Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Representative WB analysis of ADAM9 and MMP7 target genes (left); cell growth proliferation analysis (right). B) WB analysis of ADAM9 and MMP7 (left), cell growth proliferation (middle) and migration (right). Actin was utilized as internal loading control.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Migration

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative Western blot of A) ADAM9, MMP7 and OPN in normal human melanocytes (NHEM) and in a panel of melanoma cell lines, B) ADAM9 isoforms (left), MMP7 and OPN (middle) and corresponding secreted forms (right) in miR-126&126* versus empty vector-transduced Me665/1 and A375M cell lines. C) Representative Real-time PCR analysis of ADAM9 (left), MMP7 (middle) and OPN (right) mRNAs in the A375M cell line. The unresponsive short isoform of ADAM9 mRNA does not carry miR-126&126* binding sites in its 3′UTR. D) Relative expression values obtained by western blot analysis of ADAM9 (left), MMP7 (middle) and OPN (right) in A375M cells transfected with oligomers mimicking mature miR-126 or miR-126* vs non targeting (no Targ); GAPDH and actin were internal loading controls in RT-PCR and WB, respectively. Columns, of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Luciferase reporter assays performed by transfecting a Luc reporter gene (psiCHECK2) linked to 3′-UTR of ADAM9 or MMP7 or OPN or PI3KR2 in miR- versus empty vector-transduced A375M cell lines. B) Schematic presentation of predicted miR-126 and miR-126* target sites identified in the ADAM9 3′UTR (left) and relative miR-126&126*-dependent luciferase activities (right) in presence of wild-type (WT) or mutant (mut) binding sites in the-ADAM9 3′UTR. C) The same schematic representation (left) and luciferase experiments (right) carried out on MMP7 3′UTR. Columns, of minimum of 5 experiments per group; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Western blot analyses showing the effectiveness of stable si-ADAM9, si-MMP7 and scrambled control (SCR) transduction. B) Invasion and migration assays in si-ADAM9- or si-MMP7-infected melanoma cell lines compared with scrambled control. C) Invasion (left) and migration (right) in presence of either ADAM9 or MMP7 recombinant proteins in miR-126&126*-transduced A375M cell lines compared with control cells. Columns, mean±SD of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Transduction, Migration, Infection, Recombinant

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative WB of A) pro-HB-EGF (bottom) and relative densitometric analysis (top) in miR-126&126*- versus empty vector-transduced Me665/1 melanoma cell line treated or not with PMA. B ) pro-HB-EGF and HB-EGF-C levels in si-ADAM9- or si-MMP7-infected melanoma compared with si-scrambled control. C) Real time PCR analysis of ADAM9 and MMP7 in the same silenced cells.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development *

doi: 10.1074/jbc.M110.126714

Figure Lengend Snippet: KDM6B directly regulates histone H3K27 methylation in WT cultured neurospheres. A, transfection of scrambled negative control shRNA-RFP (TR30015, OriGene) into cells from WT neurospheres and immunostaining for KDM6B; B, transfection with KDM6B shRNA-RFP and immunostaining for KDM6B; C, transfection of scrambled negative control shRNA-RFP and immunostaining for H3K27me3; D, transfection with KDM6B shRNA-RFP and immunostaining for H3K27me3. Experiments were performed in quadruplicate. A representative of 4 separate experiments is shown. These results demonstrate that H3K27 methylation is directly regulated by KDM6B in cultured neurospheres.

Article Snippet: A scrambled negative control-shRNA-RFP from OriGene (TR30015) did not silence KDM6B levels ( A ) or increase H3K27 methylation ( C ).

Techniques: Methylation, Cell Culture, Transfection, Negative Control, shRNA, Immunostaining

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 < 0.0001) (G) . (H–K) Images of HIP1R-shRNA transfected neurons at DIV21 immunostained with anti-GFP and used for counting dendritic spines. Scale bar = 10 μm (H) The spine density, spine width and length were also significantly reduced in the HIP1R-KD group compared to the control with spine number per μm: 0.12 ± 0.01, n = 48 vs. 0.48 ± 0.01, n = 48 (I) ; spine width: 0.56 ± 0.02 μm, n = 62 vs. 0.73 ± 0.03 μm, n = 62 (J) ; and spine length: 0.79 ± 0.03 μm, n = 62 vs. 1.09 ± 0.04 μm, n = 62 (K) respectively. All data are presented as mean ± SEM, # P < 0.0001. Uncropped images of blots are shown in Supplementary Figure .

Article Snippet: 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

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 < 0.05, ** P < 0.01, # P < 0.0001. Uncropped images of blots are shown in Supplementary Figure .

Article Snippet: 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

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 < 0.01, # P < 0.0001.

Article Snippet: 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

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 < 0.01, # P < 0.0001.

Article Snippet: 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

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 < 0.0001.

Article Snippet: 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, Staining