Structured Review

Genechem control vector gfp
Overexpression of <t>PNUTS</t> induces EMT and significantly increases the mobility and invasiveness of CNE-2 cells. Notes: ( A ) CNE-2 cells were infected with <t>ad-GFP</t> or ad-PNUTS at an efficiency measured by Western blotting. Western blotting was used to detect the expression of vimentin, N-cadherin, and E-cadherin. The internal control was GAPDH. ( B , C ) After overexpressing PNUTS, wound-healing assays (100× magnification) and Transwell assays (200× magnification) were used to detect the mobility and invasiveness of CNE-2 cells. Mean ± SD. n=3. * P
Control Vector Gfp, supplied by Genechem, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "PNUTS mediates ionizing radiation-induced CNE-2 nasopharyngeal carcinoma cell migration, invasion, and epithelial–mesenchymal transition via the PI3K/AKT signaling pathway"

Article Title: PNUTS mediates ionizing radiation-induced CNE-2 nasopharyngeal carcinoma cell migration, invasion, and epithelial–mesenchymal transition via the PI3K/AKT signaling pathway

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S188571

Overexpression of PNUTS induces EMT and significantly increases the mobility and invasiveness of CNE-2 cells. Notes: ( A ) CNE-2 cells were infected with ad-GFP or ad-PNUTS at an efficiency measured by Western blotting. Western blotting was used to detect the expression of vimentin, N-cadherin, and E-cadherin. The internal control was GAPDH. ( B , C ) After overexpressing PNUTS, wound-healing assays (100× magnification) and Transwell assays (200× magnification) were used to detect the mobility and invasiveness of CNE-2 cells. Mean ± SD. n=3. * P
Figure Legend Snippet: Overexpression of PNUTS induces EMT and significantly increases the mobility and invasiveness of CNE-2 cells. Notes: ( A ) CNE-2 cells were infected with ad-GFP or ad-PNUTS at an efficiency measured by Western blotting. Western blotting was used to detect the expression of vimentin, N-cadherin, and E-cadherin. The internal control was GAPDH. ( B , C ) After overexpressing PNUTS, wound-healing assays (100× magnification) and Transwell assays (200× magnification) were used to detect the mobility and invasiveness of CNE-2 cells. Mean ± SD. n=3. * P

Techniques Used: Over Expression, Infection, Western Blot, Expressing

Related Articles

Infection:

Article Title: PNUTS mediates ionizing radiation-induced CNE-2 nasopharyngeal carcinoma cell migration, invasion, and epithelial–mesenchymal transition via the PI3K/AKT signaling pathway
Article Snippet: .. Adenovirus infection An adenovirus expressing PNUTS and its control vector GFP were packaged and synthesized by Genechem (Shanghai, China). ..

Over Expression:

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling
Article Snippet: .. Lentivirus vector (GV358) mediated MSI2 overexpression (MSI2-GFP) and corresponding control vector (GFP) were also synthesized from Genechem. .. SW1990 cells with MSI2 low expression was used to construct MSI2 overexpressing stable PC cells following puromycin selection.

Expressing:

Article Title: PNUTS mediates ionizing radiation-induced CNE-2 nasopharyngeal carcinoma cell migration, invasion, and epithelial–mesenchymal transition via the PI3K/AKT signaling pathway
Article Snippet: .. Adenovirus infection An adenovirus expressing PNUTS and its control vector GFP were packaged and synthesized by Genechem (Shanghai, China). ..

Plasmid Preparation:

Article Title: PNUTS mediates ionizing radiation-induced CNE-2 nasopharyngeal carcinoma cell migration, invasion, and epithelial–mesenchymal transition via the PI3K/AKT signaling pathway
Article Snippet: .. Adenovirus infection An adenovirus expressing PNUTS and its control vector GFP were packaged and synthesized by Genechem (Shanghai, China). ..

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling
Article Snippet: .. Lentivirus vector (GV358) mediated MSI2 overexpression (MSI2-GFP) and corresponding control vector (GFP) were also synthesized from Genechem. .. SW1990 cells with MSI2 low expression was used to construct MSI2 overexpressing stable PC cells following puromycin selection.

Synthesized:

Article Title: PNUTS mediates ionizing radiation-induced CNE-2 nasopharyngeal carcinoma cell migration, invasion, and epithelial–mesenchymal transition via the PI3K/AKT signaling pathway
Article Snippet: .. Adenovirus infection An adenovirus expressing PNUTS and its control vector GFP were packaged and synthesized by Genechem (Shanghai, China). ..

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling
Article Snippet: .. Lentivirus vector (GV358) mediated MSI2 overexpression (MSI2-GFP) and corresponding control vector (GFP) were also synthesized from Genechem. .. SW1990 cells with MSI2 low expression was used to construct MSI2 overexpressing stable PC cells following puromycin selection.

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    Genechem pgcsil gfp vector
    Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: <t>pGCsil-GFP</t> Vector *P
    Pgcsil Gfp Vector, supplied by Genechem, used in various techniques. Bioz Stars score: 91/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Shanghai Genechem hippocampal neurons hip1r shrna gfp lentivirus
    <t>HIP1R</t> knockdown reduces expression of NMDARs, AMPARs and PSD95, but not GABA A . Cultured hippocampal neurons were infected with <t>HIP1R-shRNA</t> or Scrambled <t>lentivirus</t> at DIV6 and analyzed by western blotting at DIV13. (A) A representative western blot for total GluN2A, GluN2B, GluA1 and GABA A -α1 expression. (B) Quantification of blots from repeated independent experiments showed that the total expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control with 0.52 ± 0.11, n = 7 vs. 1.02 ± 0.18, n = 7 for GluN2A; 0.46 ± 0.07, n = 6 vs. CTL, 1.00 ± 0.20, n = 6 for GluN2B; and 0.52 ± 0.06, n = 6 vs. 1.00 ± 0.08, n = 6 for GluA1, respectively. (C) A representative western blot for surface GluN2A, GluN2B, GluA1 and GABA A -α1 expression. (D) The ratio of surface to total expression levels of GluN2A, GluN2B, GluA1 and GABA A -α1 did not differ between the HIP1R-KD group and the control. (E) Surface expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control, when normalized for GABA A -α1. There was no significant change in the total and surface expression levels of GABA A -α1 in the HIP1R-KD group. The ratio of HIP1R-KD vs. the control: 0.47 ± 0.05, n = 4 for GluN2A, 0.66 ± 0.06, n = 3 for GluN2B; 0.36 ± 0.01, n = 5 for GluA1 vs. 1.00 ± 0.05, n = 5 for GABA A -α1, respectively. (F) Representative images of dendrites immunostained using anti-PSD95 antibody at DIV16 in cultured hippocampal neurons transfected at DIV6 with HIP1R-shRNA and Scrambled vectors, respectively. (G) Quantification showed a significant decrease in the cluster density of PSD95 in the HIP1R-KD group compared to the control with 0.12 ± 0.01, n = 55 vs. 0.48 ± 0.02, n = 55, respectively. (H) Representative western blots for PSD95 of lysates from cultured hippocampal neurons at DIV13 6 days after infection with HIP1R-shRNA or Scrambled lentivirus. (I) Quantification of western blots from three independent experiments showed decreased expression of PSD95 in the HIP1R-KD group compared to the control with 0.68 ± 0.06, n = 3 vs. 1.00 ± 0.07, n = 3, respectively. All data are presented as mean ± SEM. * P
    Hippocampal Neurons Hip1r Shrna Gfp Lentivirus, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem asic2a knockdown
    TFCP2 inversely regulates <t>ASIC2a</t> expression. ( a ) Representative immunoblot and densitometric analyses showing that TFCP2 siRNA-transfected PC12 cells had 27% lower TFCP2 expression than negative siRNA-treated cells and consequently elevated ASIC2a expression. (b ) Representative immunoblot and densitometric analyses showing that TFCP2 OE plasmid-transfected PC12 cells had 1.76 ± 0.45-fold greater TFCP2 expression than negative control cells and consequently suppressed ASIC2a expression. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P
    Asic2a Knockdown, supplied by Genechem, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem wt idh1
    <t>IDH1</t> R132H mutation induces proliferation and migration of NSCLC cells. ( a ) WB analysis of IDH1 and IDH1 R132H protein levels in H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H. ( b ) Proliferation of H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H assessed by using the MTS assay. ( c ) Migration of H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H was assessed by Transwell migration assay. ( d ) WB of IDH1 protein in H460 cells after transfection with IDH1 siRNA. ( e ) Proliferation of H460 cells after transfection with IDH1 siRNA was assessed by MTS assay. ( f ) Migration of H460 cells after transfection with IDH1 siRNA was assessed by Transwell migration assay. ( g ) Proliferation of H1299 cells expressing mutant IDH1 R132H and treated with 20 µM IDH1 inhibitor (AGI-5198) was assessed by MTS assay. ( h ) Migration of H1299 cells treated as in ( g ) was assessed by Transwell migration assay.
    Wt Idh1, supplied by Genechem, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: Over Expression, In Vivo, Negative Control, Plasmid Preparation

    Overexpression of miR-675-5p inhibited proliferation and colony formation of NSCLC cells. (A) the level of miR-675-5p in A549 and HTB-182 cells was significantly up-regulated after transfection with miR-675-precursor. (B) miR-675-5p reduced cell proliferation in NSCLC cells. Cell proliferation was determined using MTT assays. (C) miR-675-5p induced cell cycle arrest at the G1/S phase. (D) miR-675-5p suppressed colony formation compared with controls. The number of colonies were calculated and depicted by the ban graph. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: Overexpression of miR-675-5p inhibited proliferation and colony formation of NSCLC cells. (A) the level of miR-675-5p in A549 and HTB-182 cells was significantly up-regulated after transfection with miR-675-precursor. (B) miR-675-5p reduced cell proliferation in NSCLC cells. Cell proliferation was determined using MTT assays. (C) miR-675-5p induced cell cycle arrest at the G1/S phase. (D) miR-675-5p suppressed colony formation compared with controls. The number of colonies were calculated and depicted by the ban graph. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: Over Expression, Transfection, MTT Assay, Negative Control, Plasmid Preparation

    The effect of miR-675-5p on in vitro migration and invasiveness of NSCLC cells. (A) The number of migrating or invading cells in the miR-675-precursor group was significantly decreased compared with the control. (B) The wound healing rate in cells transfected with miR-675-precursor was significantly decreased. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: The effect of miR-675-5p on in vitro migration and invasiveness of NSCLC cells. (A) The number of migrating or invading cells in the miR-675-precursor group was significantly decreased compared with the control. (B) The wound healing rate in cells transfected with miR-675-precursor was significantly decreased. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: In Vitro, Migration, Transfection, Negative Control, Plasmid Preparation

    GPR55 is a direct downstream target of miR-675-5p. (A) Schematic of the construction of wild-type or mutant pGL3-GPR55 3′-UTR vectors is illustrated. (B) Relative luciferase activity was analyzed in A549 cells. Firefly luciferase vector was used as an internal control. (C and D) Related expression of GPR55 protein in A549 and HTB-182 cells treated with miR-675 precursor was determined by western blot analysis. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: GPR55 is a direct downstream target of miR-675-5p. (A) Schematic of the construction of wild-type or mutant pGL3-GPR55 3′-UTR vectors is illustrated. (B) Relative luciferase activity was analyzed in A549 cells. Firefly luciferase vector was used as an internal control. (C and D) Related expression of GPR55 protein in A549 and HTB-182 cells treated with miR-675 precursor was determined by western blot analysis. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: Mutagenesis, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Western Blot, Negative Control

    HIP1R knockdown reduces expression of NMDARs, AMPARs and PSD95, but not GABA A . Cultured hippocampal neurons were infected with HIP1R-shRNA or Scrambled lentivirus at DIV6 and analyzed by western blotting at DIV13. (A) A representative western blot for total GluN2A, GluN2B, GluA1 and GABA A -α1 expression. (B) Quantification of blots from repeated independent experiments showed that the total expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control with 0.52 ± 0.11, n = 7 vs. 1.02 ± 0.18, n = 7 for GluN2A; 0.46 ± 0.07, n = 6 vs. CTL, 1.00 ± 0.20, n = 6 for GluN2B; and 0.52 ± 0.06, n = 6 vs. 1.00 ± 0.08, n = 6 for GluA1, respectively. (C) A representative western blot for surface GluN2A, GluN2B, GluA1 and GABA A -α1 expression. (D) The ratio of surface to total expression levels of GluN2A, GluN2B, GluA1 and GABA A -α1 did not differ between the HIP1R-KD group and the control. (E) Surface expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control, when normalized for GABA A -α1. There was no significant change in the total and surface expression levels of GABA A -α1 in the HIP1R-KD group. The ratio of HIP1R-KD vs. the control: 0.47 ± 0.05, n = 4 for GluN2A, 0.66 ± 0.06, n = 3 for GluN2B; 0.36 ± 0.01, n = 5 for GluA1 vs. 1.00 ± 0.05, n = 5 for GABA A -α1, respectively. (F) Representative images of dendrites immunostained using anti-PSD95 antibody at DIV16 in cultured hippocampal neurons transfected at DIV6 with HIP1R-shRNA and Scrambled vectors, respectively. (G) Quantification showed a significant decrease in the cluster density of PSD95 in the HIP1R-KD group compared to the control with 0.12 ± 0.01, n = 55 vs. 0.48 ± 0.02, n = 55, respectively. (H) Representative western blots for PSD95 of lysates from cultured hippocampal neurons at DIV13 6 days after infection with HIP1R-shRNA or Scrambled lentivirus. (I) Quantification of western blots from three independent experiments showed decreased expression of PSD95 in the HIP1R-KD group compared to the control with 0.68 ± 0.06, n = 3 vs. 1.00 ± 0.07, n = 3, respectively. All data are presented as mean ± SEM. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons

    doi: 10.3389/fnmol.2017.00186

    Figure Lengend Snippet: HIP1R knockdown reduces expression of NMDARs, AMPARs and PSD95, but not GABA A . Cultured hippocampal neurons were infected with HIP1R-shRNA or Scrambled lentivirus at DIV6 and analyzed by western blotting at DIV13. (A) A representative western blot for total GluN2A, GluN2B, GluA1 and GABA A -α1 expression. (B) Quantification of blots from repeated independent experiments showed that the total expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control with 0.52 ± 0.11, n = 7 vs. 1.02 ± 0.18, n = 7 for GluN2A; 0.46 ± 0.07, n = 6 vs. CTL, 1.00 ± 0.20, n = 6 for GluN2B; and 0.52 ± 0.06, n = 6 vs. 1.00 ± 0.08, n = 6 for GluA1, respectively. (C) A representative western blot for surface GluN2A, GluN2B, GluA1 and GABA A -α1 expression. (D) The ratio of surface to total expression levels of GluN2A, GluN2B, GluA1 and GABA A -α1 did not differ between the HIP1R-KD group and the control. (E) Surface expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control, when normalized for GABA A -α1. There was no significant change in the total and surface expression levels of GABA A -α1 in the HIP1R-KD group. The ratio of HIP1R-KD vs. the control: 0.47 ± 0.05, n = 4 for GluN2A, 0.66 ± 0.06, n = 3 for GluN2B; 0.36 ± 0.01, n = 5 for GluA1 vs. 1.00 ± 0.05, n = 5 for GABA A -α1, respectively. (F) Representative images of dendrites immunostained using anti-PSD95 antibody at DIV16 in cultured hippocampal neurons transfected at DIV6 with HIP1R-shRNA and Scrambled vectors, respectively. (G) Quantification showed a significant decrease in the cluster density of PSD95 in the HIP1R-KD group compared to the control with 0.12 ± 0.01, n = 55 vs. 0.48 ± 0.02, n = 55, respectively. (H) Representative western blots for PSD95 of lysates from cultured hippocampal neurons at DIV13 6 days after infection with HIP1R-shRNA or Scrambled lentivirus. (I) Quantification of western blots from three independent experiments showed decreased expression of PSD95 in the HIP1R-KD group compared to the control with 0.68 ± 0.06, n = 3 vs. 1.00 ± 0.07, n = 3, respectively. All data are presented as mean ± SEM. * P

    Article Snippet: Lentivirus- or Plasmid-Mediated RNAi in Primary Cultured Hippocampal Neurons HIP1R-shRNA/GFP lentivirus mentioned above and scrambled control-shRNA/GFP lentivirus were made by the Shanghai GeneChem Company (Shanghai, China).

    Techniques: Expressing, Cell Culture, Infection, shRNA, Western Blot, CTL Assay, Transfection

    Expression of the HIP1R C-terminus and its proline-rich region confer a dominant negative effect on dendrite development. (A) A schematic illustration showing full-length and several truncated forms of HIP1R cDNA in the myc-tagged expressing vector. (B) Cultured hippocampal neurons were transfected at DIV6 with each of the myc-tagged HIP1R mutants. An empty vector was taken as a negative control and GFP-shRNA as a positive control. Representative images of transfected neurons are shown immunostained at DIV12 with myc and MAP2 antibodies. Scale bar=20 μm. (C) ANOVA with repeated measures for Sholl analysis showed a significant decrease in dendrite complexity in HIP1R 766–1068 and HIP1R 1018–1068 transfected neurons. (D,E) Quantitative analysis with unpaired two-tailed t -tests showed a partial decrease in dendrite number and total length in HIP1R 766–1068 and HIP1R 1018–1068 transfected neurons compared to the negative and positive controls. Dendrite number: 44.69 ± 2.18, n = 42 for the negative control (CTL); 43.70 ± 3.30, n = 46 for myc-HIP1R 1–350 ; 42.30 ± 2.95, n = 44 for myc-HIP1R 350–655 ; 30.21 ± 2.03, n = 48 for myc-HIP1R 766–1068 ; 42.98 ± 2.23, n = 42 for myc-HIP1R 766–1017 ; 29.70 ± 1.59, n = 40 for myc-HIP1R 1018–1068 ; 12.17 ± 0.68, n = 42 for the positive control (KD). Dendrite total length: 1041.91 ± 29.58 μm, n = 42 for the negative control (CTL); 957.21 ± 42.99 μm, n = 46 for myc-HIP1R 1–350 ; 965.05 ± 47.24 μm, n = 44 for myc-HIP1R 350–655 ; 642.64 ± 34.09 μm, n = 48 for myc-HIP1R 766–1068 ; 942.51 ± 37.93 μm, n = 42 for myc-HIP1R 766–1017 ; 711.39 ± 30.27 μm, n = 40 for myc-HIP1R 1018–1068 ; 401.25 ± 21.66 μm, n = 42 for the positive control (KD). (F,G) Myc-vector or myc-HIP1R1018–1068 plasmid was transfected into DIV6–7 hippocampal neurons. Transfected neurons were stained with anti-myc antibody to analyze the spine density at DIV18–21. Spine density per μm: 0.18 ± 0.01, n = 55 for myc-HIP1R1018–1068 vs. 0.49 ± 0.01, n = 55 for the vector control. All data are presented as mean ± SEM. # P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons

    doi: 10.3389/fnmol.2017.00186

    Figure Lengend Snippet: Expression of the HIP1R C-terminus and its proline-rich region confer a dominant negative effect on dendrite development. (A) A schematic illustration showing full-length and several truncated forms of HIP1R cDNA in the myc-tagged expressing vector. (B) Cultured hippocampal neurons were transfected at DIV6 with each of the myc-tagged HIP1R mutants. An empty vector was taken as a negative control and GFP-shRNA as a positive control. Representative images of transfected neurons are shown immunostained at DIV12 with myc and MAP2 antibodies. Scale bar=20 μm. (C) ANOVA with repeated measures for Sholl analysis showed a significant decrease in dendrite complexity in HIP1R 766–1068 and HIP1R 1018–1068 transfected neurons. (D,E) Quantitative analysis with unpaired two-tailed t -tests showed a partial decrease in dendrite number and total length in HIP1R 766–1068 and HIP1R 1018–1068 transfected neurons compared to the negative and positive controls. Dendrite number: 44.69 ± 2.18, n = 42 for the negative control (CTL); 43.70 ± 3.30, n = 46 for myc-HIP1R 1–350 ; 42.30 ± 2.95, n = 44 for myc-HIP1R 350–655 ; 30.21 ± 2.03, n = 48 for myc-HIP1R 766–1068 ; 42.98 ± 2.23, n = 42 for myc-HIP1R 766–1017 ; 29.70 ± 1.59, n = 40 for myc-HIP1R 1018–1068 ; 12.17 ± 0.68, n = 42 for the positive control (KD). Dendrite total length: 1041.91 ± 29.58 μm, n = 42 for the negative control (CTL); 957.21 ± 42.99 μm, n = 46 for myc-HIP1R 1–350 ; 965.05 ± 47.24 μm, n = 44 for myc-HIP1R 350–655 ; 642.64 ± 34.09 μm, n = 48 for myc-HIP1R 766–1068 ; 942.51 ± 37.93 μm, n = 42 for myc-HIP1R 766–1017 ; 711.39 ± 30.27 μm, n = 40 for myc-HIP1R 1018–1068 ; 401.25 ± 21.66 μm, n = 42 for the positive control (KD). (F,G) Myc-vector or myc-HIP1R1018–1068 plasmid was transfected into DIV6–7 hippocampal neurons. Transfected neurons were stained with anti-myc antibody to analyze the spine density at DIV18–21. Spine density per μm: 0.18 ± 0.01, n = 55 for myc-HIP1R1018–1068 vs. 0.49 ± 0.01, n = 55 for the vector control. All data are presented as mean ± SEM. # P

    Article Snippet: Lentivirus- or Plasmid-Mediated RNAi in Primary Cultured Hippocampal Neurons HIP1R-shRNA/GFP lentivirus mentioned above and scrambled control-shRNA/GFP lentivirus were made by the Shanghai GeneChem Company (Shanghai, China).

    Techniques: Expressing, Dominant Negative Mutation, Plasmid Preparation, Cell Culture, Transfection, Negative Control, shRNA, Positive Control, Two Tailed Test, CTL Assay, Staining

    Transfection of mouse HIP1R rescues the dendrite growth defects in HIP1R-knockeddown neurons. (A) A schematic illustration showing mouse full-length and several truncated forms of HIP1R cDNA in the myc-tagged expressing vector. (B) Cultured hippocampal neurons at DIV6 were co-transfected with HIP1R-shRNA for knocking down endogenous HIP1R expression, and full-length or truncated HIP1R vectors for re-expressing exogenous cognates. Transfected neurons were immunostained with myc and MAP2 antibodies at DIV12. GFP-expressing and positively myc stained neurons were used for morphological analysis. (C) ANOVA analysis with repeated measures of Sholl analysis showed that full-length HIP1R re-expression fully rescued the complexity impairment in HIP1R KD neurons. HIP1R 1–350/766–1068 had a partial rescue effect. (D,E) Unpaired two-tailed t -test analysis of total dendrite length and number. Total dendrite length: 1076.65 ± 36.67 μm, n = 31 for the control group (CTL); 1098.99 ± 56.12 μm, n = 32 for KD+myc-HIP1R; 425.94 ± 20.50 μm, n = 33 for KD+myc-HIP1R 350–1068 ; 817.70 ± 42.27 μm, n = 36 for KD+myc -HIP1R 1–350/766–1068 ; 437.96 ± 28.07 μm, n = 37 for KD+myc-HIP1R 1–655 ; and 374.80 ± 26.39 μm, n = 31 for KD + Vector. Total dendrite number: 44.45 ± 2.99, n = 31 for the control group (CTL); 43.80 ± 1.81, n = 32 for KD+myc-HIP1R; 13.85 ± 0.93, n = 33 for KD+myc-HIP1R 350–1068 ; 32.53 ± 2.56, n = 36 for KD+myc-HIP1R 1–350/766–1068 ; 15.19 ± 1.22, n = 37 for KD+myc-HIP1R 1–655 ; and 11.83 ± 0.83, n = 31 for KD + Vector. All data are presented as mean ± SEM. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons

    doi: 10.3389/fnmol.2017.00186

    Figure Lengend Snippet: Transfection of mouse HIP1R rescues the dendrite growth defects in HIP1R-knockeddown neurons. (A) A schematic illustration showing mouse full-length and several truncated forms of HIP1R cDNA in the myc-tagged expressing vector. (B) Cultured hippocampal neurons at DIV6 were co-transfected with HIP1R-shRNA for knocking down endogenous HIP1R expression, and full-length or truncated HIP1R vectors for re-expressing exogenous cognates. Transfected neurons were immunostained with myc and MAP2 antibodies at DIV12. GFP-expressing and positively myc stained neurons were used for morphological analysis. (C) ANOVA analysis with repeated measures of Sholl analysis showed that full-length HIP1R re-expression fully rescued the complexity impairment in HIP1R KD neurons. HIP1R 1–350/766–1068 had a partial rescue effect. (D,E) Unpaired two-tailed t -test analysis of total dendrite length and number. Total dendrite length: 1076.65 ± 36.67 μm, n = 31 for the control group (CTL); 1098.99 ± 56.12 μm, n = 32 for KD+myc-HIP1R; 425.94 ± 20.50 μm, n = 33 for KD+myc-HIP1R 350–1068 ; 817.70 ± 42.27 μm, n = 36 for KD+myc -HIP1R 1–350/766–1068 ; 437.96 ± 28.07 μm, n = 37 for KD+myc-HIP1R 1–655 ; and 374.80 ± 26.39 μm, n = 31 for KD + Vector. Total dendrite number: 44.45 ± 2.99, n = 31 for the control group (CTL); 43.80 ± 1.81, n = 32 for KD+myc-HIP1R; 13.85 ± 0.93, n = 33 for KD+myc-HIP1R 350–1068 ; 32.53 ± 2.56, n = 36 for KD+myc-HIP1R 1–350/766–1068 ; 15.19 ± 1.22, n = 37 for KD+myc-HIP1R 1–655 ; and 11.83 ± 0.83, n = 31 for KD + Vector. All data are presented as mean ± SEM. ** P

    Article Snippet: Lentivirus- or Plasmid-Mediated RNAi in Primary Cultured Hippocampal Neurons HIP1R-shRNA/GFP lentivirus mentioned above and scrambled control-shRNA/GFP lentivirus were made by the Shanghai GeneChem Company (Shanghai, China).

    Techniques: Transfection, Expressing, Plasmid Preparation, Cell Culture, shRNA, Staining, Two Tailed Test, CTL Assay

    Knockdown of HIP1R expression suppresses dendrite growth and spine formation. (A) Cultured hippocampal neurons were infected by HIP1R-shRNA or scrambled control lentivirus and harvested for western blotting at DIV13. A representative western blot shows that the expression level of HIP1R was dramatically reduced in the shRNA group (shRNA) compared to the scrambled control (Scrambled), and viral infection itself had no effect on HIP1R expression (Blank). (B) Densitometric analysis of western blotting from 4 independent experiments; Blank, 1.00 ± 0.07; Scrambled, 1.04 ± 0.12; shRNA, 0.17 ± 0.03. (C) Cultured hippocampal neurons were transfected at DIV6 with HIP1R-shRNA or scrambled plasmid vectors. Immunofluorescence staining at DIV13 showed a significant decrease in HIPIR immunoreactivity in HIP1R-KD neurons (shRNA) but not in the control (Scrambled). Arrows indicate transfected neurons marked by GFP expression. Scale bar = 10 μm. (D–G) Images of HIP1R-shRNA transfected neurons at DIV13 immunostained with anti-MAP2 and used for counting dendrites. Scale bar = 10 μm (D) . Unpaired two-tailed t -test analysis showed a significant decrease in the total dendritic branch number and total dendritic length in the HIP1R-KD group compared to the scrambled control; 11.69 ± 0.67, n = 45 vs. 41.64 ± 2.15, n = 45 (E) and 388.98 ± 16.63 μm, n = 45 vs. 989.91 ± 28.38 μm, n = 45 (F), respectively. Sholl analysis revealed dendritic complexity dramatically decreased in the HIP1R-KD group compared to the scrambled control ( n > 40, # P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons

    doi: 10.3389/fnmol.2017.00186

    Figure Lengend Snippet: Knockdown of HIP1R expression suppresses dendrite growth and spine formation. (A) Cultured hippocampal neurons were infected by HIP1R-shRNA or scrambled control lentivirus and harvested for western blotting at DIV13. A representative western blot shows that the expression level of HIP1R was dramatically reduced in the shRNA group (shRNA) compared to the scrambled control (Scrambled), and viral infection itself had no effect on HIP1R expression (Blank). (B) Densitometric analysis of western blotting from 4 independent experiments; Blank, 1.00 ± 0.07; Scrambled, 1.04 ± 0.12; shRNA, 0.17 ± 0.03. (C) Cultured hippocampal neurons were transfected at DIV6 with HIP1R-shRNA or scrambled plasmid vectors. Immunofluorescence staining at DIV13 showed a significant decrease in HIPIR immunoreactivity in HIP1R-KD neurons (shRNA) but not in the control (Scrambled). Arrows indicate transfected neurons marked by GFP expression. Scale bar = 10 μm. (D–G) Images of HIP1R-shRNA transfected neurons at DIV13 immunostained with anti-MAP2 and used for counting dendrites. Scale bar = 10 μm (D) . Unpaired two-tailed t -test analysis showed a significant decrease in the total dendritic branch number and total dendritic length in the HIP1R-KD group compared to the scrambled control; 11.69 ± 0.67, n = 45 vs. 41.64 ± 2.15, n = 45 (E) and 388.98 ± 16.63 μm, n = 45 vs. 989.91 ± 28.38 μm, n = 45 (F), respectively. Sholl analysis revealed dendritic complexity dramatically decreased in the HIP1R-KD group compared to the scrambled control ( n > 40, # P

    Article Snippet: Lentivirus- or Plasmid-Mediated RNAi in Primary Cultured Hippocampal Neurons HIP1R-shRNA/GFP lentivirus mentioned above and scrambled control-shRNA/GFP lentivirus were made by the Shanghai GeneChem Company (Shanghai, China).

    Techniques: Expressing, Cell Culture, Infection, shRNA, Western Blot, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Two Tailed Test

    Amplitude and frequency of miniature excitatory post-synaptic current (mEPSC), but not miniature inhibitory post-synaptic current (mIPSC), are reduced in HIP1R-knockeddown neurons. Cultured hippocampal neurons were infected by HIP1R-shRNA or Scrambled lentivirus at DIV6, and whole-cell patch-clamp recording was carried out at DIV13. (A) Example traces of mEPSC. (B) Graph of cumulative probability and bar graph of amplitude mEPSC (HIP1R-KD, 17.93 ± 1.13 pA, n = 19 vs. the control, 22.38 ± 1.03 pA, n = 22) and (C) Graph of cumulative probability and bar graph of inter-event interval of mEPSC (HIP1R-KD, 1.95 ± 0.28 s, n = 19 vs. the control, 0.74 ± 0.17 s, n = 22) showed a significant reduction in mEPSC amplitude and frequency in HIP1R-KD neurons compared to the control. (D) Example traces of mIPSC. (E) Graph of cumulative probability and bar graph of amplitude mIPSC (HIP1R-KD, 53.33 ± 2.32 pA, n = 25 vs. the control, 50.98 ± 3.36 pA, n = 21), and (F) graph of cumulative probability and bar graph of inter-event interval of mIPSC (HIP1R-KD, 1.33 ± 0.27 s, n = 25 vs. the control, 1.37 ± 0.31 s, n = 21) did not show marked differences in mIPSC amplitude and frequency between the two groups. All data are presented as mean ± SEM, unpaired two-tailed t -test. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons

    doi: 10.3389/fnmol.2017.00186

    Figure Lengend Snippet: Amplitude and frequency of miniature excitatory post-synaptic current (mEPSC), but not miniature inhibitory post-synaptic current (mIPSC), are reduced in HIP1R-knockeddown neurons. Cultured hippocampal neurons were infected by HIP1R-shRNA or Scrambled lentivirus at DIV6, and whole-cell patch-clamp recording was carried out at DIV13. (A) Example traces of mEPSC. (B) Graph of cumulative probability and bar graph of amplitude mEPSC (HIP1R-KD, 17.93 ± 1.13 pA, n = 19 vs. the control, 22.38 ± 1.03 pA, n = 22) and (C) Graph of cumulative probability and bar graph of inter-event interval of mEPSC (HIP1R-KD, 1.95 ± 0.28 s, n = 19 vs. the control, 0.74 ± 0.17 s, n = 22) showed a significant reduction in mEPSC amplitude and frequency in HIP1R-KD neurons compared to the control. (D) Example traces of mIPSC. (E) Graph of cumulative probability and bar graph of amplitude mIPSC (HIP1R-KD, 53.33 ± 2.32 pA, n = 25 vs. the control, 50.98 ± 3.36 pA, n = 21), and (F) graph of cumulative probability and bar graph of inter-event interval of mIPSC (HIP1R-KD, 1.33 ± 0.27 s, n = 25 vs. the control, 1.37 ± 0.31 s, n = 21) did not show marked differences in mIPSC amplitude and frequency between the two groups. All data are presented as mean ± SEM, unpaired two-tailed t -test. ** P

    Article Snippet: Lentivirus- or Plasmid-Mediated RNAi in Primary Cultured Hippocampal Neurons HIP1R-shRNA/GFP lentivirus mentioned above and scrambled control-shRNA/GFP lentivirus were made by the Shanghai GeneChem Company (Shanghai, China).

    Techniques: Cell Culture, Infection, shRNA, Patch Clamp, Two Tailed Test

    TFCP2 inversely regulates ASIC2a expression. ( a ) Representative immunoblot and densitometric analyses showing that TFCP2 siRNA-transfected PC12 cells had 27% lower TFCP2 expression than negative siRNA-treated cells and consequently elevated ASIC2a expression. (b ) Representative immunoblot and densitometric analyses showing that TFCP2 OE plasmid-transfected PC12 cells had 1.76 ± 0.45-fold greater TFCP2 expression than negative control cells and consequently suppressed ASIC2a expression. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: TFCP2 inversely regulates ASIC2a expression. ( a ) Representative immunoblot and densitometric analyses showing that TFCP2 siRNA-transfected PC12 cells had 27% lower TFCP2 expression than negative siRNA-treated cells and consequently elevated ASIC2a expression. (b ) Representative immunoblot and densitometric analyses showing that TFCP2 OE plasmid-transfected PC12 cells had 1.76 ± 0.45-fold greater TFCP2 expression than negative control cells and consequently suppressed ASIC2a expression. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Expressing, Transfection, Plasmid Preparation, Negative Control

    Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in pilocarpine-treated rats. ( a ) Representative coronal view microscopic positron emission tomography images in the different phases of epileptogenesis. ( b ) Hippocampal glucose uptake in the different phases after pilocarpine injection. ( c ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression at different post-seizure time points. ( d ) Bar graph showing decreased hippocampal TFCP2 expression in pilocarpine-treated rats. ( e ) Bar graph showing increased hippocampal ASIC2a expression in pilocarpine-treated rats. The experiments were repeated at least 3 times with at least 3 rats in each group. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in pilocarpine-treated rats. ( a ) Representative coronal view microscopic positron emission tomography images in the different phases of epileptogenesis. ( b ) Hippocampal glucose uptake in the different phases after pilocarpine injection. ( c ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression at different post-seizure time points. ( d ) Bar graph showing decreased hippocampal TFCP2 expression in pilocarpine-treated rats. ( e ) Bar graph showing increased hippocampal ASIC2a expression in pilocarpine-treated rats. The experiments were repeated at least 3 times with at least 3 rats in each group. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Expressing, Positron Emission Tomography, Injection, Western Blot

    Hippocampal ASIC2a overexpression increased seizure susceptibility. ( a ) Representative images showing GFP immunoreactivity in the hippocampal CA1 region following adeno-associated virus (AAV) vector infusion. Scale bar = 100 μm. ( b ) The time interval from pilocarpine injection to Racine IV seizures was significantly shorter in the ASIC2a overexpression group (26.3 ± 1.3 min) than in the negative control AAV group (31.9 ± 1.9) (n = 30 rats/group). ( c ) The proportion of rats with Racine IV seizures after pilocarpine treatment was significantly higher in the ASIC2a overexpression group (93.3 ± 3.33%) than in the negative control AAV group (66.7 ± 3.33%). Data are presented as means ± standard errors and were analysed using unpaired t-tests or Chi-square tests. *P

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: Hippocampal ASIC2a overexpression increased seizure susceptibility. ( a ) Representative images showing GFP immunoreactivity in the hippocampal CA1 region following adeno-associated virus (AAV) vector infusion. Scale bar = 100 μm. ( b ) The time interval from pilocarpine injection to Racine IV seizures was significantly shorter in the ASIC2a overexpression group (26.3 ± 1.3 min) than in the negative control AAV group (31.9 ± 1.9) (n = 30 rats/group). ( c ) The proportion of rats with Racine IV seizures after pilocarpine treatment was significantly higher in the ASIC2a overexpression group (93.3 ± 3.33%) than in the negative control AAV group (66.7 ± 3.33%). Data are presented as means ± standard errors and were analysed using unpaired t-tests or Chi-square tests. *P

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Over Expression, Plasmid Preparation, Injection, Negative Control

    Glucose deficiency influenced TFCP2 and ASIC2a expression in PC12 cells. ( a ) Representative immunoblot and densitometric analyses showing that cells grown in low-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 12 and 24 h of growth. ( b ) Representative immunoblot and densitometric analyses showing that cells grown in no-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 6, 12, and 24 h of growth. ( c ) Representative immunoblot and densitometric analyses showing that STF-31-treated PC12 exhibited significant downregulation of TFCP2 and significant upregulation of ASIC2a relative to DMSO-treated control cells. ( d ) Representative immunoblot and densitometric analyses showing TFCP2 and ASIC2a expression in 2-deoxy-D-glucose-treated PC12 cells cultured in no-glucose media. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: Glucose deficiency influenced TFCP2 and ASIC2a expression in PC12 cells. ( a ) Representative immunoblot and densitometric analyses showing that cells grown in low-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 12 and 24 h of growth. ( b ) Representative immunoblot and densitometric analyses showing that cells grown in no-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 6, 12, and 24 h of growth. ( c ) Representative immunoblot and densitometric analyses showing that STF-31-treated PC12 exhibited significant downregulation of TFCP2 and significant upregulation of ASIC2a relative to DMSO-treated control cells. ( d ) Representative immunoblot and densitometric analyses showing TFCP2 and ASIC2a expression in 2-deoxy-D-glucose-treated PC12 cells cultured in no-glucose media. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Expressing, Cell Culture

    Cellular localisation of TFCP2 and ASIC2a in the epileptic rats’ CA1 regions and glucose-deficient cells. (a ) Double immunofluorescence labelling for TFCP2 and ASIC2a in the epileptic rats’ CA1 regions during acute and latent post-seizure phases. ( b ) Cellular localisation of TFCP2 and ASIC2a expression in PC12 cells grown for 24 h in high-glucose media (control), low-glucose media, no-glucose media, and STF-31 (10 µM)-loaded media. Proteins were probed with anti-ASIC2a (green) and anti-TFCP2 (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DAPI: 4′,6-diamidino-2-phenylindole.

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: Cellular localisation of TFCP2 and ASIC2a in the epileptic rats’ CA1 regions and glucose-deficient cells. (a ) Double immunofluorescence labelling for TFCP2 and ASIC2a in the epileptic rats’ CA1 regions during acute and latent post-seizure phases. ( b ) Cellular localisation of TFCP2 and ASIC2a expression in PC12 cells grown for 24 h in high-glucose media (control), low-glucose media, no-glucose media, and STF-31 (10 µM)-loaded media. Proteins were probed with anti-ASIC2a (green) and anti-TFCP2 (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DAPI: 4′,6-diamidino-2-phenylindole.

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Immunofluorescence, Expressing

    Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in patients with TLE. ( a ) Patient 4’s pre-surgical assessment results: magnetic resonance imaging (left) was negative, electroencephalography (middle) showed spike waves in the temporal lobe, and fluorodeoxyglucose positron emission tomography (right) revealed hypometabolic lesions in the right hippocampus. ( b ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression in patients with TLE (n = 13) and control patients (n = 10). β-actin was used as a loading control. ( c , d ) Normalised densitometry bar graphs of TFCP2 and ASIC2a for the control subjects and patients with TLE. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in patients with TLE. ( a ) Patient 4’s pre-surgical assessment results: magnetic resonance imaging (left) was negative, electroencephalography (middle) showed spike waves in the temporal lobe, and fluorodeoxyglucose positron emission tomography (right) revealed hypometabolic lesions in the right hippocampus. ( b ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression in patients with TLE (n = 13) and control patients (n = 10). β-actin was used as a loading control. ( c , d ) Normalised densitometry bar graphs of TFCP2 and ASIC2a for the control subjects and patients with TLE. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Expressing, Magnetic Resonance Imaging, Positron Emission Tomography, Western Blot

    Changes in ASIC2a expression affected the intrinsic excitability of CA1 pyramidal neurons. ( a ) Confocal images of CA1 pyramidal neurons expressing GFP (green) and labelled with neurobiotin (blue). Scale bar = 10 μm. ( b ) Representative traces of action potential firing in response to 200 pA current injections in CA1 pyramidal neurons of the negative control, ASIC2a overexpression, and ASIC2a knockdown groups, respectively. ( c ) Number of action potentials in CA1 pyramidal neurons from the various groups at different current injection steps. Data are presented as means ± standard errors and were analysed using 1- or 2-way ANOVA and Dunnett's multiple comparisons test. *P

    Journal: Scientific Reports

    Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

    doi: 10.1038/s41598-017-05038-0

    Figure Lengend Snippet: Changes in ASIC2a expression affected the intrinsic excitability of CA1 pyramidal neurons. ( a ) Confocal images of CA1 pyramidal neurons expressing GFP (green) and labelled with neurobiotin (blue). Scale bar = 10 μm. ( b ) Representative traces of action potential firing in response to 200 pA current injections in CA1 pyramidal neurons of the negative control, ASIC2a overexpression, and ASIC2a knockdown groups, respectively. ( c ) Number of action potentials in CA1 pyramidal neurons from the various groups at different current injection steps. Data are presented as means ± standard errors and were analysed using 1- or 2-way ANOVA and Dunnett's multiple comparisons test. *P

    Article Snippet: The TFCP2 siRNA’s sense strand was 5′-GACCUGGAGACAGAAUUCUTT-3′, and its antisense strand was 5′-AGAAUUCUGUCUCCAGGUCTT-3′.We bought GFP-only, ASIC2a knockdown, and ASIC2a overexpression lentiviruses from Genechem (Shanghai, China).

    Techniques: Expressing, Negative Control, Over Expression, Injection

    IDH1 R132H mutation induces proliferation and migration of NSCLC cells. ( a ) WB analysis of IDH1 and IDH1 R132H protein levels in H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H. ( b ) Proliferation of H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H assessed by using the MTS assay. ( c ) Migration of H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H was assessed by Transwell migration assay. ( d ) WB of IDH1 protein in H460 cells after transfection with IDH1 siRNA. ( e ) Proliferation of H460 cells after transfection with IDH1 siRNA was assessed by MTS assay. ( f ) Migration of H460 cells after transfection with IDH1 siRNA was assessed by Transwell migration assay. ( g ) Proliferation of H1299 cells expressing mutant IDH1 R132H and treated with 20 µM IDH1 inhibitor (AGI-5198) was assessed by MTS assay. ( h ) Migration of H1299 cells treated as in ( g ) was assessed by Transwell migration assay.

    Journal: Open Biology

    Article Title: IDH1 mutation promotes lung cancer cell proliferation through methylation of Fibulin-5

    doi: 10.1098/rsob.180086

    Figure Lengend Snippet: IDH1 R132H mutation induces proliferation and migration of NSCLC cells. ( a ) WB analysis of IDH1 and IDH1 R132H protein levels in H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H. ( b ) Proliferation of H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H assessed by using the MTS assay. ( c ) Migration of H1299 cells transduced with an empty vector, IDH1 WT or mutant IDH1 R132H was assessed by Transwell migration assay. ( d ) WB of IDH1 protein in H460 cells after transfection with IDH1 siRNA. ( e ) Proliferation of H460 cells after transfection with IDH1 siRNA was assessed by MTS assay. ( f ) Migration of H460 cells after transfection with IDH1 siRNA was assessed by Transwell migration assay. ( g ) Proliferation of H1299 cells expressing mutant IDH1 R132H and treated with 20 µM IDH1 inhibitor (AGI-5198) was assessed by MTS assay. ( h ) Migration of H1299 cells treated as in ( g ) was assessed by Transwell migration assay.

    Article Snippet: Vectors encoding mutant IDH1 R132H (LV-GFP-IDH1R132H), WT IDH1 (LV-GFP-IDH1WT) or a control empty vector (LV-GFP-VECTOR) were obtained from Genechem.

    Techniques: Mutagenesis, Migration, Western Blot, Transduction, Plasmid Preparation, MTS Assay, Transwell Migration Assay, Transfection, Expressing

    IDH1 mutation promotes binding of DNMT1 to Fibulin-5 promoter. ( a ) Binding of DNMT1 to Fibulin-5 promoter in H1299 cells treated with 2 mM 2-HG was evaluated by CHIP assay. GAPDH served as negative control. ( b ) WB analysis of DNMT1 and Fibulin-5 in H1299 cells transfected with control or DNMT1 siRNA and treated with 2 mM 2-HG. ( c ) RT-PCR analysis of Fibulin-5 expression in H1299 cells treated as in ( b ). ( d ) WB analysis of TET2 and Fibulin-5 in H1299 cells transfected with control or TET2 siRNA and treated with 2 mM 2-HG. ( e ) WB analysis of Fibulin-5 expression in H1299 cells expressing an empty vector, WT IDH1 or mutant IDH1 R132H and treated with a DNMT1 inhibitor, disulfiram (DSF, 200 nM). ( f ) Fibulin-5 mRNA levels in H1299 cells treated as in ( e ).

    Journal: Open Biology

    Article Title: IDH1 mutation promotes lung cancer cell proliferation through methylation of Fibulin-5

    doi: 10.1098/rsob.180086

    Figure Lengend Snippet: IDH1 mutation promotes binding of DNMT1 to Fibulin-5 promoter. ( a ) Binding of DNMT1 to Fibulin-5 promoter in H1299 cells treated with 2 mM 2-HG was evaluated by CHIP assay. GAPDH served as negative control. ( b ) WB analysis of DNMT1 and Fibulin-5 in H1299 cells transfected with control or DNMT1 siRNA and treated with 2 mM 2-HG. ( c ) RT-PCR analysis of Fibulin-5 expression in H1299 cells treated as in ( b ). ( d ) WB analysis of TET2 and Fibulin-5 in H1299 cells transfected with control or TET2 siRNA and treated with 2 mM 2-HG. ( e ) WB analysis of Fibulin-5 expression in H1299 cells expressing an empty vector, WT IDH1 or mutant IDH1 R132H and treated with a DNMT1 inhibitor, disulfiram (DSF, 200 nM). ( f ) Fibulin-5 mRNA levels in H1299 cells treated as in ( e ).

    Article Snippet: Vectors encoding mutant IDH1 R132H (LV-GFP-IDH1R132H), WT IDH1 (LV-GFP-IDH1WT) or a control empty vector (LV-GFP-VECTOR) were obtained from Genechem.

    Techniques: Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation

    IDH1 mutation induces methylation of Fibulin-5 promoter. ( a ) mRNA levels of fibulin proteins in H1299 cells treated with 2 mM 2-HG. ( b ) Genomic PCR of unmethylated promoters of Fibulin-1 and Fibulin-5 genes in H1299 cells treated with 2 mM 2-HG. ( c ) Genomic PCR of unmethylated promoters of Fibulin-5 gene in H460 and H1299 cells. ( d ) WB analysis of Fibulin-5 in H1299 and H460 cells. ( e ) Fibulin-1 and Fibulin-5 mRNA levels in H460 cells transduced with IDH1 siRNA. ( f ) Fibulin-1 and Fibulin-5 mRNA levels in H1299 cells expressing an empty vector, IDH1 WT or mutant IDH1 R132H.

    Journal: Open Biology

    Article Title: IDH1 mutation promotes lung cancer cell proliferation through methylation of Fibulin-5

    doi: 10.1098/rsob.180086

    Figure Lengend Snippet: IDH1 mutation induces methylation of Fibulin-5 promoter. ( a ) mRNA levels of fibulin proteins in H1299 cells treated with 2 mM 2-HG. ( b ) Genomic PCR of unmethylated promoters of Fibulin-1 and Fibulin-5 genes in H1299 cells treated with 2 mM 2-HG. ( c ) Genomic PCR of unmethylated promoters of Fibulin-5 gene in H460 and H1299 cells. ( d ) WB analysis of Fibulin-5 in H1299 and H460 cells. ( e ) Fibulin-1 and Fibulin-5 mRNA levels in H460 cells transduced with IDH1 siRNA. ( f ) Fibulin-1 and Fibulin-5 mRNA levels in H1299 cells expressing an empty vector, IDH1 WT or mutant IDH1 R132H.

    Article Snippet: Vectors encoding mutant IDH1 R132H (LV-GFP-IDH1R132H), WT IDH1 (LV-GFP-IDH1WT) or a control empty vector (LV-GFP-VECTOR) were obtained from Genechem.

    Techniques: Mutagenesis, Methylation, Polymerase Chain Reaction, Western Blot, Transduction, Expressing, Plasmid Preparation

    IDH1 mutation promotes 2-HG production in NSCLC cells. ( a ) 2-HG levels in H1299 cells steadily expressing an empty vector, IDH1 WT or mutant IDH1 R132H. ( b ) 2-HG levels in H460 cells after transfection with IDH1 siRNA. ( c ) 2-HG levels in H1299 cells expressing IDH1 R132H after treatment with 20 µM AGI-5198. ( d ) Proliferation of H1299 cells supplemented with 2 mM 2-HG was assessed by MTS assay. ( e ) Migration of H1299 cells supplemented with 2 mM 2-HG was assessed by Transwell migration assay.

    Journal: Open Biology

    Article Title: IDH1 mutation promotes lung cancer cell proliferation through methylation of Fibulin-5

    doi: 10.1098/rsob.180086

    Figure Lengend Snippet: IDH1 mutation promotes 2-HG production in NSCLC cells. ( a ) 2-HG levels in H1299 cells steadily expressing an empty vector, IDH1 WT or mutant IDH1 R132H. ( b ) 2-HG levels in H460 cells after transfection with IDH1 siRNA. ( c ) 2-HG levels in H1299 cells expressing IDH1 R132H after treatment with 20 µM AGI-5198. ( d ) Proliferation of H1299 cells supplemented with 2 mM 2-HG was assessed by MTS assay. ( e ) Migration of H1299 cells supplemented with 2 mM 2-HG was assessed by Transwell migration assay.

    Article Snippet: Vectors encoding mutant IDH1 R132H (LV-GFP-IDH1R132H), WT IDH1 (LV-GFP-IDH1WT) or a control empty vector (LV-GFP-VECTOR) were obtained from Genechem.

    Techniques: Mutagenesis, Expressing, Plasmid Preparation, Transfection, MTS Assay, Migration, Transwell Migration Assay

    Fibulin-5 expression inhibits the effect of IDH1 mutation on NSCLC cells. ( a ) WB analysis was used to assess the expression of Fibulin-5 in H460 cells treated with 2 µM 5-aza for 4 days. ( b ) Proliferation of H460 cells treated as in ( a ) was assessed by MTS assay. ( c ) Migration of H460 cells treated as in ( a ) was assessed by Transwell migration assay. ( d ) WB analysis of Fibulin-5 expression in H1299 cells harbouring IDH1 mutation and transfected with the Fibulin-5 plasmid. ( e ) Proliferation of H1299 cells treated as in ( d ) was assessed by MTS assay. ( f ) Migration of H1299 cells treated as in ( d ) was assessed by Transwell migration assay. ( g ) WB analysis of Fibulin-5 expression in WT H1299 cells transfected with Fibulin-5 siRNA. ( h ) Proliferation of H1299 cells treated as in ( g ) was assessed by MTS assay. ( i ) Migration of H1299 cells treated as in ( g ) was assessed by Transwell migration assay.

    Journal: Open Biology

    Article Title: IDH1 mutation promotes lung cancer cell proliferation through methylation of Fibulin-5

    doi: 10.1098/rsob.180086

    Figure Lengend Snippet: Fibulin-5 expression inhibits the effect of IDH1 mutation on NSCLC cells. ( a ) WB analysis was used to assess the expression of Fibulin-5 in H460 cells treated with 2 µM 5-aza for 4 days. ( b ) Proliferation of H460 cells treated as in ( a ) was assessed by MTS assay. ( c ) Migration of H460 cells treated as in ( a ) was assessed by Transwell migration assay. ( d ) WB analysis of Fibulin-5 expression in H1299 cells harbouring IDH1 mutation and transfected with the Fibulin-5 plasmid. ( e ) Proliferation of H1299 cells treated as in ( d ) was assessed by MTS assay. ( f ) Migration of H1299 cells treated as in ( d ) was assessed by Transwell migration assay. ( g ) WB analysis of Fibulin-5 expression in WT H1299 cells transfected with Fibulin-5 siRNA. ( h ) Proliferation of H1299 cells treated as in ( g ) was assessed by MTS assay. ( i ) Migration of H1299 cells treated as in ( g ) was assessed by Transwell migration assay.

    Article Snippet: Vectors encoding mutant IDH1 R132H (LV-GFP-IDH1R132H), WT IDH1 (LV-GFP-IDH1WT) or a control empty vector (LV-GFP-VECTOR) were obtained from Genechem.

    Techniques: Expressing, Mutagenesis, Western Blot, MTS Assay, Migration, Transwell Migration Assay, Transfection, Plasmid Preparation

    Fibulin-5 expression is reversely linked with IDH1 mutation in patients with NSCLC. ( a ) WB analysis of IDH1 R132H in malignant specimens from 40 patients with NSCLC. ( b ) Representative IHC staining for IDH1 R132H in IDH1 WT and mutant IDH1 specimens. ( c ) 2-HG levels in malignant specimens from the 40 patients with NSCLC. ( d ) Fibulin-5 mRNA levels in malignant specimens from the 40 patients with NSCLC.

    Journal: Open Biology

    Article Title: IDH1 mutation promotes lung cancer cell proliferation through methylation of Fibulin-5

    doi: 10.1098/rsob.180086

    Figure Lengend Snippet: Fibulin-5 expression is reversely linked with IDH1 mutation in patients with NSCLC. ( a ) WB analysis of IDH1 R132H in malignant specimens from 40 patients with NSCLC. ( b ) Representative IHC staining for IDH1 R132H in IDH1 WT and mutant IDH1 specimens. ( c ) 2-HG levels in malignant specimens from the 40 patients with NSCLC. ( d ) Fibulin-5 mRNA levels in malignant specimens from the 40 patients with NSCLC.

    Article Snippet: Vectors encoding mutant IDH1 R132H (LV-GFP-IDH1R132H), WT IDH1 (LV-GFP-IDH1WT) or a control empty vector (LV-GFP-VECTOR) were obtained from Genechem.

    Techniques: Expressing, Mutagenesis, Western Blot, Immunohistochemistry, Staining