on tumor proliferation after in vivo irradiation in mice. (A) Immunohistochemistry of Ki67 and BRCA-1 in the negative control, HIF-1α overexpression and β-catenin silenced groups of the LNCaP orthotopic model using BALB/c-nu mice following irradiation. (B) Ratio of Ki67-positive cells in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP orthotopic model using BALB/c-nu mice following irradiation. (C) Ratio of BRCA-1-positive cells in the negative control, HIF-1α overexpression and β-catenin silenced groups of the LNCaP orthotopic model using BALB/c-nu mice after irradiation. (D) Immunohistochemistry of Ki67 and BRCA-1 in the negative control, HIF-1α overexpression and β-catenin silenced groups of the C4-2B subcutaneous model using SCID mice following irradiation. (E) Ratio of Ki67-positive cells in the negative control, HIF-1α overexpression and β-catenin silenced groups of the C4-2B subcutaneous model using SCID mice following irradiation. (F) Ratio of BRCA-1-positive cells in the negative control, HIF-1α overexpression and β-catenin silenced groups of the C4-2B subcutaneous model using SCID mice following irradiation (n=15/group).

Article Snippet: The cells were incubated at 37°C for 1 h with mouse anti-human β-catenin monoclonal antibody (sc7963, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:200.

Techniques: Translocation Assay, In Vivo, Irradiation, Immunohistochemistry, Negative Control, Over Expression

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: CRISPR/Cas9-disruption of Mss51 locus in C2C12 myoblasts. (A) Schematic of the Mss51 genomic locus with the CRISPR guide RNA (gRNA) target sequence enlarged, predicted cut site marked in red, exons marked in black, UTRs in white. At the cut site, a double-strand break occurred and was re-sealed by non-homologous end joining. (B) Production of Mss51 -disrupted cells was achieved by co-transfection of a dual-expression plasmid encoding the CRISPR gRNA (red) and the Cas9 endonuclease (blue), and a reporter plasmid encoding a disrupted GFP with two homology arms (dark green) flanking the CRISPR target site (red), which was cleaved by Cas9, re-sealed by homology-directed repair, and expressed functional GFP. Two populations of GFP-positive cells were collected by FACS sorting and expanded for further analysis – Mss51 -disrupted cells were collected as shown and in parallel, control cells were transfected with a GFP expression plasmid and subjected to the same FACS process. (C) Mss51 expression measured by qRT-PCR in control and Mss51 -disrupted populations over 6 days of differentiation, normalized to expression in proliferating control cells (Day 0) using reference genes Tfam and TBP ( n = 3). ** p < 0.01, *** p < 0.001.

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: CRISPR, Sequencing, Non-Homologous End Joining, Cotransfection, Expressing, Plasmid Preparation, Functional Assay, Transfection, Quantitative RT-PCR

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: Mss51 tissue expression. (A) Mss51 mRNA expression measured by qRT-PCR in brain, heart, kidney, liver, small intestine, diaphragm, and quadriceps of 10– 12 week-old female C57BL/6J mice, normalized to the quantity found in the brain using the reference gene Pgk1 ( n = 3). (B) Mss51 mRNA expression in soleus, diaphragm, long head of triceps brachii, extensor digitorum longus, and white vastus lateralis of the quadriceps of 10– 12 week-old female C57BL/6J mice, normalized to the expression levels found in the soleus using reference genes Pgk1 and TBP ( n = 3). (C) MSS51 expression across human tissue types determined by RNA-seq from the Genotype-Tissue Expression (GTEx) Portal showing MSS51 reads per kilobase per million reads mapped (RPKM). Data were downloaded from GTEx Portal on June 17, 2015. For qRT-PCR experiments, groups were significantly different from each other as determined by a one-way ANOVA ( n = 3, p < 0.01) and Bonferroni post hoc comparisons. Different letters above each bar signify statistically significant differences between the means in each group at p < 0.05.

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: Expressing, Quantitative RT-PCR, RNA Sequencing Assay

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: Mss51 expression in vitro . (A) Mss51 mRNA expression measured by qRT-PCR in proliferating and differentiating C2C12 cells. Expression level is normalized to proliferating (Day 0) cells using the reference gene TBP . (B) Mss51 expression in differentiated C2C12 myotubes treated with various growth factors and inhibitors: 300 ng/mL myostatin, 5 μ g/mL ActRIIB-Fc, 50 ng/mL TGF-β1, 10 μ g/mL TGF-β neutralizing antibody 1D11, 20 ng/mL Activin A, and 100 ng/mL IGF-1. Expression level is normalized to control (untreated) cells using reference genes β2 m and TBP . For (A) and (B), significant differences between groups were determined by a one-way ANOVA ( n = 3, p < 0.01) with Bonferroni post hoc comparisons. The annotation above each bar indicates statistically significant differences between the means in each group at p < 0.05: Groups sharing a letter designation were not significantly different from each other, while groups not sharing a letter were significantly different.

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: Expressing, In Vitro, Quantitative RT-PCR

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: Subcellular localization of MSS51 . Subcellular fractionation was performed on human deltoid samples from a biopsy and an autopsy and resulting fractions were subjected to SDS-PAGE. Immunoblotting was performed with the only current antibody specific to MSS51 (anti-human MSS51 , Acris Antibodies, San Diego, CA, USA) showing a band in the mitochondrial fraction of the predicted protein product size, 51 kDa. Loading controls were VDAC (mitochondrial), GAPDH (cytoplasmic), and Histone H3 (nuclear).

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: Fractionation, SDS Page, Western Blot

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: Mss51 -disrupted myoblasts proliferate and differentiate normally. (A) Proliferating control and Mss51 -disrupted myoblasts labeled with EdU (red) and Hoeschst 33342 (blue). (B) Quantification of EdU staining ( n = 6 wells imaged per sample). (C) Representative myosin heavy chain (MF20, green) staining of control and Mss51 -disrupted myotubes 2 days after induction of differentiation with nuclei stained by DAPI (blue). (D) Quantification of fusion index (percentage of nuclei found in MF20+ myotubes, n = 6). (E) Creatine kinase (CK) activity in myotubes after 6 days of differentiation. (F) Protein synthesis rates as measured by puromycin incorporation on the left and total protein stained by SYPRO Ruby on the right after 6 days of differentiation. (G) Densitometric analysis of SUnSET assay as shown in F, normalized to SYPRO Ruby-stained total protein ( n = 3). Differences between groups were not statistically significant.

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: Labeling, Staining, Activity Assay

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: Gene and protein expression in Mss51 -disrupted differentiated myotubes (A) Myosin heavy chain expression measured by qRT-PCR in control and Mss51 -disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP ( n = 3). (B) Expression of metabolic genes in control and Mss51 -disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP ( n = 3). (C) Western blots of control and Mss51 -disrupted C2C12 myotubes examining expression and phosphorylation of AMPK α . (D) Densitometric analysis of AMPK α expression normalized to GAPDH and of AMPK α phosphorylation normalized to both GAPDH and total AMPK α ( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Neuromuscular Diseases

Article Title: Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

doi: 10.3233/JND-150119

Figure Lengend Snippet: Glycolysis and Oxidative Phosphorylation in Mss51 -disrupted cells. (A) ATP production in control and Mss51 -disrupted myotubes ( n = 6). (B) Activity of C 16 :0 fatty acid β-oxidation in control and Mss51 -disrupted myotubes ( n = 6). (C) Glycolysis stress test measuring the extracellular acidification rate (ECAR) in control and Mss51 -disrupted myotubes treated with glucose, oligomycin, and 2-deoxy-D-glucose. (D) Glycolysis, glycolytic capacity, and glycolytic reserve calculated from the glycolysis stress test ( n = 10). (E) Mitochondrial stress test results comparing oxygen consumption rates (OCR) between control and Mss51 -disrupted myotubes treated with oligomycin, carbonyl cyanide-p-trifluoromethoxyphen (FCCP), and Antimycin A/Rotenone. (F) Basal respiration, ATP production, proton leak, maximum respiration, and spare respiratory capacity as calculated from the mitochondrial stress test ( n = 10). * p < 0.05, *** p < 0.001.

Article Snippet: At the time of publication, the only Mss51 antibody available is a human-specific, “anti-Zmynd17” (AP54659PU-N; Acris Antibodies, San Diego, CA, USA; 1:500).

Techniques: Activity Assay