Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: CD105(+) ccRCC tumor cells are mesenchymal cells with enhanced motility and invasion capability. (a) Western blot and (b) qRT-PCR of EMT markers of CD105(+) cells and parental cells. (c) Migration assay and (d) modified 3D transwell assay of CD105(+) cells and parental cells ( ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Western Blot, Quantitative RT-PCR, Migration, Modification, Transwell Assay

Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: CD105 knockdown induces loss of mesenchymal markers and inhibition of motility and invasion. (a) Western blot and (b) qRT-PCR of EMT markers of CD105(+) cells and shRNA-mediated CD105 knockdown CD105(+) cells. (c) Migration assay and (d) modified 3D transwell assay of CD105(+) cells and shRNA-mediated CD105 knockdown CD105(+) cells ( ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Knockdown, Inhibition, Western Blot, Quantitative RT-PCR, shRNA, Migration, Modification, Transwell Assay

Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: MYC overexpression can reverse this process. (a) Western blot of MYC in CD105(+) cells, shRNA-mediated CD105 knockdown CD105(+) cells, and MYC overexpressed cells. (b) qRT-PCR of EMT markers of shRNA-mediated CD105 knockdown CD105(+) cells before and after MYC overexpression. (c) Migration assay of shRNA-mediated CD105 knockdown CD105(+) cells and MYC overexpressed cells. (d) Migration assay of shRNA-mediated CD105 knockdown CD105(+) cells and NANOG overexpressed cells. (e) qRT-PCR analysis of EMT markers of CD105(+) cells before and after the treatment of TGF- β type I receptor kinase inhibitor LY-364947 (50 nM) ( ∗∗ p < 0.01).

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Over Expression, Western Blot, shRNA, Knockdown, Quantitative RT-PCR, Migration

Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: CD105 knockdown does not change the metastasis of ccRCC in the tail vein injection mouse model. (a) The lung weight analysis of mice received the CD105(+) cells and shRNA-mediated CD105 knockdown CD105(+) cells. Representative images of lung metastasis in both gross view (b) and H&E staining (c) established by tail vein injection of cancer cells.

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Knockdown, Injection, shRNA, Staining

Journal: Experimental Neurobiology

Article Title: Tweety-homolog ( Ttyh ) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC swell ) in the Brain

doi: 10.5607/en.2019.28.2.183

Figure Lengend Snippet: Ttyh family of genes encode the essential components of the astrocytic VRAC swell . (A) RT-PCR results confirming the efficiency of each Ttyh1/2/3 shRNA in cultured astrocytes. Lentivirus containing each pSicoR Ttyh1/2/3 shRNA were infected in cultured astrocytes. (B) Schematic diagram of the design of shRNA-insensitive clone of TTYH1. The upper sequence represents endogenous Ttyh1 mRNA sequence that is complemented with Ttyh1 shRNA. The lower sequence represents shRNA-insensitive clone of TTYH1 sequence that is not complemented with Ttyh1 shRNA due to changing every third single nucleotide of Ttyh1 shRNA target site to another nucleotide that makes same amino acid due to the codon redundancy. (C) RT-PCR results confirming the efficiency for shRNA-insensitivity using shRNA-insensitive clone of TTYH1 with control or Ttyh1 shRNA compared to TTYH1-GFP with control or Ttyh1 shRNA overexpressing HEK293T cells. (D) Fluorescence images of TTYH1-GFP and shRNA-insensitive clone of TTYH1 with control or Ttyh1 shRNA expressing HEK-293T cells. (E) Representative I Cl,swell from control shRNA, Ttyh1/3 shRNAs, Ttyh1/2/3 shRNAs, Ttyh1/2/3 shRNAs with shRNA-insensitive clone of TTYH1 ( Ttyh1 sh-insens.) expressing cultured astrocytes. (F) Summary bar graph showing the maximal amplitude of I Cl,swell from +100 mV to −100 mV in the presence of single Tty h shRNA or any combination of double Ttyh shRNAs ( T1, T2, T3, T1/2 , T1/3 , T2/3 shRNAs). Data are represented as mean±SEM (n=10 for Naïve, n=14 for 7mg of control shRNA, n=10, 12 and 8 for 7mg of single Ttyh1 , Ttyh2 , and Ttyh3 shRNA, n=8 for 14mg of control shRNA, n= 10, 10 and 10 for 14mg of double Ttyh1/2 , Ttyh1/3 , and Ttyh2/3 shRNAs; NS>0.05, Kruskal-Wallis test). (G) Summary bar graph showing the maximal amplitude of I Cl,swell from +100 mV to −100 mV in naïve astrocytes and in the presence of control or Ttyh1/2/3 shRNAs and Ttyh1/2/3 shRNAs with shRNA-insensitive clone of TTYH1 expressing cultured astrocytes. Data are represented as mean±SEM (n=10 for Naïve, n=11 for control shRNA, n=13 for Ttyh1/2/3 shRNAs, and n=18 for Ttyh1/2/3 shRNAs with shRNA-insensitive clone of TTYH1; *** <0.001, NS>0.05, Ordinary one-way ANOVA). (H) Averaged I~V curves for I Cl,swell in Control shRNA (grey), Ttyh1/2/3 shRNAs ( T1/2/3 shRNAs, blue) and T1/2/3 shRNAs with shRNA-insensitive clone of TTYH1 ( Ttyh1 sh-insens; red). (I, K) Averaged current density values at ±100mV in every minute after HOS treatment in control (black), Ttyh1/2/3 shRNAs (blue) and Lrrc8a (orange) expressing cultured astrocytes. Dot points are represented as mean±SEM. Lines (dark grey, sky blue, and orange) between each value were drawn by the fitting of sigmoidal 4 th order function. (n=11 for both control shRNA (grey), n=13 for Ttyh1/2/3 shRNA (sky blue), and n=9 for Lrrc8a shRNA (orange)). (J, L) Normalized current density from +100 mV to −100 mV by maximum current amplitude in every minute after HOS treatment. Dot points are represented as mean±SEM. Lines (dark grey, sky blue, and orange) between each value were drawn by the fitting of sigmoidal 4 th order function. (n=11 for both control shRNA (grey), n=13 for Ttyh1/2/3 shRNA (blue), and n=9 for Lrrc8a shRNA(orange)). Inset: The averaged time to 50% of peak. Data are represented as mean±SEM (n=11 for both control shRNAs, n=13 for Ttyh1/2/3 shRNA, and n=9 for Lrrc8a shRNA).

Article Snippet: The cocktail of lentiviruses carrying either (pSicoR- Ttyh1 -shRNA-GFP, pSicoR- Ttyh2 -shRNA-mcherry, and pSicoR- Ttyh3 -shRNA-mcherry) or (pSicoR-scrambled-shRNA-GFP and pSicoR-scrambled-shRNA-mcherry) were bilaterally injected into hippocampal CA1 stratum radiatum of naïve C57BL/6J mice for slice patch-clamp recording or tamoxifen-treated hGFAP-creERT2 mice for intrinsic optical signal (IOS) imaging.

Techniques: Reverse Transcription Polymerase Chain Reaction, shRNA, Cell Culture, Infection, Sequencing, Control, Fluorescence, Expressing

Journal: Experimental Neurobiology

Article Title: Tweety-homolog ( Ttyh ) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC swell ) in the Brain

doi: 10.5607/en.2019.28.2.183

Figure Lengend Snippet: The astrocytic Ttyh1/2/3 is necessary and sufficient for RVD in the hippocampus. (A) Schematic diagram of intrinsic optical signal (IOS) imaging experiment in SR of the hippocampus. (B) Representative bright-field image for IOS recording in SR after stimulating the Schaffer-collateral pathway. A white arrowhead indicates an electrical stimulation electrode in the Schaffer-collateral pathway. A white dotted lined circle indicates regions of interest (ROI). (C) (Left) Schematic diagram for electrical stimulation protocol for inducing RVD in SR of the hippocampus. (Right) The representative trace of IOS change by prolonged 1Hz for 30 min electrical stimulation on the Schaffer-collateral pathway in CA1 hippocampus. The percentage of RVD is calculated with a portion of decreased transmittance (a) from the peak transmittance (b) during 1Hz, 30min stimulation. (D) Upper panels for the representative traces for IOS change indicated as astrocytic volume change before and after treatment of DCPIB (grey line) by brief 20Hz for 1 sec electrical stimulation on the Schaffer-collateral pathway in CA1 hippocampus. Lower representative trace for IOS change by 100Hz for 1 sec electrical stimulation on the Schaffer-collateral pathway in CA1 hippocampus. (E) Representative traces of IOS induced by low-frequency stimulation (LFS) for 30 min in control (black), with 10 mM DCPIB (green) and with 50 mM Genistein (pink). The ‘a’ indicates the difference between the peak of response and the value of end of stimulation. The ‘b’ indicates the value of the peak for calculating RVD % (as described in Fig. 11C, a/b * 100). (F) Averaged traces for normalized transmittance by the peak of each trace. (G, L) Averaged bar graph of RVD percentage (%). RVD% is calculated by a/b * 100. Data are represented as mean ± SEM (n=12, 7 and 7 for control and after DCPIB and Genistein treatment; * <0.05, ** <0.01, Kruskal-Wallis test; n=10, 10 and 9 for control and Ttyh1/2/3 shRNA injected C57BL/6J mice and pSicoR Ttyh1/2/3 shRNA injected in tamoxifen-treated hGFAP-creERT2 mouse, respectively; * <0.05, ** <0.01, NS>0.05, Kruskal-Wallis test) (H) Timeline from tamoxifen injection to IOS imaging. Tamoxifen was into hGFAP-CreERT2 by intraperitoneal ( i.p. ) injection for 6 days. On 7 day, lentivirus containing pSicoR Ttyh1/2/3 shRNA were injected into CA1 stratum radiatum of the hippocampus. IOS imaging was done 2 weeks after virus injection. (I) In the presence of tamoxifen, the inactivated Cre (CreER) were converted to the activated form of Cre (CreERT), which were translocated into Nucleus. CreERT recognized loxP sites in pSicoR Ttyh1/2/3 shRNA construct and cleaved the shRNA sequence. (J) Representative fluorescence images of lenti-viral carrying control or Ttyh1/2/3 shRNA injected into SR of CA1 hippocampus (upper panel). Representative traces of 30 min LFS-induced IOS recording from control shRNA (black), Ttyh1/2/3 shRNAs (red) injected C57BL/6J mice and glial rescue condition by pSicoR Ttyh1/2/3 shRNAs injected in tamoxifen-treated hGFAP-creERT2 mouse (blue) (bottom panel). (K) Averaged traces for 30 min LFS-induced IOS recording.

Article Snippet: The cocktail of lentiviruses carrying either (pSicoR- Ttyh1 -shRNA-GFP, pSicoR- Ttyh2 -shRNA-mcherry, and pSicoR- Ttyh3 -shRNA-mcherry) or (pSicoR-scrambled-shRNA-GFP and pSicoR-scrambled-shRNA-mcherry) were bilaterally injected into hippocampal CA1 stratum radiatum of naïve C57BL/6J mice for slice patch-clamp recording or tamoxifen-treated hGFAP-creERT2 mice for intrinsic optical signal (IOS) imaging.

Techniques: Imaging, Control, shRNA, Injection, Virus, Construct, Sequencing, Fluorescence

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: Electrical properties of DGGCs

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: shRNA, Membrane

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a The averaged current – voltage (I–V) relationship of the whole-cell currents from DGGCs infected with Ad-Sc or Ad-TASK-3 shRNAs or both Ad-TASK-3 shRNA and TWIK-1 shRNA as well as from naive DGGCs was measured in standard artificial cerebrospinal fluid in the presence of Cs + /TEA/4-AP (1 mM/5 mM/5 mM). Whole-cell currents were elicited by 1-s-duration ramp pulses descending from 50 mV to −150 mV from a holding potential of −70 mV. b A summary bar graph for a . The mean values of the current density in naive DGGCs ( n = 14 cells, N = 4 mice) or DGGCs expressing Sc shRNA ( n = 12 cells, N = 3 mice), TASK-3 shRNA ( n = 15 cells, N = 3 mice), or both TASK-3 and TWIK-1 shRNAs ( n = 15 cells, N = 3 mice) measured in the presence of Cs + /TEA/4-AP (1 mM/5 mM/5 mM) are shown. The current density values are depicted at +50 mV. c The TASK-3 shRNA- or both TASK-3 shRNA- and TWIK-1 shRNA-sensitive currents were determined by subtracting each of the shRNA averaged currents from the Sc shRNA averaged currents a . ** P < 0.01

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Infection, shRNA, Expressing

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a Representative traces of the membrane potential to stepwise current injections recorded from naive DGGCs ( n = 27, N = 3) or DGGCs infected with Ad-Sc shRNA ( n = 21, N = 3), Ad-TWIK-1 shRNA ( n = 30, N = 3), Ad-TASK-3 shRNA ( n = 22, N = 3), or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA ( n = 32, N = 3). The resting membrane potentials of the cells was maintained at −70 mV by constant current injections, and the depolarizing current was then injected stepwise in 5-pA increments. b The number of spikes indicated that the neuron infected with Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA were more excitable compared to control mice. The recordings were performed in artificial cerebrospinal fluid containing 50 µM D-AP5, 10 µM CNQX, 10 µM bicuculline, 10 µM CGP 55845, 2 mM TEA, and 0.5 mM NiCl 2 , with a pipette solution containing 5 mM QX314. c Averaged values of rheobase currents in naive cells ( n = 27 cells, N = 3 mice) and cells expressing Ad-Sc shRNA ( n = 21 cells, N = 3 mice), Ad-TWIK-1 shRNA ( n = 30 cells, N = 3 mice), Ad-TASK-3 shRNA ( n = 12), or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA ( n = 32 cells, N = 3 mice). All values are means ± SEM. * P < 0.05, *** P < 0.001

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Membrane, Infection, shRNA, Injection, Control, Transferring, Expressing

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a Bath application of NT-mediated membrane depolarization of granule cells. A representative response of the membrane potential to stepwise current injections was recorded from naive DGGCs ( n = 15, N = 3) or DGGCs infected with Ad-Sc shRNA ( n = 16, N = 3), Ad-TWIK-1 shRNA ( n = 17, N = 3), Ad-TASK-3 shRNA ( n = 15, N = 3), or both Ad-TWIK-shRNA and Ad-TASK-3 shRNA ( n = 14, N = 3). The resting membrane potential of these cells was maintained at −70 mV by constant current injections, and depolarizing currents were then injected stepwise at 5-pA increments until the membrane potential reached the firing threshold. b , c Analyzed bar charts of spike numbers at 105 pA for a . b Resting membrane potential values of the whole-cell currents in naive dentate gyrus granule cells ( n = 15 cells, N = 3 mice) and cells expressing Sc shRNA ( n = 16 cells, N = 3 mice), TWIK-1 shRNA ( n = 17 cells, N = 3 mice), TASK-3 shRNA ( n = 15 cells, N = 3 mice), or TWIK-1/TASK-3 shRNAs ( n = 14 cells, N = 3 mice). c The extent of changes in the number of spikes following NT application in naive and Ad-Sc shRNA-infected DGGCs was larger than those in DGGCs transfected with Ad-TASK-1 shRNA alone, Ad-TWIK-1 shRNA alone, or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNAs. The data for Ad-TASK-3 shRNA alone, Ad-TWIK-1 shRNA alone, and both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNAs showed no change in the numbers of spikes. The recordings were obtained in artificial cerebrospinal fluid containing 50 µM D-AP5, 10 µM CNQX, 10 µM bicuculline, 10 µM CGP55845, 2 mM TEA, and 0.5 mM NiCl 2 , with a pipette solution containing 5 mM QX314. All the data are presented as the means ± SEM. *** P < 0.001 was considered statistically significant

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Membrane, Infection, shRNA, Injection, Expressing, Transfection, Transferring

Journal: Molecular Brain

Article Title: Channel-mediated astrocytic glutamate modulates hippocampal synaptic plasticity by activating postsynaptic NMDA receptors

doi: 10.1186/s13041-015-0097-y

Figure Lengend Snippet: Astrocytic Best1 is responsible for PAR1 activation-induced synaptic potentiation. A , A schedule for tamoxifen and lentivirus injection into hGFAP-CreERT2, wild-type mice (naïve), or CaMKIIα-Cre mice. For acute Cre expression in hGFAP-CreERT2 mice, tamoxifen was pre-injected for 5 days before lentivirus injection. Sunflower oil, a control for tamoxifen. B , Diagram showing Cre-loxP regulation of shRNA expression in cell-type specific gene silencing system. In hGFAP-Cre-ERT2 mice, pretreatment of tamoxifen induces Cre expression, leading to neither shRNA nor EGFP expression in astrocytes through Cre/loxP-mediated deletion. Due to the same mechanism, CA1 pyramidal neurons cannot express both lentiviral shRNA and EGFP expression in CaMKIIα-Cre mice. C , Left : Representative immunohistochemistry results showing Cre-dependent regulation of lentiviral shRNA expression. Cre-dependent shRNA expression in astrocytes or neurons was indicated by co-labeling of EGFP with GFAP or NeuN, respectively. s.p., stratum pyramidale; s.r., stratum radiatum. Scale bar = 50 μm. Right : Bar graphs demonstrate the % of GFAP/EGFP double positive cells (hGFAP-CreERT2) or EGFP positive cells from GFAP- or NeuN positive cells (CaMKIIα-Cre) among total EGFP expressing cells. Mean (%) ± s.e.m. ***, p < 0.001, unpaired t-test. Numbers of counted slices at least from three mice were indicated within each bar. D , eEPSP responses from each recording shown as in Figure . Graphs represent averaged eEPSP responses (mean ± s.e.m.) recorded from loxP-floxed scrambled-shRNA expressing slices from all genotypes. (Sc-sh, empty circles), loxP-floxed Best1 -shRNA expressing slices from hGFAP-Cre ERT2 without [ Best1 -shRNA (−Tam), black circles] or with tamoxifen [ Best1 -shRNA (+Tam), light grey circles], and loxP-floxed Best1 -shRNA expressing slices from CaMKIIα-Cre mice [ Best1 -shRNA (CamKIIα-Cre), dark gray circles]. E , Bar graph representing mean eEPSPs (mean ± s.e.m), analyzed during the period covered by the gray box in D . Same bar colors as in D . *, P < 0.01, one-way ANOVA with post-hoc test. n.s., not significant.

Article Snippet: We injected a lentivirus containing loxP-floxed Best1 short-hairpin RNA (shRNA) construct (pSicoR- Best1 -shRNA) into the hippocampal CA1 area of transgenic mice expressing tamoxifen-inducible glial fibrillary acid protein ( Gfap ) promoter-driven Cre recombinase, Cre (hGFAP-Cre ERT2 ) or mice expressing Ca 2+ /calmodulin-dependent kinase II α promoter-driven Cre (CaMKIIα-Cre).

Techniques: Activation Assay, Injection, Expressing, shRNA, Immunohistochemistry, Labeling

Journal: Molecular Brain

Article Title: Channel-mediated astrocytic glutamate modulates hippocampal synaptic plasticity by activating postsynaptic NMDA receptors

doi: 10.1186/s13041-015-0097-y

Figure Lengend Snippet: A combination of subthreshold synaptic stimulation and Best1-mediated astrocytic glutamate produces NMDAR-dependent LTP at SC-CA1 synapses. A , Summary of LTP recordings in hippocampal slices when subthreshold stimulation (40 Hz) with TFLLR (40 Hz + TFLLR; black circles), 40 Hz alone (40 Hz; white circles), TFLLR alone (TFLLR; gray circles), 40 Hz with TFLLR in the presence of APV (40 Hz + TFLLR + APV; green circles) or niflumic acid (40 Hz + TFLLR + NFA; red circles), and 40 Hz with endothelin (Endothelin; blue circles) was applied. B , Bar graph representing the averaged % baseline of fEPSPs (mean ± s.e.m.) over the time period indicated by the gray bar in A . *, p < 0.05, one-way ANOVA with post-hoc test. Same colors as A . Numbers of tested slices from at least three mice are indicated within each bar. C, Summary of LTP recording from loxP-floxed scrambled-shRNA expressing hippocampal slices (Sc-shRNA, black circles), loxP-floxed Best1-shRNA expressing slices from hGFAP-Cre ERT2 mice without [ Best1 -shRNA (−Tam), white circles] or with tamoxifen pretreatment [ Best1 -shRNA (+Tam), gray circles]. D , Bar graph representing the averaged % baseline of fEPSPs (mean ± s.e.m.) over the time period indicated by the gray bar in C . *, p < 0.05, one-way ANOVA with post-hoc test. Same colors as C . Numbers of tested slices from at least three mice are indicated within each bar. E , Summary of LTP recording from Sc-shRNA expressing hippocampal slices (black circles) or Best1 -shRNA expressing slices from CaMKIIα-Cre mice (white circles). F , Bar graph representing the averaged % baseline of fEPSPs (mean ± s.e.m.) over the time period indicated by the gray bar in E . *, p < 0.05, unpaired t-test. Same colors as E . Numbers of tested slices from at least independent mice are indicated within each bar.

Article Snippet: We injected a lentivirus containing loxP-floxed Best1 short-hairpin RNA (shRNA) construct (pSicoR- Best1 -shRNA) into the hippocampal CA1 area of transgenic mice expressing tamoxifen-inducible glial fibrillary acid protein ( Gfap ) promoter-driven Cre recombinase, Cre (hGFAP-Cre ERT2 ) or mice expressing Ca 2+ /calmodulin-dependent kinase II α promoter-driven Cre (CaMKIIα-Cre).

Techniques: shRNA, Expressing