progressive deletions Search Results


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  • 99
    ATCC ags cells
    Comparison of in-vitro adherence of H. pylori <t>26695,</t> R.hpyAII, R.hpyAII-M2.hpyAII gene deletion strains on <t>AGS</t> cells. Adhesion of H. pylori 26695 and deletion strains to AGS cells was quantified by alamarBlue assay. Varying bacterial dilutions were added to constant number of AGS cells per well. The P-values were calculated using two-way ANOVA followed by Bonferroni's post-test. * P
    Ags Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pmir report luciferase vector
    Comparison of in-vitro adherence of H. pylori <t>26695,</t> R.hpyAII, R.hpyAII-M2.hpyAII gene deletion strains on <t>AGS</t> cells. Adhesion of H. pylori 26695 and deletion strains to AGS cells was quantified by alamarBlue assay. Varying bacterial dilutions were added to constant number of AGS cells per well. The P-values were calculated using two-way ANOVA followed by Bonferroni's post-test. * P
    Pmir Report Luciferase Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pmir report vector
    miR-1a-3p, miR-24-3p and miR-486-5p regulate MRTF-A via recognition sites on its 3′UTR. ( A ) Schematic of the 3′UTR of murine MRTF-A with the STOP-codon at position 1 and the polyadenylation signal at 985. Deleted miRNA binding sites are indicated by red boxes. ( B–F ) COS-7 cells were transiently transfected with <t>pMIR-REPORT</t> luciferase reporters, harboring the 3′UTR of murine MRTF-A or mutants thereof with a deletion of the predicted binding sites. The indicated miRNAs were co-transfected and their effects compared to a control miRNA ( miCtrl .). After 48 h luciferase activity was determined and normalized to co-transfected galactosidase activity. ( G ) Decrease of 3′UTR-controlled luciferase activity upon differentiation of C2C12. Cells were transiently transfected with the indicated reporters 48 h prior to myogenic differentiation. Shown is the reporter activity over the course of differentiation, normalized to the vector control ( pMIR empty ). ( H ) Relative 3′UTR luciferase reporter activity of the deletion constructs in C2C12 cells following myogenic differentiation for four days. Shown in comparison to MRTF-A wild type 3′UTR reporter which is set as 1. Error bars, SD ( n = 3); * P
    Pmir Report Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 4287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 6 ohda
    Injection of <t>6-OHDA</t> increases the power of spontaneous LFP oscillations in the OB in all frequency bands. ( a ) Sample ongoing LFP signals from one mouse before (left) and seven days after (right) injection of 6-OHDA. The first row shows the raw trace (8 s), the second to the fifth rows show filtered signals: θ (2 to 12 Hz), β (15 to 35 Hz), low-gamma (36 to 65 Hz), and high-gamma (66 to 95 Hz). ( b ) Averaged power spectra of theta (b1), beta (b2), low-gamma (b3), and high-gamma (b4) bands across the group of mice recorded. ( c ) Statistical analysis of the power in the different bands (theta (c1), beta (c2), low-gamma (c3), and high-gamma (c4)) before and seven days after 6-OHDA injection. A two-way ANOVA (with 6-OHDA/sham and frequency as the two factors) was performed to test the significance of the effect of 6-OHDA treatment on the LFP. Error bars represent s.e. ** P
    6 Ohda, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Funakoshi lymperopoulos a
    Injection of <t>6-OHDA</t> increases the power of spontaneous LFP oscillations in the OB in all frequency bands. ( a ) Sample ongoing LFP signals from one mouse before (left) and seven days after (right) injection of 6-OHDA. The first row shows the raw trace (8 s), the second to the fifth rows show filtered signals: θ (2 to 12 Hz), β (15 to 35 Hz), low-gamma (36 to 65 Hz), and high-gamma (66 to 95 Hz). ( b ) Averaged power spectra of theta (b1), beta (b2), low-gamma (b3), and high-gamma (b4) bands across the group of mice recorded. ( c ) Statistical analysis of the power in the different bands (theta (c1), beta (c2), low-gamma (c3), and high-gamma (c4)) before and seven days after 6-OHDA injection. A two-way ANOVA (with 6-OHDA/sham and frequency as the two factors) was performed to test the significance of the effect of 6-OHDA treatment on the LFP. Error bars represent s.e. ** P
    Lymperopoulos A, supplied by Funakoshi, used in various techniques. Bioz Stars score: 90/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Aviva Systems twist1
    <t>Twist1</t> deletion in basal keratinocytes suppresses TPA-induced epidermal proliferation
    Twist1, supplied by Aviva Systems, used in various techniques. Bioz Stars score: 93/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cxcr4  (Abcam)
    92
    Abcam cxcr4
    <t>miR-23a/CXCR4</t> regulates neuropathic pain via TXNIP. a , b Increased spinal TXNIP protein expression in pSNL mice was reversed by intrathecal injection of miR-23a mimics ( a ) or Lenti-miR-23a ( b ). Intrathecal injections began on day 7 after pSNL. TXNIP was measured at 24 h after 2-day miR-23a mimics injections or at 48 h after 3-day Lenti-miR-23a injections. One-way ANOVA (expression versus treatment groups) followed by post hoc Tukey test a F (3, 16) = 12.54; b F (3, 16) = 17.27, * p
    Cxcr4, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 3526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam caspr2
    Patients’ antibodies react with wild-type but not <t>Caspr2</t> -null mouse brains
    Caspr2, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Addgene inc nfatc1
    Patients’ antibodies react with wild-type but not <t>Caspr2</t> -null mouse brains
    Nfatc1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    c1qbp  (Abcam)
    90
    Abcam c1qbp
    Complementation Studies Using <t>C1qbp</t> −/− Mouse Fibroblasts (A) Quantification of C1QBP, COXI, and COXIII levels. Top: WT or C1qbp KO MEFs were transfected with the pcDNA3-C1qbp-IRES-GFP plasmid for 48 hr. Western blotting on total cell extracts was performed with anti-C1QBP, anti-COXI, anti-COXIII, anti-GFP, and anti-VDAC antibodies. VDAC was used as the loading control. Bottom: the mean ratios of band densities in transfected MEFs from blots are shown. Cells transfected with expression constructs for p.Gly244Trp and p.Leu272Pro variants show significantly lower amounts of mature C1QBP and mitochondrial-DNA-encoded proteins (COXI and COXIII). The results represent the mean ± SD of three independent experiments. ∗∗∗ p
    C1qbp, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Astex at13148
    <t>AT13148</t> reduces physical force and migration on pliable substrates. A, Directed scratch wound migration of AT13148, Y27632 or H1152 drug-treated cells was monitored over 48 hours. Left: Representative images at 0 and 24 hours for DMSO and 1 µm AT13148 treated cells. Upper right graph shows mean ± SEM percentage wound confluence acquired every 2 hours over 48 hours for n=6 replicates. Lower right graph shows mean ± SEM percentage wound confluence at 24 hours for n=3 independent experiments. B, Random migration of cells treated with AT13148, Y27632 or H1152 was monitored over 10 hours. Left panels show representative images at 0, 5 and 10 hours. Means ± SEM are shown from n = 3 independent experiments. C, Schematic diagram and representative images of cells on nanopillars with DMSO, AT13148 or Y27632 treatments as indicated. D, Forces leading to nanopillar deflection were determined for ≥10 pillars under 16 cells for AT13148 and Y27632 treatments, and for 32 cells for DMSO. Mean force values determined at each 2 minute interval over 120 minutes are plotted for each condition, with indicated means ± SEM. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p
    At13148, supplied by Astex, used in various techniques. Bioz Stars score: 90/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc anti mycn
    <t>AT13148</t> reduces physical force and migration on pliable substrates. A, Directed scratch wound migration of AT13148, Y27632 or H1152 drug-treated cells was monitored over 48 hours. Left: Representative images at 0 and 24 hours for DMSO and 1 µm AT13148 treated cells. Upper right graph shows mean ± SEM percentage wound confluence acquired every 2 hours over 48 hours for n=6 replicates. Lower right graph shows mean ± SEM percentage wound confluence at 24 hours for n=3 independent experiments. B, Random migration of cells treated with AT13148, Y27632 or H1152 was monitored over 10 hours. Left panels show representative images at 0, 5 and 10 hours. Means ± SEM are shown from n = 3 independent experiments. C, Schematic diagram and representative images of cells on nanopillars with DMSO, AT13148 or Y27632 treatments as indicated. D, Forces leading to nanopillar deflection were determined for ≥10 pillars under 16 cells for AT13148 and Y27632 treatments, and for 32 cells for DMSO. Mean force values determined at each 2 minute interval over 120 minutes are plotted for each condition, with indicated means ± SEM. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p
    Anti Mycn, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Johnson & Johnson van goethem
    <t>AT13148</t> reduces physical force and migration on pliable substrates. A, Directed scratch wound migration of AT13148, Y27632 or H1152 drug-treated cells was monitored over 48 hours. Left: Representative images at 0 and 24 hours for DMSO and 1 µm AT13148 treated cells. Upper right graph shows mean ± SEM percentage wound confluence acquired every 2 hours over 48 hours for n=6 replicates. Lower right graph shows mean ± SEM percentage wound confluence at 24 hours for n=3 independent experiments. B, Random migration of cells treated with AT13148, Y27632 or H1152 was monitored over 10 hours. Left panels show representative images at 0, 5 and 10 hours. Means ± SEM are shown from n = 3 independent experiments. C, Schematic diagram and representative images of cells on nanopillars with DMSO, AT13148 or Y27632 treatments as indicated. D, Forces leading to nanopillar deflection were determined for ≥10 pillars under 16 cells for AT13148 and Y27632 treatments, and for 32 cells for DMSO. Mean force values determined at each 2 minute interval over 120 minutes are plotted for each condition, with indicated means ± SEM. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p
    Van Goethem, supplied by Johnson & Johnson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    4Gene olfactomedin 4 gene
    <t>AT13148</t> reduces physical force and migration on pliable substrates. A, Directed scratch wound migration of AT13148, Y27632 or H1152 drug-treated cells was monitored over 48 hours. Left: Representative images at 0 and 24 hours for DMSO and 1 µm AT13148 treated cells. Upper right graph shows mean ± SEM percentage wound confluence acquired every 2 hours over 48 hours for n=6 replicates. Lower right graph shows mean ± SEM percentage wound confluence at 24 hours for n=3 independent experiments. B, Random migration of cells treated with AT13148, Y27632 or H1152 was monitored over 10 hours. Left panels show representative images at 0, 5 and 10 hours. Means ± SEM are shown from n = 3 independent experiments. C, Schematic diagram and representative images of cells on nanopillars with DMSO, AT13148 or Y27632 treatments as indicated. D, Forces leading to nanopillar deflection were determined for ≥10 pillars under 16 cells for AT13148 and Y27632 treatments, and for 32 cells for DMSO. Mean force values determined at each 2 minute interval over 120 minutes are plotted for each condition, with indicated means ± SEM. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p
    Olfactomedin 4 Gene, supplied by 4Gene, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Covance icam 5
    sICAM-5 does not affect Th17—APC interactions. (A) FACS staining for LFA-1 of naïve T-cells, Th17 cells, and Th17 cells after 24 h of stimulation with anti-CD3/CD28 ( n = 3 for each condition). (B) Representative depiction of CFSE stainings of naïve 2d2 T cells cocultured with APCs and sICAM-5 (10 μg/ml) or IgG (10 μg/ml) for 72 h. (C) FACS staining of Th17 cells stimulated for 4 and 24 h with anti-CD3/CD28 and with or without <t>ICAM-5</t> treatment (10 μg/ml). (D,E) Naïve T cells were differentiated into Th17 cells in the presence of or without sICAM-5 treatment (10 μg/ml) and stained for extracellular (D) and intracellular (E) markers. Data shown are mean ± SEM. (A) One-way ANOVA followed by Tukey's multiple comparison test, n.s. no significance; (C–E) Student's t -test.
    Icam 5, supplied by Covance, used in various techniques. Bioz Stars score: 93/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mimetics bh3 mimetics
    sICAM-5 does not affect Th17—APC interactions. (A) FACS staining for LFA-1 of naïve T-cells, Th17 cells, and Th17 cells after 24 h of stimulation with anti-CD3/CD28 ( n = 3 for each condition). (B) Representative depiction of CFSE stainings of naïve 2d2 T cells cocultured with APCs and sICAM-5 (10 μg/ml) or IgG (10 μg/ml) for 72 h. (C) FACS staining of Th17 cells stimulated for 4 and 24 h with anti-CD3/CD28 and with or without <t>ICAM-5</t> treatment (10 μg/ml). (D,E) Naïve T cells were differentiated into Th17 cells in the presence of or without sICAM-5 treatment (10 μg/ml) and stained for extracellular (D) and intracellular (E) markers. Data shown are mean ± SEM. (A) One-way ANOVA followed by Tukey's multiple comparison test, n.s. no significance; (C–E) Student's t -test.
    Bh3 Mimetics, supplied by Mimetics, used in various techniques. Bioz Stars score: 92/100, based on 7612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    AstraZeneca osimertinib
    sICAM-5 does not affect Th17—APC interactions. (A) FACS staining for LFA-1 of naïve T-cells, Th17 cells, and Th17 cells after 24 h of stimulation with anti-CD3/CD28 ( n = 3 for each condition). (B) Representative depiction of CFSE stainings of naïve 2d2 T cells cocultured with APCs and sICAM-5 (10 μg/ml) or IgG (10 μg/ml) for 72 h. (C) FACS staining of Th17 cells stimulated for 4 and 24 h with anti-CD3/CD28 and with or without <t>ICAM-5</t> treatment (10 μg/ml). (D,E) Naïve T cells were differentiated into Th17 cells in the presence of or without sICAM-5 treatment (10 μg/ml) and stained for extracellular (D) and intracellular (E) markers. Data shown are mean ± SEM. (A) One-way ANOVA followed by Tukey's multiple comparison test, n.s. no significance; (C–E) Student's t -test.
    Osimertinib, supplied by AstraZeneca, used in various techniques. Bioz Stars score: 91/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Ipsen Group cdk5
    Complementation of cell stress, polarity, and cell cycle phenotypes in a pho85 Δ strain by <t>CDK5</t> expression. ( A ) Salt sensitivity. Serial dilutions of log phase cultures of pho85 or wild-type cells expressing either CDK5 or CDK5-I plasmids were plated on SG-URA medium with 0.75 M NaCl or on an SD-URA plate (control) and incubated for 5 days at 30°C. ( B ) Overexpression of CDK5 in a cln1 Δ cln2 Δ pho85 Δ strain. A cln1Δ cln2Δ pho85Δ strain carrying a pho85 mutant allele ( pho85 ts17 ) expressed from the MET25 promoter (BY794) and either pCDK5 (right side of plates) or pCDK5-I (left side of plates) was streaked on a galactose-containing plate plus methionine (+ Gal + Met, Right ; repress pho85 ts17 , induce CDK5 ) or a galactose-containing plate minus methionine (control, Left ) and incubated for 5 days at 30°C. Two independent isolates are shown. ( C ) Overexpression of CDK5 in a slt2 Δ pho85 Δ strain. A slt2 Δ pho85 Δ strain expressing either PHO85 (left side of plates) or CDK5 (right side of plates) from the GAL promoter was streaked onto a yeast extract/peptone/dextrose (YPD) or yeast extract/peptone/galactose (YPG) plate as indicated and incubated for 5 days at 25°C. ( D ) Pho4 kinase activity associated with HA-Pcl2-Cdk5. Ha-Pcl2 was immunoprecipitated from yeast extracts and used in kinase assays with Pho4 as a substrate. The following strains were used for extract preparation: lane 1, pho85 Δ HA-PCL2 with pCDK5; lane 2, pho85 Δ HA-PCL2 with pCDK5-I; lane 3, pho85 Δ HA-PCL2 with pPHO85 (positive control).
    Cdk5, supplied by Ipsen Group, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AstraZeneca gefitinib
    Complementation of cell stress, polarity, and cell cycle phenotypes in a pho85 Δ strain by <t>CDK5</t> expression. ( A ) Salt sensitivity. Serial dilutions of log phase cultures of pho85 or wild-type cells expressing either CDK5 or CDK5-I plasmids were plated on SG-URA medium with 0.75 M NaCl or on an SD-URA plate (control) and incubated for 5 days at 30°C. ( B ) Overexpression of CDK5 in a cln1 Δ cln2 Δ pho85 Δ strain. A cln1Δ cln2Δ pho85Δ strain carrying a pho85 mutant allele ( pho85 ts17 ) expressed from the MET25 promoter (BY794) and either pCDK5 (right side of plates) or pCDK5-I (left side of plates) was streaked on a galactose-containing plate plus methionine (+ Gal + Met, Right ; repress pho85 ts17 , induce CDK5 ) or a galactose-containing plate minus methionine (control, Left ) and incubated for 5 days at 30°C. Two independent isolates are shown. ( C ) Overexpression of CDK5 in a slt2 Δ pho85 Δ strain. A slt2 Δ pho85 Δ strain expressing either PHO85 (left side of plates) or CDK5 (right side of plates) from the GAL promoter was streaked onto a yeast extract/peptone/dextrose (YPD) or yeast extract/peptone/galactose (YPG) plate as indicated and incubated for 5 days at 25°C. ( D ) Pho4 kinase activity associated with HA-Pcl2-Cdk5. Ha-Pcl2 was immunoprecipitated from yeast extracts and used in kinase assays with Pho4 as a substrate. The following strains were used for extract preparation: lane 1, pho85 Δ HA-PCL2 with pCDK5; lane 2, pho85 Δ HA-PCL2 with pCDK5-I; lane 3, pho85 Δ HA-PCL2 with pPHO85 (positive control).
    Gefitinib, supplied by AstraZeneca, used in various techniques. Bioz Stars score: 92/100, based on 1603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tead1  (Abcam)
    90
    Abcam tead1
    Loss of <t>Tead1</t> leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p
    Tead1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher cd133
    Loss of <t>Tead1</t> leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p
    Cd133, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Orexigen agrp
    Loss of <t>Tead1</t> leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p
    Agrp, supplied by Orexigen, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Mimotopes autoreactive t cells
    Loss of <t>Tead1</t> leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p
    Autoreactive T Cells, supplied by Mimotopes, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega luciferase reporter plasmid
    Loss of <t>Tead1</t> leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p
    Luciferase Reporter Plasmid, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd68  (Abcam)
    92
    Abcam cd68
    Loss of SOCS3 in mature myofibers in vivo enhances the inflammatory response after myotoxic injury. Control (SOCS3 fl/fl MCK-Cre − ) and SOCS3 MKO (SOCS3 fl/fl MCK-Cre + ) mice were either left uninjured (UN) or received a single 40 μL injection of notexin (10 μg/ml) into the right TA muscle and then killed for analysis at 1 day (D1), 2 days (D2), 3 days (D3), 5 days (D5), 7 days (D7), or 14 days (D14) post-notexin injury. a Western immunoblotting for phosphorylated and total STAT3 protein levels was performed on protein extracted from remaining OCT embedded muscles following tissue sectioning. Representative immunoblots for phosphorylated ( top ) and total ( bottom ) STAT3 protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories) and the ratio of phosphorylated/total STAT3 protein levels was determined. b Representative sections immunostained with F4/80 ( green ) and DAPI ( blue ) of the TA muscle from uninjured or day 1, 2, 3, 5, 7, or 14 injured control and SOCS3 MKO mice. c Representative sections immunostained with <t>CD68</t> ( red ) and DAPI ( blue ) of the TA muscle from uninjured or day 1, 2, 3, 5, 7, or 14 injured control and SOCS3 MKO mice. qRT-PCR using primers to detect IL-6 ( d ), IFN-γ ( e ), TNF-α ( f ), F4/80 ( g ), and CD68 ( h ) was performed on RNA extracted from snap frozen muscles following dissection. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-way ANOVA with a Fisher’s LSD post hoc multiple comparisons test to determine the effects of genotype and time. n = 8 mice/time-point/genotype. ** P
    Cd68, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 3483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of in-vitro adherence of H. pylori 26695, R.hpyAII, R.hpyAII-M2.hpyAII gene deletion strains on AGS cells. Adhesion of H. pylori 26695 and deletion strains to AGS cells was quantified by alamarBlue assay. Varying bacterial dilutions were added to constant number of AGS cells per well. The P-values were calculated using two-way ANOVA followed by Bonferroni's post-test. * P

    Journal: Nucleic Acids Research

    Article Title: N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori

    doi: 10.1093/nar/gky126

    Figure Lengend Snippet: Comparison of in-vitro adherence of H. pylori 26695, R.hpyAII, R.hpyAII-M2.hpyAII gene deletion strains on AGS cells. Adhesion of H. pylori 26695 and deletion strains to AGS cells was quantified by alamarBlue assay. Varying bacterial dilutions were added to constant number of AGS cells per well. The P-values were calculated using two-way ANOVA followed by Bonferroni's post-test. * P

    Article Snippet: Twenty four hours coculture of H. pylori 26695 strain and deletion strains with AGS cells were carried out to monitor the effect of m4C on H. pylori -induced AGS cell apoptosis.

    Techniques: In Vitro, Alamar Blue Assay

    miR-1a-3p, miR-24-3p and miR-486-5p regulate MRTF-A via recognition sites on its 3′UTR. ( A ) Schematic of the 3′UTR of murine MRTF-A with the STOP-codon at position 1 and the polyadenylation signal at 985. Deleted miRNA binding sites are indicated by red boxes. ( B–F ) COS-7 cells were transiently transfected with pMIR-REPORT luciferase reporters, harboring the 3′UTR of murine MRTF-A or mutants thereof with a deletion of the predicted binding sites. The indicated miRNAs were co-transfected and their effects compared to a control miRNA ( miCtrl .). After 48 h luciferase activity was determined and normalized to co-transfected galactosidase activity. ( G ) Decrease of 3′UTR-controlled luciferase activity upon differentiation of C2C12. Cells were transiently transfected with the indicated reporters 48 h prior to myogenic differentiation. Shown is the reporter activity over the course of differentiation, normalized to the vector control ( pMIR empty ). ( H ) Relative 3′UTR luciferase reporter activity of the deletion constructs in C2C12 cells following myogenic differentiation for four days. Shown in comparison to MRTF-A wild type 3′UTR reporter which is set as 1. Error bars, SD ( n = 3); * P

    Journal: Nucleic Acids Research

    Article Title: Post-transcriptional regulation of MRTF-A by miRNAs during myogenic differentiation of myoblasts

    doi: 10.1093/nar/gkaa596

    Figure Lengend Snippet: miR-1a-3p, miR-24-3p and miR-486-5p regulate MRTF-A via recognition sites on its 3′UTR. ( A ) Schematic of the 3′UTR of murine MRTF-A with the STOP-codon at position 1 and the polyadenylation signal at 985. Deleted miRNA binding sites are indicated by red boxes. ( B–F ) COS-7 cells were transiently transfected with pMIR-REPORT luciferase reporters, harboring the 3′UTR of murine MRTF-A or mutants thereof with a deletion of the predicted binding sites. The indicated miRNAs were co-transfected and their effects compared to a control miRNA ( miCtrl .). After 48 h luciferase activity was determined and normalized to co-transfected galactosidase activity. ( G ) Decrease of 3′UTR-controlled luciferase activity upon differentiation of C2C12. Cells were transiently transfected with the indicated reporters 48 h prior to myogenic differentiation. Shown is the reporter activity over the course of differentiation, normalized to the vector control ( pMIR empty ). ( H ) Relative 3′UTR luciferase reporter activity of the deletion constructs in C2C12 cells following myogenic differentiation for four days. Shown in comparison to MRTF-A wild type 3′UTR reporter which is set as 1. Error bars, SD ( n = 3); * P

    Article Snippet: Murine MRTF-A 3′UTR region was obtained by PCR from cDNA and cloned into pMIR-REPORT vector (Thermo Fisher, Waltham, MA, USA).

    Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Construct

    Injection of 6-OHDA increases the power of spontaneous LFP oscillations in the OB in all frequency bands. ( a ) Sample ongoing LFP signals from one mouse before (left) and seven days after (right) injection of 6-OHDA. The first row shows the raw trace (8 s), the second to the fifth rows show filtered signals: θ (2 to 12 Hz), β (15 to 35 Hz), low-gamma (36 to 65 Hz), and high-gamma (66 to 95 Hz). ( b ) Averaged power spectra of theta (b1), beta (b2), low-gamma (b3), and high-gamma (b4) bands across the group of mice recorded. ( c ) Statistical analysis of the power in the different bands (theta (c1), beta (c2), low-gamma (c3), and high-gamma (c4)) before and seven days after 6-OHDA injection. A two-way ANOVA (with 6-OHDA/sham and frequency as the two factors) was performed to test the significance of the effect of 6-OHDA treatment on the LFP. Error bars represent s.e. ** P

    Journal: Scientific Reports

    Article Title: Partial depletion of dopaminergic neurons in the substantia nigra impairs olfaction and alters neural activity in the olfactory bulb

    doi: 10.1038/s41598-018-36538-2

    Figure Lengend Snippet: Injection of 6-OHDA increases the power of spontaneous LFP oscillations in the OB in all frequency bands. ( a ) Sample ongoing LFP signals from one mouse before (left) and seven days after (right) injection of 6-OHDA. The first row shows the raw trace (8 s), the second to the fifth rows show filtered signals: θ (2 to 12 Hz), β (15 to 35 Hz), low-gamma (36 to 65 Hz), and high-gamma (66 to 95 Hz). ( b ) Averaged power spectra of theta (b1), beta (b2), low-gamma (b3), and high-gamma (b4) bands across the group of mice recorded. ( c ) Statistical analysis of the power in the different bands (theta (c1), beta (c2), low-gamma (c3), and high-gamma (c4)) before and seven days after 6-OHDA injection. A two-way ANOVA (with 6-OHDA/sham and frequency as the two factors) was performed to test the significance of the effect of 6-OHDA treatment on the LFP. Error bars represent s.e. ** P

    Article Snippet: A dental drill was used to make a craniotomy and animals received unilateral or bilateral injections of either 1 μl 6-OHDA (H4381, Sigma; 2 mg/ml 6-OHDA dissolved in 0.9% saline containing 0.02% ascorbic acid) or equivoluminal 0.9% saline containing 0.02% ascorbic acid (sham group).

    Techniques: Injection, Mouse Assay

    Injection of 6-OHDA decreases the odor-evoked LFP response in the OB. ( a ) Raw LFP traces (top row), filtered beta band (middle row), and filtered high-gamma bands (bottom row) in response to odor (isoamyl acetate) stimulation from one mouse before (left) and seven days after (right) 6-OHDA application. ( b , e ) Normalized odor-evoked beta ( b ) and high-gamma ( e ) responses before and seven days after 6-OHDA injection for all animals and odors ( n = 38). The dotted line shows the diagonal, where the response amplitude before 6-OHDA injection is equal to that seven days after 6-OHDA application. O1–O6 indicate isoamyl acetate, 2-heptanone, phenyl acetate, benzaldehyde, dimethylbutyric acid, and n-Heptane acid, respectively. ( c , f ) Comparison of odor-evoked normalized beta responses ( c ) and high-gamma responses ( f ) before and seven days after 6-OHDA injection across the group of mice recorded. * P

    Journal: Scientific Reports

    Article Title: Partial depletion of dopaminergic neurons in the substantia nigra impairs olfaction and alters neural activity in the olfactory bulb

    doi: 10.1038/s41598-018-36538-2

    Figure Lengend Snippet: Injection of 6-OHDA decreases the odor-evoked LFP response in the OB. ( a ) Raw LFP traces (top row), filtered beta band (middle row), and filtered high-gamma bands (bottom row) in response to odor (isoamyl acetate) stimulation from one mouse before (left) and seven days after (right) 6-OHDA application. ( b , e ) Normalized odor-evoked beta ( b ) and high-gamma ( e ) responses before and seven days after 6-OHDA injection for all animals and odors ( n = 38). The dotted line shows the diagonal, where the response amplitude before 6-OHDA injection is equal to that seven days after 6-OHDA application. O1–O6 indicate isoamyl acetate, 2-heptanone, phenyl acetate, benzaldehyde, dimethylbutyric acid, and n-Heptane acid, respectively. ( c , f ) Comparison of odor-evoked normalized beta responses ( c ) and high-gamma responses ( f ) before and seven days after 6-OHDA injection across the group of mice recorded. * P

    Article Snippet: A dental drill was used to make a craniotomy and animals received unilateral or bilateral injections of either 1 μl 6-OHDA (H4381, Sigma; 2 mg/ml 6-OHDA dissolved in 0.9% saline containing 0.02% ascorbic acid) or equivoluminal 0.9% saline containing 0.02% ascorbic acid (sham group).

    Techniques: Injection, Mouse Assay

    No significant locomotor impairments in mice with partial depletion of SN dopaminergic neurons after injection of 6-OHDA. ( a , c ) TH immunostaining in SN ( a ) and OB ( c ) from a representative mouse. Left and right images are contralateral and ipsilateral to the 6-OHDA SN injection site, respectively. ( b , d ) Comparison of the number of TH + cells in the ipsilateral and contralateral SN ( b ) and OB ( d ) after unilateral SN 6-OHDA across all the 6 mice. ( e , f ) Time on the rod in the rotarod test for 6-OHDA treated mice and sham mice injected unilaterally ( e ) or bilaterally ( f ). ( g ) Number of apomorphine-induced rotations in 30 minutes in 6-OHDA treated mice and sham mice. ( h ) Time on the rod in the rotarod test in 6-OHDA treated mice and sham mice 28 days after drug injection. Error bars represent s.e. ** P

    Journal: Scientific Reports

    Article Title: Partial depletion of dopaminergic neurons in the substantia nigra impairs olfaction and alters neural activity in the olfactory bulb

    doi: 10.1038/s41598-018-36538-2

    Figure Lengend Snippet: No significant locomotor impairments in mice with partial depletion of SN dopaminergic neurons after injection of 6-OHDA. ( a , c ) TH immunostaining in SN ( a ) and OB ( c ) from a representative mouse. Left and right images are contralateral and ipsilateral to the 6-OHDA SN injection site, respectively. ( b , d ) Comparison of the number of TH + cells in the ipsilateral and contralateral SN ( b ) and OB ( d ) after unilateral SN 6-OHDA across all the 6 mice. ( e , f ) Time on the rod in the rotarod test for 6-OHDA treated mice and sham mice injected unilaterally ( e ) or bilaterally ( f ). ( g ) Number of apomorphine-induced rotations in 30 minutes in 6-OHDA treated mice and sham mice. ( h ) Time on the rod in the rotarod test in 6-OHDA treated mice and sham mice 28 days after drug injection. Error bars represent s.e. ** P

    Article Snippet: A dental drill was used to make a craniotomy and animals received unilateral or bilateral injections of either 1 μl 6-OHDA (H4381, Sigma; 2 mg/ml 6-OHDA dissolved in 0.9% saline containing 0.02% ascorbic acid) or equivoluminal 0.9% saline containing 0.02% ascorbic acid (sham group).

    Techniques: Mouse Assay, Injection, Immunostaining

    Injection of 6-OHDA enhances odor-evoked mitral cell calcium responses in the OB. ( a ) Schematic for recording calcium signals by fiber photometry from mitral cells expressing GCaMP6s in Thy1-cre mice. ( b ) Expression of GCaMP6s in the MCL. ( c ) Representative calcium transient hit maps in response to six different odors from one mouse before (upper row) and seven days after 6-OHDA injection (middle row). The plots (bottom row) show the average odor-evoked calcium response from ten trials before (black line) and seven days after 6-OHDA application (red line). ( d ) Normalized odor-evoked calcium responses before 6-OHDA injection and seven days after 6-OHDA injection for all animals and odors ( n = 48, 8 mice with 6 odors). The dotted line shows the diagonal, where response amplitude before 6-OHDA application is equal to that seven days after 6-OHDA application. ( e ) Odor-evoked calcium responses before and seven days after 6-OHDA treatment across the group of mice recorded. Error bars represent s.e. *** P

    Journal: Scientific Reports

    Article Title: Partial depletion of dopaminergic neurons in the substantia nigra impairs olfaction and alters neural activity in the olfactory bulb

    doi: 10.1038/s41598-018-36538-2

    Figure Lengend Snippet: Injection of 6-OHDA enhances odor-evoked mitral cell calcium responses in the OB. ( a ) Schematic for recording calcium signals by fiber photometry from mitral cells expressing GCaMP6s in Thy1-cre mice. ( b ) Expression of GCaMP6s in the MCL. ( c ) Representative calcium transient hit maps in response to six different odors from one mouse before (upper row) and seven days after 6-OHDA injection (middle row). The plots (bottom row) show the average odor-evoked calcium response from ten trials before (black line) and seven days after 6-OHDA application (red line). ( d ) Normalized odor-evoked calcium responses before 6-OHDA injection and seven days after 6-OHDA injection for all animals and odors ( n = 48, 8 mice with 6 odors). The dotted line shows the diagonal, where response amplitude before 6-OHDA application is equal to that seven days after 6-OHDA application. ( e ) Odor-evoked calcium responses before and seven days after 6-OHDA treatment across the group of mice recorded. Error bars represent s.e. *** P

    Article Snippet: A dental drill was used to make a craniotomy and animals received unilateral or bilateral injections of either 1 μl 6-OHDA (H4381, Sigma; 2 mg/ml 6-OHDA dissolved in 0.9% saline containing 0.02% ascorbic acid) or equivoluminal 0.9% saline containing 0.02% ascorbic acid (sham group).

    Techniques: Injection, Expressing, Mouse Assay

    Injection of 6-OHDA into the SN impairs odor discrimination and odor spatial memory in mice. ( a1 ) Schematic for the odor habituation/dishabituation test in C57BL/6 J mice. ( a2 , a3 ) Odor habituation (the same odor stimulation repeated four times with 2 min interval, 1 st –4 th trial) and dishabituation (novel odorant on the 5 th trial) in sham mice ( a2 , n = 14 ) and 6-OHDA treated mice ( a3 , n = 14 ). ( b1 ) Schematic for the olfactory spatial memory test in C57BL/6 J mice. ( b2 , b3 ) Number of visits to the control and switched odors in the training trials (trial 1 and 2) and the recall trial (trial 3) in sham mice ( b2 , n = 10 ) and 6-OHDA treated mice ( b3 , n = 10 ). ( c ) Latency to locate a food pellet in sham and 6-OHDA treated mice for a buried food pellet (Buried) or food on the surface of the bedding (Visual). * P

    Journal: Scientific Reports

    Article Title: Partial depletion of dopaminergic neurons in the substantia nigra impairs olfaction and alters neural activity in the olfactory bulb

    doi: 10.1038/s41598-018-36538-2

    Figure Lengend Snippet: Injection of 6-OHDA into the SN impairs odor discrimination and odor spatial memory in mice. ( a1 ) Schematic for the odor habituation/dishabituation test in C57BL/6 J mice. ( a2 , a3 ) Odor habituation (the same odor stimulation repeated four times with 2 min interval, 1 st –4 th trial) and dishabituation (novel odorant on the 5 th trial) in sham mice ( a2 , n = 14 ) and 6-OHDA treated mice ( a3 , n = 14 ). ( b1 ) Schematic for the olfactory spatial memory test in C57BL/6 J mice. ( b2 , b3 ) Number of visits to the control and switched odors in the training trials (trial 1 and 2) and the recall trial (trial 3) in sham mice ( b2 , n = 10 ) and 6-OHDA treated mice ( b3 , n = 10 ). ( c ) Latency to locate a food pellet in sham and 6-OHDA treated mice for a buried food pellet (Buried) or food on the surface of the bedding (Visual). * P

    Article Snippet: A dental drill was used to make a craniotomy and animals received unilateral or bilateral injections of either 1 μl 6-OHDA (H4381, Sigma; 2 mg/ml 6-OHDA dissolved in 0.9% saline containing 0.02% ascorbic acid) or equivoluminal 0.9% saline containing 0.02% ascorbic acid (sham group).

    Techniques: Injection, Mouse Assay

    Twist1 deletion in basal keratinocytes suppresses TPA-induced epidermal proliferation

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 deletion in basal keratinocytes suppresses TPA-induced epidermal proliferation

    Article Snippet: Previous investigations from our laboratory suggested that Stat3 functionally regulates skin tumor progression in the two-stage skin carcinogenesis model, in part, by modulating the expression of Twist1 ( ).

    Techniques:

    Twist1 expression regulates p53 localization and p21 transcription in the epidermis

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 expression regulates p53 localization and p21 transcription in the epidermis

    Article Snippet: Previous investigations from our laboratory suggested that Stat3 functionally regulates skin tumor progression in the two-stage skin carcinogenesis model, in part, by modulating the expression of Twist1 ( ).

    Techniques: Expressing

    Twist1 is a novel regulator of the cell cycle in epidermal keratinocytes

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 is a novel regulator of the cell cycle in epidermal keratinocytes

    Article Snippet: Previous investigations from our laboratory suggested that Stat3 functionally regulates skin tumor progression in the two-stage skin carcinogenesis model, in part, by modulating the expression of Twist1 ( ).

    Techniques:

    Induction of skin tumors in Twist1 KO mice in response to initiation with 7,12-dimenthylbenz[a]anthracene (DMBA) and promotion with 12- O -tetradecanoylphorbol-13-acetate (TPA)

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Induction of skin tumors in Twist1 KO mice in response to initiation with 7,12-dimenthylbenz[a]anthracene (DMBA) and promotion with 12- O -tetradecanoylphorbol-13-acetate (TPA)

    Article Snippet: Previous investigations from our laboratory suggested that Stat3 functionally regulates skin tumor progression in the two-stage skin carcinogenesis model, in part, by modulating the expression of Twist1 ( ).

    Techniques: Mouse Assay

    Twist1 deficiency in epidermal keratinocytes hinders proliferative capacity of the bulge region stem cell compartment

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 deficiency in epidermal keratinocytes hinders proliferative capacity of the bulge region stem cell compartment

    Article Snippet: Previous investigations from our laboratory suggested that Stat3 functionally regulates skin tumor progression in the two-stage skin carcinogenesis model, in part, by modulating the expression of Twist1 ( ).

    Techniques:

    miR-23a/CXCR4 regulates neuropathic pain via TXNIP. a , b Increased spinal TXNIP protein expression in pSNL mice was reversed by intrathecal injection of miR-23a mimics ( a ) or Lenti-miR-23a ( b ). Intrathecal injections began on day 7 after pSNL. TXNIP was measured at 24 h after 2-day miR-23a mimics injections or at 48 h after 3-day Lenti-miR-23a injections. One-way ANOVA (expression versus treatment groups) followed by post hoc Tukey test a F (3, 16) = 12.54; b F (3, 16) = 17.27, * p

    Journal: Journal of Neuroinflammation

    Article Title: miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

    doi: 10.1186/s12974-018-1073-0

    Figure Lengend Snippet: miR-23a/CXCR4 regulates neuropathic pain via TXNIP. a , b Increased spinal TXNIP protein expression in pSNL mice was reversed by intrathecal injection of miR-23a mimics ( a ) or Lenti-miR-23a ( b ). Intrathecal injections began on day 7 after pSNL. TXNIP was measured at 24 h after 2-day miR-23a mimics injections or at 48 h after 3-day Lenti-miR-23a injections. One-way ANOVA (expression versus treatment groups) followed by post hoc Tukey test a F (3, 16) = 12.54; b F (3, 16) = 17.27, * p

    Article Snippet: However, CXCR4 is rarely observed in spinal microglial and astrocyte cells of sham rats [ , ] and significantly increased in spinal microglial and astrocyte cells of ischemia-reperfusion-induced pain rats, further blocking CXCR4 changes in glial membrane receptor, such as TLR4, and inflammatory cytokine release [ ], indicating the activation of rat spinal glial cells are associated with the CXCR4.

    Techniques: Expressing, Mouse Assay, Injection

    The schematic of miR-23a targeting CXCR4 regulates neuropathic pain via TXNIP/NLRP3 inflammasome in spinal glial cells of mice. In pSNL-induced chronic neuropathic pain, spinal miR-23a expression was significantly reduced, which increased the expression of spinal CXCR4, and subsequently the expression of TXNIP and NLRP3 inflammasome including NLRP3, ASC, Caspase-1, and IL-1β

    Journal: Journal of Neuroinflammation

    Article Title: miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

    doi: 10.1186/s12974-018-1073-0

    Figure Lengend Snippet: The schematic of miR-23a targeting CXCR4 regulates neuropathic pain via TXNIP/NLRP3 inflammasome in spinal glial cells of mice. In pSNL-induced chronic neuropathic pain, spinal miR-23a expression was significantly reduced, which increased the expression of spinal CXCR4, and subsequently the expression of TXNIP and NLRP3 inflammasome including NLRP3, ASC, Caspase-1, and IL-1β

    Article Snippet: However, CXCR4 is rarely observed in spinal microglial and astrocyte cells of sham rats [ , ] and significantly increased in spinal microglial and astrocyte cells of ischemia-reperfusion-induced pain rats, further blocking CXCR4 changes in glial membrane receptor, such as TLR4, and inflammatory cytokine release [ ], indicating the activation of rat spinal glial cells are associated with the CXCR4.

    Techniques: Mouse Assay, Expressing

    CXCR4 modulates neuropathic pain. a – c CXCR4 immunofluorescent co-staining with NeuN (a neuron marker) ( a , a′ ), GFAP (an astrocyte marker) ( b , b′ ) or IBA1 (a microglial marker) ( c , c′ ) in the lumbar segment of the spinal cord at 7 days after pSNL surgery. * p

    Journal: Journal of Neuroinflammation

    Article Title: miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

    doi: 10.1186/s12974-018-1073-0

    Figure Lengend Snippet: CXCR4 modulates neuropathic pain. a – c CXCR4 immunofluorescent co-staining with NeuN (a neuron marker) ( a , a′ ), GFAP (an astrocyte marker) ( b , b′ ) or IBA1 (a microglial marker) ( c , c′ ) in the lumbar segment of the spinal cord at 7 days after pSNL surgery. * p

    Article Snippet: However, CXCR4 is rarely observed in spinal microglial and astrocyte cells of sham rats [ , ] and significantly increased in spinal microglial and astrocyte cells of ischemia-reperfusion-induced pain rats, further blocking CXCR4 changes in glial membrane receptor, such as TLR4, and inflammatory cytokine release [ ], indicating the activation of rat spinal glial cells are associated with the CXCR4.

    Techniques: Staining, Marker

    miR-23a regulates CXCR4 expression in spinal astrocyte in neuropathic pain. a The shared miRNA numbers to targeting CXCR4 predicted by the use of TargetScan and MicroRNA program. b The informatics analysis of miR-23a binding the 3′UTR in CXCR4 mRNA. c Time course of spinal miR-23a expression in pSNL-induced chronic neuropathic pain mice. One-way ANOVA (expression versus time point) followed by post hoc Tukey test, F time (5, 24) = 19.66, * p

    Journal: Journal of Neuroinflammation

    Article Title: miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

    doi: 10.1186/s12974-018-1073-0

    Figure Lengend Snippet: miR-23a regulates CXCR4 expression in spinal astrocyte in neuropathic pain. a The shared miRNA numbers to targeting CXCR4 predicted by the use of TargetScan and MicroRNA program. b The informatics analysis of miR-23a binding the 3′UTR in CXCR4 mRNA. c Time course of spinal miR-23a expression in pSNL-induced chronic neuropathic pain mice. One-way ANOVA (expression versus time point) followed by post hoc Tukey test, F time (5, 24) = 19.66, * p

    Article Snippet: However, CXCR4 is rarely observed in spinal microglial and astrocyte cells of sham rats [ , ] and significantly increased in spinal microglial and astrocyte cells of ischemia-reperfusion-induced pain rats, further blocking CXCR4 changes in glial membrane receptor, such as TLR4, and inflammatory cytokine release [ ], indicating the activation of rat spinal glial cells are associated with the CXCR4.

    Techniques: Expressing, Binding Assay, Mouse Assay

    TXNIP is involved in the process of neuropathic pain by the CXCR4-dependent regulation. a Quantitative expression of Txnip mRNA at 1, 3, 7, 14, and 21 days after pSNL surgery. One-way ANOVA (expression versus time point) followed by post hoc Tukey test, F time (5, 24) = 40.4, * p

    Journal: Journal of Neuroinflammation

    Article Title: miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

    doi: 10.1186/s12974-018-1073-0

    Figure Lengend Snippet: TXNIP is involved in the process of neuropathic pain by the CXCR4-dependent regulation. a Quantitative expression of Txnip mRNA at 1, 3, 7, 14, and 21 days after pSNL surgery. One-way ANOVA (expression versus time point) followed by post hoc Tukey test, F time (5, 24) = 40.4, * p

    Article Snippet: However, CXCR4 is rarely observed in spinal microglial and astrocyte cells of sham rats [ , ] and significantly increased in spinal microglial and astrocyte cells of ischemia-reperfusion-induced pain rats, further blocking CXCR4 changes in glial membrane receptor, such as TLR4, and inflammatory cytokine release [ ], indicating the activation of rat spinal glial cells are associated with the CXCR4.

    Techniques: Expressing

    Spinal miR-23a regulates pain behavior via CXCR4. a , b Daily intrathecal injections of miR-23a mimics ( a ) or Lenti-miR-23a ( b ) for 2 or 3 consecutive days, respectively, reversed pSNL-induced thermal hyperalgesia and mechanical allodynia. Two-way ANOVA (effect versus group × time interaction) followed by post hoc Tukey test, a PWL F group (18, 112) = 10.24, PWT F group (18, 112) = 12.02; b PWL F group (18, 112) = 10.57, PWT F group (18, 112) = 8.12, * p

    Journal: Journal of Neuroinflammation

    Article Title: miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

    doi: 10.1186/s12974-018-1073-0

    Figure Lengend Snippet: Spinal miR-23a regulates pain behavior via CXCR4. a , b Daily intrathecal injections of miR-23a mimics ( a ) or Lenti-miR-23a ( b ) for 2 or 3 consecutive days, respectively, reversed pSNL-induced thermal hyperalgesia and mechanical allodynia. Two-way ANOVA (effect versus group × time interaction) followed by post hoc Tukey test, a PWL F group (18, 112) = 10.24, PWT F group (18, 112) = 12.02; b PWL F group (18, 112) = 10.57, PWT F group (18, 112) = 8.12, * p

    Article Snippet: However, CXCR4 is rarely observed in spinal microglial and astrocyte cells of sham rats [ , ] and significantly increased in spinal microglial and astrocyte cells of ischemia-reperfusion-induced pain rats, further blocking CXCR4 changes in glial membrane receptor, such as TLR4, and inflammatory cytokine release [ ], indicating the activation of rat spinal glial cells are associated with the CXCR4.

    Techniques:

    Patients’ antibodies react with wild-type but not Caspr2 -null mouse brains

    Journal: Annals of neurology

    Article Title: Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

    doi: 10.1002/ana.22297

    Figure Lengend Snippet: Patients’ antibodies react with wild-type but not Caspr2 -null mouse brains

    Article Snippet: Caspr2 has a critical role in concentrating VGKCs and other proteins in the juxtaparanodal region of myelinated axons in both the PNS and the CNS., One patient with homozygous deletion of CNTNAP2 (OMIM 604569), the human gene that encodes Caspr2, had history of seizures and developed over 6 months progressive painful peripheral neuropathy and neuromyotonia to the point that it interfered with the patient’s gait (K Strauss, unpublished data).

    Techniques:

    Characteristics of patients with Caspr2 antibodies

    Journal: Annals of neurology

    Article Title: Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

    doi: 10.1002/ana.22297

    Figure Lengend Snippet: Characteristics of patients with Caspr2 antibodies

    Article Snippet: Caspr2 has a critical role in concentrating VGKCs and other proteins in the juxtaparanodal region of myelinated axons in both the PNS and the CNS., One patient with homozygous deletion of CNTNAP2 (OMIM 604569), the human gene that encodes Caspr2, had history of seizures and developed over 6 months progressive painful peripheral neuropathy and neuromyotonia to the point that it interfered with the patient’s gait (K Strauss, unpublished data).

    Techniques:

    Immunoabsorption with Caspr2 abrogates the reactivity of the index patient’s antibodies with rodent brain and peripheral nerve

    Journal: Annals of neurology

    Article Title: Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

    doi: 10.1002/ana.22297

    Figure Lengend Snippet: Immunoabsorption with Caspr2 abrogates the reactivity of the index patient’s antibodies with rodent brain and peripheral nerve

    Article Snippet: Caspr2 has a critical role in concentrating VGKCs and other proteins in the juxtaparanodal region of myelinated axons in both the PNS and the CNS., One patient with homozygous deletion of CNTNAP2 (OMIM 604569), the human gene that encodes Caspr2, had history of seizures and developed over 6 months progressive painful peripheral neuropathy and neuromyotonia to the point that it interfered with the patient’s gait (K Strauss, unpublished data).

    Techniques:

    Identification of Caspr2 as a target autoantigen

    Journal: Annals of neurology

    Article Title: Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

    doi: 10.1002/ana.22297

    Figure Lengend Snippet: Identification of Caspr2 as a target autoantigen

    Article Snippet: Caspr2 has a critical role in concentrating VGKCs and other proteins in the juxtaparanodal region of myelinated axons in both the PNS and the CNS., One patient with homozygous deletion of CNTNAP2 (OMIM 604569), the human gene that encodes Caspr2, had history of seizures and developed over 6 months progressive painful peripheral neuropathy and neuromyotonia to the point that it interfered with the patient’s gait (K Strauss, unpublished data).

    Techniques:

    Patients’ antibodies label teased fibers from wild-type but not Caspr2 -null nerves

    Journal: Annals of neurology

    Article Title: Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

    doi: 10.1002/ana.22297

    Figure Lengend Snippet: Patients’ antibodies label teased fibers from wild-type but not Caspr2 -null nerves

    Article Snippet: Caspr2 has a critical role in concentrating VGKCs and other proteins in the juxtaparanodal region of myelinated axons in both the PNS and the CNS., One patient with homozygous deletion of CNTNAP2 (OMIM 604569), the human gene that encodes Caspr2, had history of seizures and developed over 6 months progressive painful peripheral neuropathy and neuromyotonia to the point that it interfered with the patient’s gait (K Strauss, unpublished data).

    Techniques:

    Patients’ antibodies react with cells expressing Caspr2

    Journal: Annals of neurology

    Article Title: Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

    doi: 10.1002/ana.22297

    Figure Lengend Snippet: Patients’ antibodies react with cells expressing Caspr2

    Article Snippet: Caspr2 has a critical role in concentrating VGKCs and other proteins in the juxtaparanodal region of myelinated axons in both the PNS and the CNS., One patient with homozygous deletion of CNTNAP2 (OMIM 604569), the human gene that encodes Caspr2, had history of seizures and developed over 6 months progressive painful peripheral neuropathy and neuromyotonia to the point that it interfered with the patient’s gait (K Strauss, unpublished data).

    Techniques: Expressing

    Complementation Studies Using C1qbp −/− Mouse Fibroblasts (A) Quantification of C1QBP, COXI, and COXIII levels. Top: WT or C1qbp KO MEFs were transfected with the pcDNA3-C1qbp-IRES-GFP plasmid for 48 hr. Western blotting on total cell extracts was performed with anti-C1QBP, anti-COXI, anti-COXIII, anti-GFP, and anti-VDAC antibodies. VDAC was used as the loading control. Bottom: the mean ratios of band densities in transfected MEFs from blots are shown. Cells transfected with expression constructs for p.Gly244Trp and p.Leu272Pro variants show significantly lower amounts of mature C1QBP and mitochondrial-DNA-encoded proteins (COXI and COXIII). The results represent the mean ± SD of three independent experiments. ∗∗∗ p

    Journal: American Journal of Human Genetics

    Article Title: Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

    doi: 10.1016/j.ajhg.2017.08.015

    Figure Lengend Snippet: Complementation Studies Using C1qbp −/− Mouse Fibroblasts (A) Quantification of C1QBP, COXI, and COXIII levels. Top: WT or C1qbp KO MEFs were transfected with the pcDNA3-C1qbp-IRES-GFP plasmid for 48 hr. Western blotting on total cell extracts was performed with anti-C1QBP, anti-COXI, anti-COXIII, anti-GFP, and anti-VDAC antibodies. VDAC was used as the loading control. Bottom: the mean ratios of band densities in transfected MEFs from blots are shown. Cells transfected with expression constructs for p.Gly244Trp and p.Leu272Pro variants show significantly lower amounts of mature C1QBP and mitochondrial-DNA-encoded proteins (COXI and COXIII). The results represent the mean ± SD of three independent experiments. ∗∗∗ p

    Article Snippet: A very recently established cardiomyocyte-specific deletion of C1qbp resulted in contractile dysfunction, cardiac dilatation, and cardiac fibrosis and thereby confirmed an important function of C1QBP in the heart.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Construct

    Western Blot Analysis of C1QBP Cell lysates isolated from (A) the skeletal muscle of affected probands S1, S3, and S4 and (B) fibroblasts from probands S1–S3 and age-matched control individuals (C1 and C2) were analyzed. Fibroblasts from proband S4 and skeletal muscle from proband S2 were not available. β-actin and α-tubulin were used as loading controls. All experiments were repeated at least two times, and representative images are shown. Number of repeats: (A) n = 3 (S1) and n = 2 (S3 and S4); (B) n = 2 (S1–S3).

    Journal: American Journal of Human Genetics

    Article Title: Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

    doi: 10.1016/j.ajhg.2017.08.015

    Figure Lengend Snippet: Western Blot Analysis of C1QBP Cell lysates isolated from (A) the skeletal muscle of affected probands S1, S3, and S4 and (B) fibroblasts from probands S1–S3 and age-matched control individuals (C1 and C2) were analyzed. Fibroblasts from proband S4 and skeletal muscle from proband S2 were not available. β-actin and α-tubulin were used as loading controls. All experiments were repeated at least two times, and representative images are shown. Number of repeats: (A) n = 3 (S1) and n = 2 (S3 and S4); (B) n = 2 (S1–S3).

    Article Snippet: A very recently established cardiomyocyte-specific deletion of C1qbp resulted in contractile dysfunction, cardiac dilatation, and cardiac fibrosis and thereby confirmed an important function of C1QBP in the heart.

    Techniques: Western Blot, Isolation

    C1QBP Variants and Gene and Protein Structure (A) Pedigrees of the investigated families (S1–S4) affected by recessively inherited C1QBP variants. Affected individuals are indicated by closed symbols. Both variants in proband S2 have been confirmed to be compound heterozygous by phasing of WES data. (B) Gene structure with exons and introns shows the localization of the investigated gene variations. Conservation of the affected amino acid residues is presented in the alignment of homologs across different species. Exons are highlighted in blue. The size of the introns was reduced 10-fold. MTS is the mitochondrial target sequence. MAM33 (mitochondrial acidic matrix protein 33) is the Saccharomyces cerevisiae homolog of C1QBP. (C) Inspection of the protein structure was performed with PyMOL (PDB: 1P32 ). A monomer is presented on the left, and the trimer is in the center. The electrostatic field of the trimer is indicated to the right (negative polarity, red; positive polarity, blue). Affected residues are colored in one of the monomers: Cys186, green; Tyr188, blue; Phe204, red; Gly247, magenta; and Leu275, orange.

    Journal: American Journal of Human Genetics

    Article Title: Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

    doi: 10.1016/j.ajhg.2017.08.015

    Figure Lengend Snippet: C1QBP Variants and Gene and Protein Structure (A) Pedigrees of the investigated families (S1–S4) affected by recessively inherited C1QBP variants. Affected individuals are indicated by closed symbols. Both variants in proband S2 have been confirmed to be compound heterozygous by phasing of WES data. (B) Gene structure with exons and introns shows the localization of the investigated gene variations. Conservation of the affected amino acid residues is presented in the alignment of homologs across different species. Exons are highlighted in blue. The size of the introns was reduced 10-fold. MTS is the mitochondrial target sequence. MAM33 (mitochondrial acidic matrix protein 33) is the Saccharomyces cerevisiae homolog of C1QBP. (C) Inspection of the protein structure was performed with PyMOL (PDB: 1P32 ). A monomer is presented on the left, and the trimer is in the center. The electrostatic field of the trimer is indicated to the right (negative polarity, red; positive polarity, blue). Affected residues are colored in one of the monomers: Cys186, green; Tyr188, blue; Phe204, red; Gly247, magenta; and Leu275, orange.

    Article Snippet: A very recently established cardiomyocyte-specific deletion of C1qbp resulted in contractile dysfunction, cardiac dilatation, and cardiac fibrosis and thereby confirmed an important function of C1QBP in the heart.

    Techniques: Sequencing

    AT13148 reduces physical force and migration on pliable substrates. A, Directed scratch wound migration of AT13148, Y27632 or H1152 drug-treated cells was monitored over 48 hours. Left: Representative images at 0 and 24 hours for DMSO and 1 µm AT13148 treated cells. Upper right graph shows mean ± SEM percentage wound confluence acquired every 2 hours over 48 hours for n=6 replicates. Lower right graph shows mean ± SEM percentage wound confluence at 24 hours for n=3 independent experiments. B, Random migration of cells treated with AT13148, Y27632 or H1152 was monitored over 10 hours. Left panels show representative images at 0, 5 and 10 hours. Means ± SEM are shown from n = 3 independent experiments. C, Schematic diagram and representative images of cells on nanopillars with DMSO, AT13148 or Y27632 treatments as indicated. D, Forces leading to nanopillar deflection were determined for ≥10 pillars under 16 cells for AT13148 and Y27632 treatments, and for 32 cells for DMSO. Mean force values determined at each 2 minute interval over 120 minutes are plotted for each condition, with indicated means ± SEM. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p

    Journal: Cancer research

    Article Title: Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

    doi: 10.1158/0008-5472.CAN-17-1339

    Figure Lengend Snippet: AT13148 reduces physical force and migration on pliable substrates. A, Directed scratch wound migration of AT13148, Y27632 or H1152 drug-treated cells was monitored over 48 hours. Left: Representative images at 0 and 24 hours for DMSO and 1 µm AT13148 treated cells. Upper right graph shows mean ± SEM percentage wound confluence acquired every 2 hours over 48 hours for n=6 replicates. Lower right graph shows mean ± SEM percentage wound confluence at 24 hours for n=3 independent experiments. B, Random migration of cells treated with AT13148, Y27632 or H1152 was monitored over 10 hours. Left panels show representative images at 0, 5 and 10 hours. Means ± SEM are shown from n = 3 independent experiments. C, Schematic diagram and representative images of cells on nanopillars with DMSO, AT13148 or Y27632 treatments as indicated. D, Forces leading to nanopillar deflection were determined for ≥10 pillars under 16 cells for AT13148 and Y27632 treatments, and for 32 cells for DMSO. Mean force values determined at each 2 minute interval over 120 minutes are plotted for each condition, with indicated means ± SEM. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p

    Article Snippet: The effects of AT13148 at concentrations from 10 nM to 10 µM, as well as 10 µM Y27632 and 10 µM H1152, on viable KPC cell numbers were examined after 18 hours of treatment.

    Techniques: Migration

    AT13148 affects cell morphology. A, Representative confocal microscope maximum intensity projection images of F-actin (green) and DAPI (blue) stained KPC cells treated with indicated concentrations of AT13148, Y27632 or H1152 for 1 h. Alternatively, cells were transfected with non-targeting control (NTC) or a mixture of Rock1 + Rock2 (Rock1/2) siRNAs and imaged on an Operetta High Content Imaging System. High content imaging of KPC cells after 1 h of AT13148, Y27632 or H1152 drug treatment, or 24 h post siRNA transfection (left graphs), or > 7 days post lentiviral transduction of TKCC5 cells with NTC or ROCK1 and ROCK2 targeted shRNAs (right graphs), was used to determine B, cell area; C, cell length; D, cell width/length ratios; E, cell roundness. Means ± SEM are shown from n = 3 independent experiments for drug treatments, n = 4 independent experiments for siRNA transfections, or n = 3 independent experiments with shRNA knockdown cells. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test for AT13148, Y276732 or H1152 treatments. Statistical significance was determined for the comparisons of corresponding NTC with Rock1/2 or ROCK1/2 knockdown by unpaired Student’s t-test. *, p

    Journal: Cancer research

    Article Title: Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

    doi: 10.1158/0008-5472.CAN-17-1339

    Figure Lengend Snippet: AT13148 affects cell morphology. A, Representative confocal microscope maximum intensity projection images of F-actin (green) and DAPI (blue) stained KPC cells treated with indicated concentrations of AT13148, Y27632 or H1152 for 1 h. Alternatively, cells were transfected with non-targeting control (NTC) or a mixture of Rock1 + Rock2 (Rock1/2) siRNAs and imaged on an Operetta High Content Imaging System. High content imaging of KPC cells after 1 h of AT13148, Y27632 or H1152 drug treatment, or 24 h post siRNA transfection (left graphs), or > 7 days post lentiviral transduction of TKCC5 cells with NTC or ROCK1 and ROCK2 targeted shRNAs (right graphs), was used to determine B, cell area; C, cell length; D, cell width/length ratios; E, cell roundness. Means ± SEM are shown from n = 3 independent experiments for drug treatments, n = 4 independent experiments for siRNA transfections, or n = 3 independent experiments with shRNA knockdown cells. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test for AT13148, Y276732 or H1152 treatments. Statistical significance was determined for the comparisons of corresponding NTC with Rock1/2 or ROCK1/2 knockdown by unpaired Student’s t-test. *, p

    Article Snippet: The effects of AT13148 at concentrations from 10 nM to 10 µM, as well as 10 µM Y27632 and 10 µM H1152, on viable KPC cell numbers were examined after 18 hours of treatment.

    Techniques: Microscopy, Staining, Transfection, Imaging, Transduction, shRNA

    Effect of ROCK inhibitors on PDAC cell metabolism. A, Normalized levels of intracellular metabolites in cells treated with 1 µM AT13148, 10 µM H1152 or 10 µM Y27632 for 24 hours as negative or positive relative differences from DMSO treated controls. Means ± SEM are shown from n = 4 independent experiments. Colored * indicates p

    Journal: Cancer research

    Article Title: Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

    doi: 10.1158/0008-5472.CAN-17-1339

    Figure Lengend Snippet: Effect of ROCK inhibitors on PDAC cell metabolism. A, Normalized levels of intracellular metabolites in cells treated with 1 µM AT13148, 10 µM H1152 or 10 µM Y27632 for 24 hours as negative or positive relative differences from DMSO treated controls. Means ± SEM are shown from n = 4 independent experiments. Colored * indicates p

    Article Snippet: The effects of AT13148 at concentrations from 10 nM to 10 µM, as well as 10 µM Y27632 and 10 µM H1152, on viable KPC cell numbers were examined after 18 hours of treatment.

    Techniques:

    Collagen matrix invasion is efficiently inhibited by AT13148. A, H E-stained sections of KPC cell invasion into fibroblast-conditioned collagen matrix after 8 days, which included treatment with AT13148, Y27632 or GM6001 as indicated. The invasion index was normalized to DMSO control conditions, with means ± SEM shown from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. *, p

    Journal: Cancer research

    Article Title: Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

    doi: 10.1158/0008-5472.CAN-17-1339

    Figure Lengend Snippet: Collagen matrix invasion is efficiently inhibited by AT13148. A, H E-stained sections of KPC cell invasion into fibroblast-conditioned collagen matrix after 8 days, which included treatment with AT13148, Y27632 or GM6001 as indicated. The invasion index was normalized to DMSO control conditions, with means ± SEM shown from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. *, p

    Article Snippet: The effects of AT13148 at concentrations from 10 nM to 10 µM, as well as 10 µM Y27632 and 10 µM H1152, on viable KPC cell numbers were examined after 18 hours of treatment.

    Techniques: Staining

    AT13148 blocks PDAC invasion and tumor growth in vivo. A, Mouse PDAC cells were injected into the peritoneum of CD-1 mice and animals were treated with vehicle or AT13148 (40 mg/kg, 3 x per week) for 14 days. Left: Representative H E-stained sections of pancreas (dark staining) and invading tumor cells (light staining). Right: Quantification of pancreas area invaded by KPC tumor cells. Means ± SEM are shown for n=7 for vehicle-treated and n=8 for AT13148-treated mice. Exact P value by Mann-Whitney test. B, Quantification of pancreas area invaded by KPC tumor cells. Means ± SEM are shown for n=8 for vehicle-treated and n=8 for mice treated daily with 2 mg/mouse fasudil. Exact P value by Mann-Whitney test. C, Representative image of BrdU staining of normal pancreas and tumor cells, with white line indicating normal pancreas from region with tumor cells. D, Quantification of percentage BrdU staining in KPC tumor cells. Means ± SEM shown for n=8 for vehicle-treated and n=8 for AT13148-treated mice. Exact P value by Mann-Whitney test. E, Volumes of subcutaneous KPC cell tumors in CD-1 mice treated with AT13148 (40 mg/kg, 3 x per week) or vehicle control for up to 25 days (dots represent individual mice). Statistical significance was determined for vehicle versus AT13148 for each day by unpaired Student’s t-test. *, p

    Journal: Cancer research

    Article Title: Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

    doi: 10.1158/0008-5472.CAN-17-1339

    Figure Lengend Snippet: AT13148 blocks PDAC invasion and tumor growth in vivo. A, Mouse PDAC cells were injected into the peritoneum of CD-1 mice and animals were treated with vehicle or AT13148 (40 mg/kg, 3 x per week) for 14 days. Left: Representative H E-stained sections of pancreas (dark staining) and invading tumor cells (light staining). Right: Quantification of pancreas area invaded by KPC tumor cells. Means ± SEM are shown for n=7 for vehicle-treated and n=8 for AT13148-treated mice. Exact P value by Mann-Whitney test. B, Quantification of pancreas area invaded by KPC tumor cells. Means ± SEM are shown for n=8 for vehicle-treated and n=8 for mice treated daily with 2 mg/mouse fasudil. Exact P value by Mann-Whitney test. C, Representative image of BrdU staining of normal pancreas and tumor cells, with white line indicating normal pancreas from region with tumor cells. D, Quantification of percentage BrdU staining in KPC tumor cells. Means ± SEM shown for n=8 for vehicle-treated and n=8 for AT13148-treated mice. Exact P value by Mann-Whitney test. E, Volumes of subcutaneous KPC cell tumors in CD-1 mice treated with AT13148 (40 mg/kg, 3 x per week) or vehicle control for up to 25 days (dots represent individual mice). Statistical significance was determined for vehicle versus AT13148 for each day by unpaired Student’s t-test. *, p

    Article Snippet: The effects of AT13148 at concentrations from 10 nM to 10 µM, as well as 10 µM Y27632 and 10 µM H1152, on viable KPC cell numbers were examined after 18 hours of treatment.

    Techniques: In Vivo, Injection, Mouse Assay, Staining, MANN-WHITNEY, BrdU Staining

    Potent inhibition of substrate phosphorylation by AT13148. A, KPC mouse PDAC cells were treated with DMSO vehicle or indicated concentrations of AT13148, Y27632 or H1152 for 1 hour, then immunoblotted for ROCK1, ROCK2, phosphorylation of MLC2 (pMLC2; T18S19), total MLC (tMLC), phosphorylation of MYPT (pMYPT1; T850) and GAPDH B, Left panel Quantifications represent the ratio of pMLC2 to total MLC as percentage relative to DMSO vehicle. Means ± SEM are shown from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p

    Journal: Cancer research

    Article Title: Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

    doi: 10.1158/0008-5472.CAN-17-1339

    Figure Lengend Snippet: Potent inhibition of substrate phosphorylation by AT13148. A, KPC mouse PDAC cells were treated with DMSO vehicle or indicated concentrations of AT13148, Y27632 or H1152 for 1 hour, then immunoblotted for ROCK1, ROCK2, phosphorylation of MLC2 (pMLC2; T18S19), total MLC (tMLC), phosphorylation of MYPT (pMYPT1; T850) and GAPDH B, Left panel Quantifications represent the ratio of pMLC2 to total MLC as percentage relative to DMSO vehicle. Means ± SEM are shown from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA and post-hoc Dunnett’s multiple comparison test. ***, p

    Article Snippet: The effects of AT13148 at concentrations from 10 nM to 10 µM, as well as 10 µM Y27632 and 10 µM H1152, on viable KPC cell numbers were examined after 18 hours of treatment.

    Techniques: Inhibition

    sICAM-5 does not affect Th17—APC interactions. (A) FACS staining for LFA-1 of naïve T-cells, Th17 cells, and Th17 cells after 24 h of stimulation with anti-CD3/CD28 ( n = 3 for each condition). (B) Representative depiction of CFSE stainings of naïve 2d2 T cells cocultured with APCs and sICAM-5 (10 μg/ml) or IgG (10 μg/ml) for 72 h. (C) FACS staining of Th17 cells stimulated for 4 and 24 h with anti-CD3/CD28 and with or without ICAM-5 treatment (10 μg/ml). (D,E) Naïve T cells were differentiated into Th17 cells in the presence of or without sICAM-5 treatment (10 μg/ml) and stained for extracellular (D) and intracellular (E) markers. Data shown are mean ± SEM. (A) One-way ANOVA followed by Tukey's multiple comparison test, n.s. no significance; (C–E) Student's t -test.

    Journal: Frontiers in Neurology

    Article Title: Neuronal ICAM-5 Plays a Neuroprotective Role in Progressive Neurodegeneration

    doi: 10.3389/fneur.2019.00205

    Figure Lengend Snippet: sICAM-5 does not affect Th17—APC interactions. (A) FACS staining for LFA-1 of naïve T-cells, Th17 cells, and Th17 cells after 24 h of stimulation with anti-CD3/CD28 ( n = 3 for each condition). (B) Representative depiction of CFSE stainings of naïve 2d2 T cells cocultured with APCs and sICAM-5 (10 μg/ml) or IgG (10 μg/ml) for 72 h. (C) FACS staining of Th17 cells stimulated for 4 and 24 h with anti-CD3/CD28 and with or without ICAM-5 treatment (10 μg/ml). (D,E) Naïve T cells were differentiated into Th17 cells in the presence of or without sICAM-5 treatment (10 μg/ml) and stained for extracellular (D) and intracellular (E) markers. Data shown are mean ± SEM. (A) One-way ANOVA followed by Tukey's multiple comparison test, n.s. no significance; (C–E) Student's t -test.

    Article Snippet: For the interpretation of results from the ICAM-5−/− mouse, it has to be kept in mind that the full knockout of ICAM-5 might have both beneficial effects (via deletion of T cell-neuron binding capacity) and detrimental effects (since the beneficial effects of sICAM-5 would also be abrogated).

    Techniques: FACS, Staining

    No significant correlation between neurofilament light chain levels and sICAM-5. (A) ICAM-5 was correlated with NfL concentrations (pg/ml) in the cerebrospinal fluid and the serum of SPMS patients ( n = 7–8). (B) ICAM-5 was correlated to NfL concentrations (pg/ml) in the cerebrospinal fluid and the serum of PPMS patients ( n = 7–8). (C) ICAM-5 was correlated to NfL concentrations (pg/ml) in the cerebrospinal fluid and the serum of pooled SPMS and PPMS patients ( n = 14–16). For all figures: Data was tested for normality using Shapiro-Wilk normality test and Pearson's or Spearman's r and p -values was determined accordingly.

    Journal: Frontiers in Neurology

    Article Title: Neuronal ICAM-5 Plays a Neuroprotective Role in Progressive Neurodegeneration

    doi: 10.3389/fneur.2019.00205

    Figure Lengend Snippet: No significant correlation between neurofilament light chain levels and sICAM-5. (A) ICAM-5 was correlated with NfL concentrations (pg/ml) in the cerebrospinal fluid and the serum of SPMS patients ( n = 7–8). (B) ICAM-5 was correlated to NfL concentrations (pg/ml) in the cerebrospinal fluid and the serum of PPMS patients ( n = 7–8). (C) ICAM-5 was correlated to NfL concentrations (pg/ml) in the cerebrospinal fluid and the serum of pooled SPMS and PPMS patients ( n = 14–16). For all figures: Data was tested for normality using Shapiro-Wilk normality test and Pearson's or Spearman's r and p -values was determined accordingly.

    Article Snippet: For the interpretation of results from the ICAM-5−/− mouse, it has to be kept in mind that the full knockout of ICAM-5 might have both beneficial effects (via deletion of T cell-neuron binding capacity) and detrimental effects (since the beneficial effects of sICAM-5 would also be abrogated).

    Techniques:

    Schematic overview of potential ICAM-5 dependent T cell—neuron and T cell—APC interactions in the CNS. Schematic illustration of the interplay of neurons, T cells, and APCs in the CNS. The adhesion molecule ICAM-5 (yellow) is exclusively expressed by neurons and can be cleaved by MMP-2 and 9 from the neuronal surface (also called shedding). The soluble form (sICAM-5) has been proposed to act as an inhibitor of ICAM1–LFA-1 interactions between T cells and APCs and T cells and neurons therefore displaying a protective function. Green highlights and arrows represent anti-inflammatory/ protective function and red highlights represent proinflammatory properties.

    Journal: Frontiers in Neurology

    Article Title: Neuronal ICAM-5 Plays a Neuroprotective Role in Progressive Neurodegeneration

    doi: 10.3389/fneur.2019.00205

    Figure Lengend Snippet: Schematic overview of potential ICAM-5 dependent T cell—neuron and T cell—APC interactions in the CNS. Schematic illustration of the interplay of neurons, T cells, and APCs in the CNS. The adhesion molecule ICAM-5 (yellow) is exclusively expressed by neurons and can be cleaved by MMP-2 and 9 from the neuronal surface (also called shedding). The soluble form (sICAM-5) has been proposed to act as an inhibitor of ICAM1–LFA-1 interactions between T cells and APCs and T cells and neurons therefore displaying a protective function. Green highlights and arrows represent anti-inflammatory/ protective function and red highlights represent proinflammatory properties.

    Article Snippet: For the interpretation of results from the ICAM-5−/− mouse, it has to be kept in mind that the full knockout of ICAM-5 might have both beneficial effects (via deletion of T cell-neuron binding capacity) and detrimental effects (since the beneficial effects of sICAM-5 would also be abrogated).

    Techniques: Activated Clotting Time Assay

    Absence of ICAM-5 worsens disease progression which can be reversed by application of sICAM-5. (A) Immunohistochemical staining of ICAM-5-AF647 on cortical neurons (scale bar = 15 μm, 3D overlay scale bar = 10 μm). Co-staining was performed with NeuN-AF488 and DAPI (blue). (B) mRNA analysis of ICAM-5 and MMP-9 were performed with cortical neurons after stimulation with LPS, IFNγ, and splenocyte supernatant ( n = 6 for each condition). Unstimulated cortical neurons served as a control. (C) Immunohistochemical staining of MAP2- AF647, Tuj1-FITC, ICAM-5-AF568, and DAPI on cortical neurons (scale bar = 20 μm. (D) EAE was induced actively in ICAM-5-KO mice and WT littermates via the injection of MOG 35−55 (two independent EAEs, untreated; WT n = 12, KO n = 12). The dotted box on the plot provides a closer look at the chronic phase of the EAE but does not agree with the x axis of the upper plot. The lower plot shows only the mean disease score of the period from day 40 after immunization. (E) EAE lesions were stained from wt ( n = 9) and ICAM5 KO mice ( n = 6) for inflammation (HE staining), demyelination (LFB-PAS), and axonal loss (APP) and were quantified accordingly. (F) EAE was induced actively in B6 mice via the injection of MOG 35−55 and mice were treated with Methylprednisolone for 5 days as soon as the mice reached a clinical score of 2. Additionally, once animals reached a clinical score of 2, 0.2 μg ICAM-5 D1-2 Fc or human IgG was applied via intrathecal (i.th) injection by lumbar puncture seven times on every second day (WT n = 7, KO n = 7). Data shown are mean ± SEM. (C, D) Mann-Whitney U -test; * p

    Journal: Frontiers in Neurology

    Article Title: Neuronal ICAM-5 Plays a Neuroprotective Role in Progressive Neurodegeneration

    doi: 10.3389/fneur.2019.00205

    Figure Lengend Snippet: Absence of ICAM-5 worsens disease progression which can be reversed by application of sICAM-5. (A) Immunohistochemical staining of ICAM-5-AF647 on cortical neurons (scale bar = 15 μm, 3D overlay scale bar = 10 μm). Co-staining was performed with NeuN-AF488 and DAPI (blue). (B) mRNA analysis of ICAM-5 and MMP-9 were performed with cortical neurons after stimulation with LPS, IFNγ, and splenocyte supernatant ( n = 6 for each condition). Unstimulated cortical neurons served as a control. (C) Immunohistochemical staining of MAP2- AF647, Tuj1-FITC, ICAM-5-AF568, and DAPI on cortical neurons (scale bar = 20 μm. (D) EAE was induced actively in ICAM-5-KO mice and WT littermates via the injection of MOG 35−55 (two independent EAEs, untreated; WT n = 12, KO n = 12). The dotted box on the plot provides a closer look at the chronic phase of the EAE but does not agree with the x axis of the upper plot. The lower plot shows only the mean disease score of the period from day 40 after immunization. (E) EAE lesions were stained from wt ( n = 9) and ICAM5 KO mice ( n = 6) for inflammation (HE staining), demyelination (LFB-PAS), and axonal loss (APP) and were quantified accordingly. (F) EAE was induced actively in B6 mice via the injection of MOG 35−55 and mice were treated with Methylprednisolone for 5 days as soon as the mice reached a clinical score of 2. Additionally, once animals reached a clinical score of 2, 0.2 μg ICAM-5 D1-2 Fc or human IgG was applied via intrathecal (i.th) injection by lumbar puncture seven times on every second day (WT n = 7, KO n = 7). Data shown are mean ± SEM. (C, D) Mann-Whitney U -test; * p

    Article Snippet: For the interpretation of results from the ICAM-5−/− mouse, it has to be kept in mind that the full knockout of ICAM-5 might have both beneficial effects (via deletion of T cell-neuron binding capacity) and detrimental effects (since the beneficial effects of sICAM-5 would also be abrogated).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Injection, MANN-WHITNEY

    Patients suffering from progressive forms of MS show lower levels of sICAM-5 in CSF. (A) ICAM-5 concentration in ng/ml in the cerebrospinal fluid of NIND patients (NIND, n = 35), RRMS ( n = 17), PPMS ( n = 19), and SPMS ( n = 12) patients. Data shown are mean ± SEM. One-way ANOVA followed by Tukey's multiple comparison test, * p

    Journal: Frontiers in Neurology

    Article Title: Neuronal ICAM-5 Plays a Neuroprotective Role in Progressive Neurodegeneration

    doi: 10.3389/fneur.2019.00205

    Figure Lengend Snippet: Patients suffering from progressive forms of MS show lower levels of sICAM-5 in CSF. (A) ICAM-5 concentration in ng/ml in the cerebrospinal fluid of NIND patients (NIND, n = 35), RRMS ( n = 17), PPMS ( n = 19), and SPMS ( n = 12) patients. Data shown are mean ± SEM. One-way ANOVA followed by Tukey's multiple comparison test, * p

    Article Snippet: For the interpretation of results from the ICAM-5−/− mouse, it has to be kept in mind that the full knockout of ICAM-5 might have both beneficial effects (via deletion of T cell-neuron binding capacity) and detrimental effects (since the beneficial effects of sICAM-5 would also be abrogated).

    Techniques: Mass Spectrometry, Concentration Assay

    Complementation of cell stress, polarity, and cell cycle phenotypes in a pho85 Δ strain by CDK5 expression. ( A ) Salt sensitivity. Serial dilutions of log phase cultures of pho85 or wild-type cells expressing either CDK5 or CDK5-I plasmids were plated on SG-URA medium with 0.75 M NaCl or on an SD-URA plate (control) and incubated for 5 days at 30°C. ( B ) Overexpression of CDK5 in a cln1 Δ cln2 Δ pho85 Δ strain. A cln1Δ cln2Δ pho85Δ strain carrying a pho85 mutant allele ( pho85 ts17 ) expressed from the MET25 promoter (BY794) and either pCDK5 (right side of plates) or pCDK5-I (left side of plates) was streaked on a galactose-containing plate plus methionine (+ Gal + Met, Right ; repress pho85 ts17 , induce CDK5 ) or a galactose-containing plate minus methionine (control, Left ) and incubated for 5 days at 30°C. Two independent isolates are shown. ( C ) Overexpression of CDK5 in a slt2 Δ pho85 Δ strain. A slt2 Δ pho85 Δ strain expressing either PHO85 (left side of plates) or CDK5 (right side of plates) from the GAL promoter was streaked onto a yeast extract/peptone/dextrose (YPD) or yeast extract/peptone/galactose (YPG) plate as indicated and incubated for 5 days at 25°C. ( D ) Pho4 kinase activity associated with HA-Pcl2-Cdk5. Ha-Pcl2 was immunoprecipitated from yeast extracts and used in kinase assays with Pho4 as a substrate. The following strains were used for extract preparation: lane 1, pho85 Δ HA-PCL2 with pCDK5; lane 2, pho85 Δ HA-PCL2 with pCDK5-I; lane 3, pho85 Δ HA-PCL2 with pPHO85 (positive control).

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

    Article Title: Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

    doi:

    Figure Lengend Snippet: Complementation of cell stress, polarity, and cell cycle phenotypes in a pho85 Δ strain by CDK5 expression. ( A ) Salt sensitivity. Serial dilutions of log phase cultures of pho85 or wild-type cells expressing either CDK5 or CDK5-I plasmids were plated on SG-URA medium with 0.75 M NaCl or on an SD-URA plate (control) and incubated for 5 days at 30°C. ( B ) Overexpression of CDK5 in a cln1 Δ cln2 Δ pho85 Δ strain. A cln1Δ cln2Δ pho85Δ strain carrying a pho85 mutant allele ( pho85 ts17 ) expressed from the MET25 promoter (BY794) and either pCDK5 (right side of plates) or pCDK5-I (left side of plates) was streaked on a galactose-containing plate plus methionine (+ Gal + Met, Right ; repress pho85 ts17 , induce CDK5 ) or a galactose-containing plate minus methionine (control, Left ) and incubated for 5 days at 30°C. Two independent isolates are shown. ( C ) Overexpression of CDK5 in a slt2 Δ pho85 Δ strain. A slt2 Δ pho85 Δ strain expressing either PHO85 (left side of plates) or CDK5 (right side of plates) from the GAL promoter was streaked onto a yeast extract/peptone/dextrose (YPD) or yeast extract/peptone/galactose (YPG) plate as indicated and incubated for 5 days at 25°C. ( D ) Pho4 kinase activity associated with HA-Pcl2-Cdk5. Ha-Pcl2 was immunoprecipitated from yeast extracts and used in kinase assays with Pho4 as a substrate. The following strains were used for extract preparation: lane 1, pho85 Δ HA-PCL2 with pCDK5; lane 2, pho85 Δ HA-PCL2 with pCDK5-I; lane 3, pho85 Δ HA-PCL2 with pPHO85 (positive control).

    Article Snippet: Thus, CDK5 is able to complement a pho85 deletion strain for the G1 progression seen in strains deleted for other G1 regulators.

    Techniques: Expressing, Incubation, Over Expression, Mutagenesis, Activity Assay, Immunoprecipitation, Positive Control

    Biochemical and genetic complementation of the acid phosphatase regulation defect in a pho85Δ mutant by Cdk5 expression. ( A ) Complementation of defect in acid phosphatase regulation. Acid phosphatase activity was measured in pho85Δ strains containing pCDK5 or pCDK5-I and a PHO80 expression plasmid (pPho80) or a control vector (p230v). Acid phosphatase activity from a wild-type strain is also shown (wt). These strains were grown in repressed (7.4 mM phosphate, shaded bars) and derepressed (3.7 μM phosphate, solid bars) conditions. Values are normalized for cell density (OD 600 ). ( B ) Pho4 kinase assays with HA-Pho80-Cdk5. HA-Pho80 was immunoprecipitated from yeast extracts and used in kinase assays with Pho4 as substrate. The following strains were used for extract preparation: lane 1, pho85Δ transformed with pCDK5 and pHA-Pho80; lane 2, pho85Δ strain with pCDK5-I and pPho80; lane 3, pho85Δ strain with pCDK5 and pBA230v; lane 4, wild-type strain with pHA-Pho80 (positive control).

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

    Article Title: Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

    doi:

    Figure Lengend Snippet: Biochemical and genetic complementation of the acid phosphatase regulation defect in a pho85Δ mutant by Cdk5 expression. ( A ) Complementation of defect in acid phosphatase regulation. Acid phosphatase activity was measured in pho85Δ strains containing pCDK5 or pCDK5-I and a PHO80 expression plasmid (pPho80) or a control vector (p230v). Acid phosphatase activity from a wild-type strain is also shown (wt). These strains were grown in repressed (7.4 mM phosphate, shaded bars) and derepressed (3.7 μM phosphate, solid bars) conditions. Values are normalized for cell density (OD 600 ). ( B ) Pho4 kinase assays with HA-Pho80-Cdk5. HA-Pho80 was immunoprecipitated from yeast extracts and used in kinase assays with Pho4 as substrate. The following strains were used for extract preparation: lane 1, pho85Δ transformed with pCDK5 and pHA-Pho80; lane 2, pho85Δ strain with pCDK5-I and pPho80; lane 3, pho85Δ strain with pCDK5 and pBA230v; lane 4, wild-type strain with pHA-Pho80 (positive control).

    Article Snippet: Thus, CDK5 is able to complement a pho85 deletion strain for the G1 progression seen in strains deleted for other G1 regulators.

    Techniques: Mutagenesis, Expressing, Activity Assay, Plasmid Preparation, Immunoprecipitation, Transformation Assay, Positive Control

    Effect of Cdk5 and p35 overexpression in pho85 Δ cells. ( A ) Growth arrest caused by coexpression of Cdk5 and p35. pho85Δ strains transformed with pCDK5 or pCDK5-I with either a plasmid expressing p35 (p35) or an empty vector (230v) were grown in SD medium, serially diluted, and plated on glucose (SD-URA-TRP, Right ) or galactose (SG-URA-TRP, Left ) medium. ( B ) Morphology of pho85 Δ cells overexpressing Cdk5 and p35. Cells were grown in raffinose-containing medium (noninducing) to an OD 600 of ≈0.1, and then galactose was added to a final concentration of 2% to induce expression of CDK5 and p35. Cells were cultured for 24 hr and viewed at ×630 magnification and photographed. 1, pCDK5 + p35; 2, pCDK5 + vector; 3, pCDK5-I + p35; 4, pCDK5-I + vector.

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

    Article Title: Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

    doi:

    Figure Lengend Snippet: Effect of Cdk5 and p35 overexpression in pho85 Δ cells. ( A ) Growth arrest caused by coexpression of Cdk5 and p35. pho85Δ strains transformed with pCDK5 or pCDK5-I with either a plasmid expressing p35 (p35) or an empty vector (230v) were grown in SD medium, serially diluted, and plated on glucose (SD-URA-TRP, Right ) or galactose (SG-URA-TRP, Left ) medium. ( B ) Morphology of pho85 Δ cells overexpressing Cdk5 and p35. Cells were grown in raffinose-containing medium (noninducing) to an OD 600 of ≈0.1, and then galactose was added to a final concentration of 2% to induce expression of CDK5 and p35. Cells were cultured for 24 hr and viewed at ×630 magnification and photographed. 1, pCDK5 + p35; 2, pCDK5 + vector; 3, pCDK5-I + p35; 4, pCDK5-I + vector.

    Article Snippet: Thus, CDK5 is able to complement a pho85 deletion strain for the G1 progression seen in strains deleted for other G1 regulators.

    Techniques: Over Expression, Transformation Assay, Plasmid Preparation, Expressing, Concentration Assay, Cell Culture

    Activators of Cdk5 bind and activate Pho85 in mammalian and insect cells. ( A ) Coimmunoprecipitation of p35/p25 with Pho85. p35 antibodies were used to generate immunoprecipitates from lysates of COS-7 cells that had been transiently transfected with various combinations of CMV-p35, CMV-p25, CMV-cdk5, and CMV-HA-PHO85 as indicated at the top. Immunoblots were reacted by blotting with either anti-cdk5 (DC17) antibodies ( Left ) or anti-HA (12CA5) antibodies to detect HA-Pho85 ( Right ). Lanes: 1, p35+cdk5; 2, p25+cdk5; 3, cdk5 alone; 4, p25+PHO85; 5, p35+PHO85. Levels of Cdk5, HA-Pho85, and p25/35 expression in the extracts used in the immunoprecipitation reactions are shown ( Middle and Bottom ). ( B ) Phosphorylation of p25 and p35 by Pho85. Immunoprecipitation-kinase reactions were performed with anti-p35 antibodies by using lysates from COS-7 cells transfected with the following vector combinations: lane 1, CMV-p35; lane 2, CMV-p25; lane 3, CMV-cdk5; lane 4, CMV-HA-Pho85; lane 5, p35+cdk5; lane 6, p35+Pho85; lane 7, p25+cdk5; lane 8, p25+Pho85. The positions of migration of phosphorylated p35 and p25 are shown to the right. Levels of protein expression were confirmed by Western blotting with anti-p35, anti-cdk5, or anti-HA (Pho85) antibodies as indicated to the left. ( C ) Activation of Pho85 by p25 in insect cells. Lysates from Hi5 insect cells infected with baculoviral vectors expressing various combinations of GST-p25, GST-Pho85, 6XHis-Pho85D151N, and GST-Cdk5 were incubated with glutathione (GSH)-Sepharose beads to pull down the GST-tagged proteins. Kinase reactions were performed on the GSH precipitates with Histone H1 as substrate. The following proteins were present in the lysates: lane 1, GST-p25; lane 2, GST-Pho85; lane 3, 6XHis-Pho85D151N; lane 4, GST-p25+GST-cdk5; lane 5, GST-p25+GST-Pho85; lane 6, GST-p25+6XHis-Pho85D151N. The positions of migration of phosphorylated GST-Pho85, GST-p25, and Histone H1 (H1) are indicated to the right. Levels of protein expression in the lysates are shown by anti-6X-HIS Western blotting ( Middle , to detect the Pho85D151N protein) and anti-GST blotting ( Top ).

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

    Article Title: Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

    doi:

    Figure Lengend Snippet: Activators of Cdk5 bind and activate Pho85 in mammalian and insect cells. ( A ) Coimmunoprecipitation of p35/p25 with Pho85. p35 antibodies were used to generate immunoprecipitates from lysates of COS-7 cells that had been transiently transfected with various combinations of CMV-p35, CMV-p25, CMV-cdk5, and CMV-HA-PHO85 as indicated at the top. Immunoblots were reacted by blotting with either anti-cdk5 (DC17) antibodies ( Left ) or anti-HA (12CA5) antibodies to detect HA-Pho85 ( Right ). Lanes: 1, p35+cdk5; 2, p25+cdk5; 3, cdk5 alone; 4, p25+PHO85; 5, p35+PHO85. Levels of Cdk5, HA-Pho85, and p25/35 expression in the extracts used in the immunoprecipitation reactions are shown ( Middle and Bottom ). ( B ) Phosphorylation of p25 and p35 by Pho85. Immunoprecipitation-kinase reactions were performed with anti-p35 antibodies by using lysates from COS-7 cells transfected with the following vector combinations: lane 1, CMV-p35; lane 2, CMV-p25; lane 3, CMV-cdk5; lane 4, CMV-HA-Pho85; lane 5, p35+cdk5; lane 6, p35+Pho85; lane 7, p25+cdk5; lane 8, p25+Pho85. The positions of migration of phosphorylated p35 and p25 are shown to the right. Levels of protein expression were confirmed by Western blotting with anti-p35, anti-cdk5, or anti-HA (Pho85) antibodies as indicated to the left. ( C ) Activation of Pho85 by p25 in insect cells. Lysates from Hi5 insect cells infected with baculoviral vectors expressing various combinations of GST-p25, GST-Pho85, 6XHis-Pho85D151N, and GST-Cdk5 were incubated with glutathione (GSH)-Sepharose beads to pull down the GST-tagged proteins. Kinase reactions were performed on the GSH precipitates with Histone H1 as substrate. The following proteins were present in the lysates: lane 1, GST-p25; lane 2, GST-Pho85; lane 3, 6XHis-Pho85D151N; lane 4, GST-p25+GST-cdk5; lane 5, GST-p25+GST-Pho85; lane 6, GST-p25+6XHis-Pho85D151N. The positions of migration of phosphorylated GST-Pho85, GST-p25, and Histone H1 (H1) are indicated to the right. Levels of protein expression in the lysates are shown by anti-6X-HIS Western blotting ( Middle , to detect the Pho85D151N protein) and anti-GST blotting ( Top ).

    Article Snippet: Thus, CDK5 is able to complement a pho85 deletion strain for the G1 progression seen in strains deleted for other G1 regulators.

    Techniques: Transfection, Western Blot, Expressing, Immunoprecipitation, Plasmid Preparation, Migration, Activation Assay, Infection, Incubation

    Loss of Tead1 leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p

    Journal: PLoS ONE

    Article Title: Tead1 is required for perinatal cardiomyocyte proliferation

    doi: 10.1371/journal.pone.0212017

    Figure Lengend Snippet: Loss of Tead1 leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal day 7 Tead F/F (n = 7) and Tead1 -cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression, normalized by Hsp90. Data are presented as mean ± s.e.m. *** p

    Article Snippet: Loss of Tead1 leads to decreased levels of critical cell cycle proteins The expression of essential cell cycle proteins was assessed in Tead1 -deleted myocardium as compared to control flox littermates, by Western blotting.

    Techniques: Expressing, Western Blot

    Cardiomyocyte-specific deletion of Tead1 leads to early neonatal death by day 9 after birth. ( A ) Generation of Tead1 cardiomyocyte-specific knockout mice ( Tead1 -cKO) using knockout-first strategy: promoter-driven selection cassette. ( B ) Representative photograph of a Tead1 -cKO breeding litter, demonstrating Tead1 -cKO were born in Mendelian manner and not overtly distinguishable from littermates. ( C ) Kaplan-Meier survival curves of Tead1 F/F littermates and Tead1 -cKO mice. n = 15 mice per group. *** p

    Journal: PLoS ONE

    Article Title: Tead1 is required for perinatal cardiomyocyte proliferation

    doi: 10.1371/journal.pone.0212017

    Figure Lengend Snippet: Cardiomyocyte-specific deletion of Tead1 leads to early neonatal death by day 9 after birth. ( A ) Generation of Tead1 cardiomyocyte-specific knockout mice ( Tead1 -cKO) using knockout-first strategy: promoter-driven selection cassette. ( B ) Representative photograph of a Tead1 -cKO breeding litter, demonstrating Tead1 -cKO were born in Mendelian manner and not overtly distinguishable from littermates. ( C ) Kaplan-Meier survival curves of Tead1 F/F littermates and Tead1 -cKO mice. n = 15 mice per group. *** p

    Article Snippet: Loss of Tead1 leads to decreased levels of critical cell cycle proteins The expression of essential cell cycle proteins was assessed in Tead1 -deleted myocardium as compared to control flox littermates, by Western blotting.

    Techniques: Knock-Out, Mouse Assay, Selection

    Tead1 is required for cardiomyocyte proliferation. ( A, B ) Cardiomyocyte proliferation (Ki67) and mitosis (phospho-Histone 3 Ser10 positive immunostaining) in postnatal day 1 Tead F/F and Tead1 -cKO hearts. ( C, D ) Quantitative analysis represents counting of multiple sections from three independent samples per group (approximately 1.2–1.5 X 10 5 cardiomyocytes were counted). ** p

    Journal: PLoS ONE

    Article Title: Tead1 is required for perinatal cardiomyocyte proliferation

    doi: 10.1371/journal.pone.0212017

    Figure Lengend Snippet: Tead1 is required for cardiomyocyte proliferation. ( A, B ) Cardiomyocyte proliferation (Ki67) and mitosis (phospho-Histone 3 Ser10 positive immunostaining) in postnatal day 1 Tead F/F and Tead1 -cKO hearts. ( C, D ) Quantitative analysis represents counting of multiple sections from three independent samples per group (approximately 1.2–1.5 X 10 5 cardiomyocytes were counted). ** p

    Article Snippet: Loss of Tead1 leads to decreased levels of critical cell cycle proteins The expression of essential cell cycle proteins was assessed in Tead1 -deleted myocardium as compared to control flox littermates, by Western blotting.

    Techniques: Immunostaining

    Tead1 deletion in perinatal cardiomyocytes leads to elevated markers of heart failure. Data are presented as mean ± s.e.m. *** p

    Journal: PLoS ONE

    Article Title: Tead1 is required for perinatal cardiomyocyte proliferation

    doi: 10.1371/journal.pone.0212017

    Figure Lengend Snippet: Tead1 deletion in perinatal cardiomyocytes leads to elevated markers of heart failure. Data are presented as mean ± s.e.m. *** p

    Article Snippet: Loss of Tead1 leads to decreased levels of critical cell cycle proteins The expression of essential cell cycle proteins was assessed in Tead1 -deleted myocardium as compared to control flox littermates, by Western blotting.

    Techniques:

    Tead1 is required for postnatal cardiac function. Echocardiography on postnatal day 1 neonatal pups. ( A ) Representative image of the echocardiography. ( B ) ejection fraction, ( C ) fractional shortening (FS), ( D ) left ventricular anterior wall at systole (LVAWs), ( E-F ) left ventricle internal diameter at systole and diastole (LVIDs and LVIDd), and ( G ) relative wall thickness (RWT, 2xLVPWd/LVIDd) of the Tead F/F and Tead1 -cKO pups (n = 4–5). ** p

    Journal: PLoS ONE

    Article Title: Tead1 is required for perinatal cardiomyocyte proliferation

    doi: 10.1371/journal.pone.0212017

    Figure Lengend Snippet: Tead1 is required for postnatal cardiac function. Echocardiography on postnatal day 1 neonatal pups. ( A ) Representative image of the echocardiography. ( B ) ejection fraction, ( C ) fractional shortening (FS), ( D ) left ventricular anterior wall at systole (LVAWs), ( E-F ) left ventricle internal diameter at systole and diastole (LVIDs and LVIDd), and ( G ) relative wall thickness (RWT, 2xLVPWd/LVIDd) of the Tead F/F and Tead1 -cKO pups (n = 4–5). ** p

    Article Snippet: Loss of Tead1 leads to decreased levels of critical cell cycle proteins The expression of essential cell cycle proteins was assessed in Tead1 -deleted myocardium as compared to control flox littermates, by Western blotting.

    Techniques:

    Tead1 loss-of-function results in heart failure associated with myocardium hypoplasia. ( A ) Heart weight ( B ) Body weight and ( C ) heart weight to body weight ratios of Tead1-cKO mice at postnatal days 1 and 7 (n = 10–13). *** p

    Journal: PLoS ONE

    Article Title: Tead1 is required for perinatal cardiomyocyte proliferation

    doi: 10.1371/journal.pone.0212017

    Figure Lengend Snippet: Tead1 loss-of-function results in heart failure associated with myocardium hypoplasia. ( A ) Heart weight ( B ) Body weight and ( C ) heart weight to body weight ratios of Tead1-cKO mice at postnatal days 1 and 7 (n = 10–13). *** p

    Article Snippet: Loss of Tead1 leads to decreased levels of critical cell cycle proteins The expression of essential cell cycle proteins was assessed in Tead1 -deleted myocardium as compared to control flox littermates, by Western blotting.

    Techniques: Mouse Assay

    Loss of SOCS3 in mature myofibers in vivo enhances the inflammatory response after myotoxic injury. Control (SOCS3 fl/fl MCK-Cre − ) and SOCS3 MKO (SOCS3 fl/fl MCK-Cre + ) mice were either left uninjured (UN) or received a single 40 μL injection of notexin (10 μg/ml) into the right TA muscle and then killed for analysis at 1 day (D1), 2 days (D2), 3 days (D3), 5 days (D5), 7 days (D7), or 14 days (D14) post-notexin injury. a Western immunoblotting for phosphorylated and total STAT3 protein levels was performed on protein extracted from remaining OCT embedded muscles following tissue sectioning. Representative immunoblots for phosphorylated ( top ) and total ( bottom ) STAT3 protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories) and the ratio of phosphorylated/total STAT3 protein levels was determined. b Representative sections immunostained with F4/80 ( green ) and DAPI ( blue ) of the TA muscle from uninjured or day 1, 2, 3, 5, 7, or 14 injured control and SOCS3 MKO mice. c Representative sections immunostained with CD68 ( red ) and DAPI ( blue ) of the TA muscle from uninjured or day 1, 2, 3, 5, 7, or 14 injured control and SOCS3 MKO mice. qRT-PCR using primers to detect IL-6 ( d ), IFN-γ ( e ), TNF-α ( f ), F4/80 ( g ), and CD68 ( h ) was performed on RNA extracted from snap frozen muscles following dissection. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-way ANOVA with a Fisher’s LSD post hoc multiple comparisons test to determine the effects of genotype and time. n = 8 mice/time-point/genotype. ** P

    Journal: Skeletal Muscle

    Article Title: Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury

    doi: 10.1186/s13395-016-0108-4

    Figure Lengend Snippet: Loss of SOCS3 in mature myofibers in vivo enhances the inflammatory response after myotoxic injury. Control (SOCS3 fl/fl MCK-Cre − ) and SOCS3 MKO (SOCS3 fl/fl MCK-Cre + ) mice were either left uninjured (UN) or received a single 40 μL injection of notexin (10 μg/ml) into the right TA muscle and then killed for analysis at 1 day (D1), 2 days (D2), 3 days (D3), 5 days (D5), 7 days (D7), or 14 days (D14) post-notexin injury. a Western immunoblotting for phosphorylated and total STAT3 protein levels was performed on protein extracted from remaining OCT embedded muscles following tissue sectioning. Representative immunoblots for phosphorylated ( top ) and total ( bottom ) STAT3 protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories) and the ratio of phosphorylated/total STAT3 protein levels was determined. b Representative sections immunostained with F4/80 ( green ) and DAPI ( blue ) of the TA muscle from uninjured or day 1, 2, 3, 5, 7, or 14 injured control and SOCS3 MKO mice. c Representative sections immunostained with CD68 ( red ) and DAPI ( blue ) of the TA muscle from uninjured or day 1, 2, 3, 5, 7, or 14 injured control and SOCS3 MKO mice. qRT-PCR using primers to detect IL-6 ( d ), IFN-γ ( e ), TNF-α ( f ), F4/80 ( g ), and CD68 ( h ) was performed on RNA extracted from snap frozen muscles following dissection. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-way ANOVA with a Fisher’s LSD post hoc multiple comparisons test to determine the effects of genotype and time. n = 8 mice/time-point/genotype. ** P

    Article Snippet: The gene expression of the pro-inflammatory cytokine TNF-α , as well as the inflammatory cell markers F4/80 and CD68 , was low in uninjured muscles from control and SOCS3 MKO mice, increased progressively to day 3, then reduced to basal levels by day 14 post-notexin injury (Fig. – ; P < 0.0001 time main effect).

    Techniques: In Vivo, Mouse Assay, Injection, Western Blot, Software, Quantitative RT-PCR, Dissection

    SOCS3 deletion does not alter regeneration or inflammation after myotoxic injury in aged mice. Twenty-four-month-old control (SOCS3 fl/fl MCK-Cre − ) and SOCS3 MKO (SOCS3 fl/fl MCK-Cre + ) mice were either left uninjured (UN) or received a single 40 μL injection of notexin (10 μg/ml) into the right TA muscle. a Representative hematoxylin and eosin-stained sections of the TA muscle from uninjured or day 7 injured control and SOCS3 MKO mice. Muscle mass relative to body mass ( b ) and muscle fiber size ( c ) was determined at day 7 post-notexin injury. d Western immunoblotting for phosphorylated and total STAT3 protein levels was performed on protein extracted from the remaining OCT embedded muscles following tissue sectioning. Representative immunoblots for phosphorylated ( top ) and total ( bottom ) STAT3 protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories), and the ratio of phosphorylated/total STAT3 protein levels was determined. Bands used for quantification are indicated by arrows . qRT-PCR using primers to detect Socs3 ( e ), IL-6 ( f ), TNF-α ( g ), IFN-γ ( h ), F4/80 ( i ), CD68 ( j ), Pax7 ( k ), MyoD ( l ), or Myogenin ( m ) was performed on RNA extracted from snap frozen muscles following dissection. Data are expressed as mean ± SEM. Statistical analysis was performed using an unpaired two-tailed Student’s t test ( n = 7–8 mice/time-point/genotype) except for analysis of muscle fiber size ( c ) which was analyzed with a two-way ANOVA and Fisher’s LSD post hoc multiple comparisons test to determine the effect of genotype and injury ( n = 4–8 mice/time-point/genotype). Scale bar = 100 μm

    Journal: Skeletal Muscle

    Article Title: Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury

    doi: 10.1186/s13395-016-0108-4

    Figure Lengend Snippet: SOCS3 deletion does not alter regeneration or inflammation after myotoxic injury in aged mice. Twenty-four-month-old control (SOCS3 fl/fl MCK-Cre − ) and SOCS3 MKO (SOCS3 fl/fl MCK-Cre + ) mice were either left uninjured (UN) or received a single 40 μL injection of notexin (10 μg/ml) into the right TA muscle. a Representative hematoxylin and eosin-stained sections of the TA muscle from uninjured or day 7 injured control and SOCS3 MKO mice. Muscle mass relative to body mass ( b ) and muscle fiber size ( c ) was determined at day 7 post-notexin injury. d Western immunoblotting for phosphorylated and total STAT3 protein levels was performed on protein extracted from the remaining OCT embedded muscles following tissue sectioning. Representative immunoblots for phosphorylated ( top ) and total ( bottom ) STAT3 protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories), and the ratio of phosphorylated/total STAT3 protein levels was determined. Bands used for quantification are indicated by arrows . qRT-PCR using primers to detect Socs3 ( e ), IL-6 ( f ), TNF-α ( g ), IFN-γ ( h ), F4/80 ( i ), CD68 ( j ), Pax7 ( k ), MyoD ( l ), or Myogenin ( m ) was performed on RNA extracted from snap frozen muscles following dissection. Data are expressed as mean ± SEM. Statistical analysis was performed using an unpaired two-tailed Student’s t test ( n = 7–8 mice/time-point/genotype) except for analysis of muscle fiber size ( c ) which was analyzed with a two-way ANOVA and Fisher’s LSD post hoc multiple comparisons test to determine the effect of genotype and injury ( n = 4–8 mice/time-point/genotype). Scale bar = 100 μm

    Article Snippet: The gene expression of the pro-inflammatory cytokine TNF-α , as well as the inflammatory cell markers F4/80 and CD68 , was low in uninjured muscles from control and SOCS3 MKO mice, increased progressively to day 3, then reduced to basal levels by day 14 post-notexin injury (Fig. – ; P < 0.0001 time main effect).

    Techniques: Mouse Assay, Injection, Staining, Western Blot, Software, Quantitative RT-PCR, Dissection, Two Tailed Test