Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Characterisation of H9C2 proliferating clones whose genome was modified within the Tmpo gene sequence by CRISPR-Cas9 to reduce or extinguish LAP2a protein expression. ( A ) Schema of the Tmpo gene and LAP2a protein domains. The following are indicated: the number of base pairs targeted by one of the SgRNAs within the Tmpo gene and the amino acid position; the mutations induced by our SgRNA (at protein level) for the four H9C2 clones selected as effectively edited to trigger a premature stop codon; the names of the WT +/+ (unedited CRISPR clones) and of the LAP2a +/- and LAP2a -/- clones considered throughout this study. ( B ) Western blots are shown for whole protein extracts of naive H9C2 and CRISPR clones (WT (21E10, 21B1, 22A11); LAP2a +/- (21H4, 22G2, 22G3) and LAP2a -/- (22B3)), as indicated. Proteins of interest were detected using anti LAP2a Ab (middle panel) and anti TMPO Ab (lower panel). Red Ponceau staining (upper panel) was used to normalize ECL signals. ( C ) The graphs show the ECL signal quantification of western blots (arbitrary units; a.u) after revelation with anti TMPO Ab as shown in 1B. The graphs present the individual values and means ± s.e.m. (N= 2 to 5 independent samples per clone). * p<0.05, ** p<0.01, **** p<0.0001 (Mann Whitney test). ( D ) Immunofluorescence of CRISPR clones (WT (22A11, 21B1), LAP2a -/- (22B3) and LAP2a +/- (21H4)), using a rabbit Ab to detect LAP2a (red), phalloidin to label cytoplasmic actin (green) and DAPI to label nuclear DNA (blue). Scale bar = 50 um. ( E ) The graphs represent the % of cells with either a negative (-) or relatively higher or lower (+, ++, +++) mean signal (a.u) as detected by immunofluorescence when using a rabbit anti LAP2a Ab, as shown in 1D. For the analysed experiment (N = 1), the total n (numbers of cell nuclei) were 184, 321, 174 and 216 for the clones 22B3, 22A11, 21H4 and 21B1, respectively. ( F ) The graph represents the cell doubling time (mean value ± s.e.m.) calculated for naive H9C2 cells, WT CRISPR clones, LAP2a +/- and LAP2a -/- CRISPR edited clones, as indicated. The individual values and means ± s.e.m are given. (N = 4 to 8 independent experiments per clone). * p<0.05; **** p<0.0001. (Mann Whitney test) ( G ) The graph represents the amount of cells that were either positively (green) or negatively (black) stained in situ by immunofluorescence for the proliferation marker Ki67. For the analysed experiment (N = 1), the total n (numbers of cells) were 499, 562 and 647 for the WT, LAP2a +/- and LAP2a -/- clones, respectively. * p<0.05. *** p<0.001 (Chi square test for a contingency table).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Clone Assay, Modification, Sequencing, CRISPR, Expressing, Western Blot, Staining, MANN-WHITNEY, Immunofluorescence, In Situ, Marker

Journal: American Journal of Translational Research

Article Title: Antitumor properties of arsenic trioxide-loaded CalliSpheres ® microspheres by transarterial chemoembolization in VX2 liver tumor rabbits: suppression of tumor growth, angiogenesis, and metastasis and elongation of survival

doi:

Figure Lengend Snippet: Antibodies

Article Snippet: CD31-positive cells were used to assess microvessel density (MVD), and any CD31-stained endothelial cell or cell cluster that was clearly separated from adjacent tissue elements was counted as a single countable microvessel [ 14 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Dilution ratio Company Country Mouse HIF-1 alpha Monoclonal Antibody 1:50 Invitrogen USA Mouse VEGF Monoclonal Antibody 1:20 Invitrogen USA Mouse MMP9 Monoclonal Antibody 1:500 Invitrogen USA Rabbit CD31 Polyclonal Antibody 1:200 Bioss Inc. USA Rabbit Twist Polyclonal Antibody 1:200 Novus Biologicals USA Rabbit E-cadherin Polyclonal Antibody 1:100 MyBioSource USA Mouse N-cadherin Monoclonal Antibody 1:20 Invitrogen USA Rabbit Vimentin Polyclonal Antibody 1:500 Abcam USA Mouse PCNA Monoclonal Antibody 1:1000 Novus Biologicals USA Goat Anti-Mouse IgG H&L 1:10000 Abcam USA Goat Anti-Rabbit IgG H&L 1:10000 Abcam USA Open in a separate window Antibodies

Techniques:

Journal: American Journal of Translational Research

Article Title: Antitumor properties of arsenic trioxide-loaded CalliSpheres ® microspheres by transarterial chemoembolization in VX2 liver tumor rabbits: suppression of tumor growth, angiogenesis, and metastasis and elongation of survival

doi:

Figure Lengend Snippet: Comparisons of the tumor apoptosis rate and PCNA-positive cells among the CSM-ATO, cTACE-ATO, TAE-CSM, and control groups. A: Comparison of the tumor apoptosis rate in VX2 liver tumor rabbits among the CSM-ATO, cTACE-ATO, TAE-CSM, and control groups. B: Comparison of tumor PCNA-positive cells in VX2 liver tumor rabbits among the CSM-ATO, cTACE-ATO, TAE-CSM, and control groups. C: Example images of the TUNEL assay (peak at 12 h) and PCNA expression detection by IHC staining (peak at 14 d) in the four groups. 12 h: 12 hours; 3 d: 3 days; 7 d: 7 days; 14 d: 14 days; CSM: CalliSpheres® microspheres; ATO: arsenic trioxide; cTACE: conventional transarterial chemoembolization; TAE: transcatheter arterial embolization; PCNA: proliferating cell nuclear antigen; TUNEL: terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling; IHC: immunohistochemical; ***, P<0.001; **, P<0.01; *, P<0.05; NS, not significant.

Article Snippet: CD31-positive cells were used to assess microvessel density (MVD), and any CD31-stained endothelial cell or cell cluster that was clearly separated from adjacent tissue elements was counted as a single countable microvessel [ 14 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Dilution ratio Company Country Mouse HIF-1 alpha Monoclonal Antibody 1:50 Invitrogen USA Mouse VEGF Monoclonal Antibody 1:20 Invitrogen USA Mouse MMP9 Monoclonal Antibody 1:500 Invitrogen USA Rabbit CD31 Polyclonal Antibody 1:200 Bioss Inc. USA Rabbit Twist Polyclonal Antibody 1:200 Novus Biologicals USA Rabbit E-cadherin Polyclonal Antibody 1:100 MyBioSource USA Mouse N-cadherin Monoclonal Antibody 1:20 Invitrogen USA Rabbit Vimentin Polyclonal Antibody 1:500 Abcam USA Mouse PCNA Monoclonal Antibody 1:1000 Novus Biologicals USA Goat Anti-Mouse IgG H&L 1:10000 Abcam USA Goat Anti-Rabbit IgG H&L 1:10000 Abcam USA Open in a separate window Antibodies

Techniques: Control, Comparison, TUNEL Assay, Expressing, Immunohistochemistry, End Labeling, Immunohistochemical staining

Journal: Oncology Letters

Article Title: Survivin and gliomas: A literature review

doi: 10.3892/ol.2016.4867

Figure Lengend Snippet: Summary of studies included in the present literature review.

Article Snippet: Mellai et al , 2008 , 20 , GBM, 20 , Rabbit polyclonal anti-survivin (catalog no., NB-500-201 K3; dilution, 1:500; Novus Biologicals LLC) , LI (each area) = percentage of positively stained cells in ≥1,000 cells. , Immunoreactive cases: 20/20 (100%). Survivin variably stained nuclei in viable areas; cytoplasmic staining inconstant and irregular, so nuclear staining considered for comparative evaluation. SI lower for survivin expression vs. Ki67. Positive association survivin vs. Ki67/MiB-1 (P=0.0001). No inverse association with AI (P=0.1547). No association with survival. , ( ) .

Techniques: Expressing, Staining, Negative Staining, Labeling, Activity Assay, Produced

Journal: BioMed Research International

Article Title: Improvement of Inflammation through Antioxidant Pathway of Gardeniae Fructus 50% EtOH Extract (GE) from Acute Reflux Esophagitis Rats

doi: 10.1155/2020/4826176

Figure Lengend Snippet: Western blot analysis of (a) survivin, (b) cytochrome c, (c) caspase-3, (d) Bax, and (e) Bcl-2 expressions. Nor: normal rats, Con: reflux esophagitis control rats, GL: GE 50 mg/kg treated reflux esophagitis rats, and GH: GE 100 mg/kg treated reflux esophagitis rats. (f) All data are expressed means ± SEM, ( n = 6) rats per group. Significance: ∗ p < 0.05, ∗∗ p < 0.01 versus RE control rat values.

Article Snippet: Mouse monoclonal antibodies against survivin (1 : 1,000, NB 500-205) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Reflux, Control

Journal: British Journal of Cancer

Article Title: Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma

doi: 10.1038/sj.bjc.6602904

Figure Lengend Snippet: Immunohistochemical staining of Survivin and fluorescent TUNEL staining in human gliomas ( A–G ). A primary glioblastoma showed a high level (++++) expression of nuclear Survivin ( A ). High staining score (++++) of cytoplasmic Survivin was detected in another primary glioblastoma ( B ). Moderate expression level (++) of cytoplasmic Survivin was detected in a pre-existing low-graded (grade II) glioma ( C ), which was obviously lower than that in its paired secondary glioblastoma with a staining score of ++++ ( D ). High-level (++++) expression of nuclear Survivin was detected in a secondary glioblastoma ( E ) and its matched pre-existing grade II glioma ( F ). Three apoptotic cells with clear nuclear staining (green color) were examined in a primary glioblastoma ( G ).

Article Snippet: The tumour slides were incubated at 4°C overnight in a moist chamber with one of the two polyclonal anti-Survivin antibodies (NB-500-201 K3, Novus Biologicals, Littleton, CO, USA, 1 : 300 dilution and SC-10811, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 400 dilution).

Techniques: Immunohistochemical staining, Staining, TUNEL Assay, Expressing

Journal: British Journal of Cancer

Article Title: Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma

doi: 10.1038/sj.bjc.6602904

Figure Lengend Snippet: Status of Survivin expression and apoptotic index in primary GBMs

Article Snippet: The tumour slides were incubated at 4°C overnight in a moist chamber with one of the two polyclonal anti-Survivin antibodies (NB-500-201 K3, Novus Biologicals, Littleton, CO, USA, 1 : 300 dilution and SC-10811, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 400 dilution).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma

doi: 10.1038/sj.bjc.6602904

Figure Lengend Snippet: Status of Survivin expression and apoptotic index in secondary GBMs

Article Snippet: The tumour slides were incubated at 4°C overnight in a moist chamber with one of the two polyclonal anti-Survivin antibodies (NB-500-201 K3, Novus Biologicals, Littleton, CO, USA, 1 : 300 dilution and SC-10811, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 400 dilution).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma

doi: 10.1038/sj.bjc.6602904

Figure Lengend Snippet: The expression of cytoplasmic and nuclear Survivin in 15 paired secondary GBMs and pre-existing lesions a

Article Snippet: The tumour slides were incubated at 4°C overnight in a moist chamber with one of the two polyclonal anti-Survivin antibodies (NB-500-201 K3, Novus Biologicals, Littleton, CO, USA, 1 : 300 dilution and SC-10811, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 400 dilution).

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 1 Semiquantitative RT-PCR and real-time PCR for Furin, TACE and AREG genes expression. (a) RT-PCR analysis of Furin, TACE and AREG mRNA extracted from SGEC treated with anti-Ro/SSA autoantibodies. M, marker; control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. RT-PCR of GADPH was used as control. Band intensities were analyzed by densitometry (b). (c) Real-time PCR for Furin, TACE and AREG genes expression. Representative histograms of mRNA levels of Furin, TACE and AREG in untreated SGEC (control), SGEC treated with healthy IgG (HIgG), anti-Ro/SSA autoantibodies (anti-Ro). The mRNA levels of the housekeeping gene, b-2 microglobulin, were quantified between untreated control cells and variously treated cells (data represent the mean±s.e. of five independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Marker, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 2 Analysis of Furin, TACE and AREG expression in anti-Ro/SSA Abs-treated SGEC. (a) Flow cytometric analysis of Furin, TACE and AREG expression in SGEC after anti-Ro/SSA Abs treatment. Examples of flow cytometric images from one representative experiment. (A) Furin expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (B) intracellular active TACE expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (C) AREG expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC. (b) Western blot analysis of Furin, TACE and AREG proteins expression in SGEC treated or not with anti-Ro/SSA. Immunoblotting gave rise to bands of the expected size (97 kDa for Furin, 80 kDa for active TACE and 50 kDa for AREG). b-Actin was used as protein loading control. (c) Detection of soluble AREG by ELISA. Secreted AREG was detected by ELISA in the conditioned medium. Control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. (Data represent the mean±s.e. of four independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Reduced-Beclin1-Expressing Mice Infected with Zika-R103451 and Viral-Associated Pathology during Pregnancy

doi: 10.3390/v12060608

Figure Lengend Snippet: ZIKV infection in Becn1 +/+ and Becn1 +/− pregnant dams. ( A ) Schematic diagram illustrating Zika virus (ZIKV)-infection in timed-pregnant dams. Prior to viral infection, pregnant dams received the antibody MAR1-5A3 at 2 mg/animal via intraperitoneal (ip) route at gestational day 8 followed by subcutaneous (sc) infection with ZIKV at 10 3 plaque-forming unit (PFU) in 50 µL of PBS or mock (PBS) injection at gestational day E9. ( B ) Representative Western Blots probed with antibodies against several autophagy proteins, and B-actin was used as an internal control. Adult Becn1 +/+ and Becn1 +/− brains were removed postmortem and minced according to the Materials and Methods. ( C ) Densitometric analysis using image J indicate the levels of p62, Beclin1, ATG5, LC3-I and LC3-II in brains of adult Becn1 +/+ (black bar) and Becn1 +/− (brown bar) mice. The error bars show mean ± SEM for N = 3 animals per treatment. The data were analyzed using GraphPad Prism and two-way analysis of variance (ANOVA) followed by Tukey’s test. * p < 0.05 and ** p < 0.01 vs. Becn1 +/+ . ( D ) Weight gain, expressed in grams, was measured using an analytical balance at gestation day 0 and throughout gestation period, at 3-day intervals. ( E ) Percent survival rate in pregnant dams infected with ZIKV or mock (PBS) was calculated by dividing the total number of live animals by the number of live + dead animals X 100. ( F ) Viral RNA detected in serum collected from ZIKV-infected dams on E13. ( G ) Viral RNA detected in organs removed postmortem from ZIKV-infected dams on E17. ( D – G ) Error bars show mean ± SEM for N = 5–8 animals per treatment. The data were analyzed using GraphPad Prism and two-way ANOVA followed by Tukey’s test. * p < 0.05 vs. Becn1 +/+ . ( F , G ) Viral RNA equivalent is expressed on a log10 scale after comparison with a standard curve produced using serial 10-fold dilutions of ZIKV RNA from known quantities of infectious virus.

Article Snippet: Immunoblots were labeled with primary antibodies against Beclin1 (Catalog# NB500–249), ATG5 (Catalog# NB110-53818), LC3-B (Catalog# NB600–1384) and P62/SQSTM1 (Catalog# NBP1–48320) purchased from Novus Biologicals (Centennial, CO, USA). β-actin (Catalog# sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as internal control.

Techniques: Infection, Virus, Injection, Western Blot, Control, Mass Measurement, Comparison, Produced

Journal: Viruses

Article Title: Reduced-Beclin1-Expressing Mice Infected with Zika-R103451 and Viral-Associated Pathology during Pregnancy

doi: 10.3390/v12060608

Figure Lengend Snippet: Potential link between ZIKV proteins and Beclin1 protein. ( A – D ) The secretions of pro-inflammatory molecules were detected in glial supernatants exposed to 50 nM of viral proteins after 8, 24- and 96-h by ELISA. Becn1 +/+ glia (black bar) and Becn1 +/− glia (brown bar). The data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05 vs. respective media control, # p < 0.05 vs. Becn1 +/+ .

Article Snippet: Immunoblots were labeled with primary antibodies against Beclin1 (Catalog# NB500–249), ATG5 (Catalog# NB110-53818), LC3-B (Catalog# NB600–1384) and P62/SQSTM1 (Catalog# NBP1–48320) purchased from Novus Biologicals (Centennial, CO, USA). β-actin (Catalog# sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as internal control.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: bioRxiv

Article Title: Delivery of loaded MR1 Monomer Results in Efficient Ligand Exchange to Host MR1 and Subsequent MR1T cell activation

doi: 10.1101/2022.07.11.499573

Figure Lengend Snippet: A . Biotinylated MR1 monomer, recombinant TCR, or HLA-A2 were immobilized on a traptavidin-coated plate before incubation with PBS at the indicated pH overnight at 4C. Binding of either anti-MR1 (clone 26.5) or anti-b2m (clone B2M-01) antibody was assess with an HRP-conjugated secondary antibody and TMB substrate. Error bars denote standard deviation. B . MR1T clone (5e3) IFN-γ response to BEAS-2B (1e4) pre-treated with Bafilomycin A1 for 2 hours, followed by addition of the MR1/5-OP-RU monomer or 5-OP-RU prodrug. Data in B are representative of three independent experiments.

Article Snippet: After removal of the acidic buffer and washing with PBS pH 7.2, either anti-human MR1 (unlabeled, clone 26.5, BioLegend 361102) or anti-human B2m (unlabeled, clone B2M-01, Novus Biologicals NB500-317) antibody was applied to the plate, followed by a rabbit anti-mouse IgG H+L, HRP-conjugated secondary antibody (Abcam ab6728).

Techniques: Recombinant, Incubation, Binding Assay, Standard Deviation