Journal: Scientific Reports

Article Title: Potential Orphan Drug Therapy of Intravesical Liposomal Onabotulinumtoxin-A for Ketamine-Induced Cystitis by Mucosal Protection and Anti-inflammation in a Rat Model

doi: 10.1038/s41598-018-24239-9

Figure Lengend Snippet: Alterations of neuroreceptors protein expression in the mucosa layer or smooth muscle layer of the bladder for all groups (n = 8). Western blot analyses with specific antibodies to the TRPV1 receptor and P2X 3 receptor of the rat mucosal layer as well as M 2 -and M 3 - mAChRs and the purinergic P2X 1 receptor of the rat detrusor layer were performed in 3 groups. ( A ) TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95 kDa. ( B ) Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65 kDa). ( C ) M 2 –mAChR of bladder detrusor layer. The M 2 –mAChR antibody produced a clear single band between 50kD and 75kD. ( D ) M 3 –mAChR of bladder detrusor layer. ( E ) Purinergic P2X 1 receptor. Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and other groups (One-way ANOVA with Dunnett’s test, p < 0.05). The grouping of blots was cropped from the same gel for each protein. The full-length gels and blots are included in the Supplementary Figure .

Article Snippet: Antibodies raised against SNAP25 (1: 1000 dilution; Cell signal), E-cadherin (1:1000 dilution; Cell Signal), nerve growth factor (1:200 dilution; Cell Signal), IL-1β (1:500 dilution; Abcam) IL-6 (1:1000 dilution; Abcam), TNF-α (1:250 dilution, Santa Cruz), NF-κB (1: 2500 dilution; Cell SiZgnal), COX-2 (1:200 dilution; Abcam), TRPV1 receptor (1:1000 dilution; Alomone), purinergic receptor P2X 1 (1:10000 dilution; Alomone), purinergic receptor P2X 3 (1: 1000 dilution; Alomone), M 2 -mAChR (1:1000 dilution; Alomone), M 3 -mAChR (1:1000 dilution; Alomone), and GAPDH (1: 10,000 dilution; Millipore) were used.

Techniques: Expressing, Western Blot, Produced

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: Involvement of LRP1 in Tat-mediated HIV-1 LTR transactivation. a The LRP1 antagonist, receptor-associated protein (RAP), concentration dependently reduced Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05). b Protein expression levels of LRP1 were significantly ( n = 3, p < 0.05) reduced in U87MG cells transfected with LRP1 siRNA (100 nM) compared to cells transfected with control (scrambled) siRNA. c siRNA knockdown of LRP1 reduced significantly ( n = 3; * p < 0.05) Tat-mediated HIV-1 LTR transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Concentration Assay, Expressing, Transfection

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE-HDL isoform dependently affected Tat-mediated HIV-1 LTR transactivation. U87MG cells stably transfected with a luciferase gene under the control of HIV-1 Tat responsive LTR promoter were incubated for 48 h with ApoE2-HDL ( a ), ApoE3-HDL ( b ), or ApoE4-HDL ( c ) in the presence of HIV-1 Tat protein (2 μg/ml) and 100 μM chloroquine (CQ). ApoE2-HDL, ApoE3-HDL, and ApoE4-HDL significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001) ( d ). In comparison to ApoE2-HDL and ApoE3-HDL, ApoE4-HDL was less potent and effective at restricting Tat-mediated HIV-1 transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Stable Transfection, Transfection, Luciferase, Incubation, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: HDL affects the ability of ApoE to restrict Tat-mediated HIV-1 LTR transactivation. a ApoE2 significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3; * p < 0.05; ** p < 0.01). b ApoE3 significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3; ** p < 0.01). c ApoE4 concentration dependently enhanced Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3, ** p < 0.01). d In the presence of HDL, ApoE2 was more potent and effective at restricting Tat-mediated HIV-1 LTR transactivation. e In the presence of HDL, ApoE3 was more potent and effective at restricting Tat-mediated HIV-1 LTR transactivation. f In presence of HDL, ApoE4 concentration dependently restricted Tat-mediated HIV-1 LTR transactivation; however, in the absence of HDL, ApoE4 concentration dependently enhanced Tat-mediated HIV-1 LTR transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE2 and ApoE3, but not ApoE4, inhibited HIV-1 Tat internalization. Representative images ( a ) and quantified internalized fluorescence intensities ( b ) show that ApoE2 and ApoE3 at 5 and 10 μg/ml inhibited HIV-1 Tat internalization as evidenced by lower levels of punctate staining of internalized FITC-labeled Tat ( n = 30; *** p < 0.001, **** p < 0.0001), whereas ApoE4 did not inhibit HIV-1 Tat internalization

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Fluorescence, Staining, Labeling

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE mimetic peptide decreased Tat-mediated HIV-1 LTR transactivation. a U87MG cells were treated with an ApoE mimetic peptide in the presence of HIV-1 Tat and chloroquine (CQ) for 48 h. ApoE mimetic peptide significantly reduced Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05). b Incubation with an ApoE4 structure corrector, that makes the structure and function of ApoE4 more like ApoE3, enhanced the ability of ApoE4 to restrict Tat-mediated LTR transactivation ( n = 3; * p < 0.05)

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Incubation

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a,b) Western blot analysis of MeCP2 S421 phosphorylation and the quantification of total MeCP2 protein level in WT and phosphor-mutant aNPCs. (p=0.718, unpaired t-test with Welch’s correction, n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in WT aNPCs under normal proliferating condition and FGF2/EGF withdrawal for 24 hours. (n=3 in each group) (e) Western blot analysis reveals that MeCP2 S421 phosphorylation is induced by synchronizing aNPCs with nocodazole (150ng/ml). (f,g) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs under conditions: 1) DMSO, 2) 36 hours of nocodazole treatment, 3) 24 hours of roscovitine (25 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) Numbers next to Western blots are molecular weight markers. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Western Blot, Phospho-proteomics, Mutagenesis, Molecular Weight

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a,b) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs: 1) DMSO, 2) 36 hours of nocodazole (150ng/ml) treatment, 3) 24 hours of hesperadin (2 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in EGFP-shRNA or AURKB-shRNA lentivirus infected aNPCs, which are treated with DMSO or nocodazole for 24 hours. (e,f) Western blot analysis reveals that MeCP2 and AURKB are co-immunoprecipitated reciprocally. (g) Western blot analysis reveals endogenous interaction between MeCP2 and AURKB in aNPCs. (h) In vitro kinase assay followed by Western blot demonstrates that AURKB can phosphorylate S421 on MeCP2. Numbers next to Western blots are molecular weight markers.

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Western Blot, Phospho-proteomics, shRNA, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Molecular Weight

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a) Representative images of aNPCs isolated from WT and Mecp2 S421A;S424A/y hippocampus with BrdU pulse labeling, followed by immunocytochemistry analysis (b) Quantification of the percentage of BrdU/Sox2/Nestin triple-labeled cells in WT and Mecp2 S421A;S424A/y aNPCs. (n=3 in each group) (c) Representative images of Tuj1+ neurons differentiated from WT and Mecp2 S421A;S424A/y aNPCs (d) Quantification of the percentage of Tuj1+ cells in WT and Mecp2 S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (e) Representative images of GFAP+ astrocyte differentiated from WT and Mecp2 S421A;S424A/y aNPCs (f) Quantification of the percentage of GFAP+ cells in WT and Mecp2 S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (g) Relative mRNA level of neuronal marker ( Tuj1 and NeuroD1 ) and astrocyte marker ( GFAP ) in WT and Mecp2 S421A;S424A/y aNPCs upon differentiation, assayed by RT-qPCR. (n=3 in each group) (h) Schematics of the design of the in vivo BrdU labeling experiment. (i) Representative images of WT and the Mecp2 S421A;S424A/y brain sections stained for BrdU immunoreactivity. (j) Quantification of relative number of BrdU+ cells obtained through stereological counting from WT and Mecp2 S421A;S424A/y mice (n=9 in each group). (k) Quantification of the relative number of Ki67+ cells obtained through stereological counting from WT and Mecp2 S421A;S424A/y mice (n=6 in each group). (l) Schematics of the design of in vivo BrdU pulse/chase experiment to examine the differentiation profile of the adult-born hippocampal cells. (m) Representative confocal microscopy images to demonstrate how each cell type is identified. Three adult-born neurons (co-stained by BrdU and NeuN) are marked by arrow. One adult-born glial cell (co-stained by BrdU and S100b) is marked by arrowhead. Two adult-born undetermined cells (stained by BrdU only) are marked by asterisk. The rectangle panel to the right of the merged channel image is the y-z view of the same optical stack. The optical size of the z-scan is 0.4μm/step. (n) Quantification of proportions of the cell fate choices made by the dividing aNPCs in the hippocampus of WT and Mecp2 S421A;S424A/y mice. All scale bars are 50 μm. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Isolation, Labeling, Immunocytochemistry, Marker, Quantitative RT-PCR, In Vivo, Staining, Pulse Chase, Confocal Microscopy

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a) RT-qPCR analysis of the relative mRNA level of Dll1 and Notch1 in WT and Mecp2 S421A;S424A/y aNPCs. (n=5 in each group for Dll1 , n=8 in each group for Notch1 ) (b) RT-qPCR analysis of the relative mRNA level of Notch target gene Hes5 and Hey1 in WT and Mecp2 S421A;S424A/y aNPCs. (n=5 in each group) (c,d) Western blot analysis and quantification of the relative protein level of DLL1 in WT and Mecp2 S421A;S424A/y aNPCs. (n=4 in each group) (e, f) Western blot analysis and quantification of NICD level in WT and Mecp2 S421A;S424A/y aNPCs (n=5 in each group) (g) RT-qPCR analysis of the relative mRNA level of Hes5 and Hey1 in WT and Mecp2 S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (h, i) Representative images and quantification of BrdU-labeled cells in WT and Mecp2 S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus, followed by BrdU pulse labeling. (n=3 in each group) (j, k) Representative images and quantification of Tuj1+ neurons differentiated from WT and Mecp2 S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (l) Relative mRNA level of neuronal marker ( Tuj1 and NeuroD1 ) and astrocyte marker ( GFAP ) in WT and Mecp2 S421A;S424A/y aNPCs, which are infected with GFP- or NICD-lentivirus and then cultured in differentiation condition. (n=3 in each group) Scale bar 50 μm. (m) ChIP-qPCR analysis of the promoter occupancy of WT and phosphor-mutant MeCP2 on Dll1 and Notch1 promoters. (n=4 in each group) Numbers next to Western blots are molecular weight markers. The bar graphs in this figure show the mean ± s.e.m * p<0.05 ** p<0.01

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Quantitative RT-PCR, Western Blot, Infection, Labeling, Marker, Cell Culture, ChIP-qPCR, Mutagenesis, Molecular Weight