Journal: The Journal of Neuroscience

Article Title: Interdependent Conductances Drive Infraslow Intrinsic Rhythmogenesis in a Subset of Accessory Olfactory Bulb Projection Neurons

doi: 10.1523/JNEUROSCI.2520-15.2016

Figure Lengend Snippet: Expression of BK and CaV2.3 channels in AOB mitral cells. A–C, Confocal laser-scanning microscopy images of the mouse AOB (parasagittal cryosections). Immunofluorescence labeling (green) of CaV2.3 (A), the BK channel subunit α-1 (Sloα1; B), and the BK channel subunit β-4 (Sloβ4; C). Nuclei are counterstained with DRAQ5 (red). Dashed lines indicate both the lateral olfactory tract (LOT) and the glomerular layer (GL) in Aiii, Biii, and Ciii. Representative single optical section images show an overview of the AOB (Ai, Bi, Ci) and a high-magnification view (Aii, Bii, Cii) of the mitral cell layer (MCL). Note the robust labeling of cells in the MCL as well as the absence of immunofluorescence following peptide preadsorption (Aiii, Biii, Ciii; no DRAQ5 counterstaining). D, E, Post hoc immunolabeling of previously recorded intrinsically rhythmic neurons. Confocal images of AOB cryosections containing biocytin-filled iAMC somata and proximal dendrites (visualized by Alexa Fluor 488/633 streptavidin conjugate). Sections were counterstained against the BK channel α-1 (D) and β-4 (E) subunits, respectively. Merged images (Diii, Eiii) reveal BK expression in these representative iAMCs. Div, Eiv, Western blot analysis of total olfactory bulb (right lanes) and posterior brain (left lanes) protein extracts. Immunoblots were probed with anti-Sloα1 (D) and anti-Sloβ4 (E) antibodies, respectively. Expected band size is indicated above both blots. d, Dorsal; FC, frontal cortex; v, ventral.

Article Snippet: Primary antibodies were anti-Slo1 (K Ca 1.1, 1:500; Alomone Labs), anti-Sloβ4 (1:100; Alomone Labs), and anti-Ca V 2.3 (1:100; Alomone Labs).

Techniques: Expressing, Confocal Laser Scanning Microscopy, Immunofluorescence, Labeling, Immunolabeling, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 2. Effects of integrin β4 knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co-culture system for 24 h. Then, the levels of integrin β4 in the VECs were examined using the immunofluorescence assay. The bar graph shows the relative intensity of integrin β4 in the single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of integrin β4 in VECs. The levels of integrin β4 were determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L integrin β4-specific siRNA or with scramble siRNA for 48 h. After the addition of SPC, the effects of the integrin β4 knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed by the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as the ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 3. Effects of Fyn knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co- culture system for 24 h. Then, the levels of Fyn in the VECs were examined using the immunofluorescence assay. The bar shows the relative intensity of Fyn in single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of Fyn in VECs. The value of Fyn was determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), the cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L Fyn-specific siRNA or with scramble siRNA for 48 h. After the SPC was added, the effects of the Fyn knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed using the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as a ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 4. Effects of integrin β4 knockdown on the changes in Fyn levels in VECs. (A) VECs were treated with 10–40 nmol/L integrin β4 siRNA or scramble siRNA for 48 h. The levels of integrin β4 in the VECs were determined by the immunofluorescence assay, and the bar graph shows the relative fluorescent intensity of integrin β4 per cell as determined by laser scanning confocal microscopy (bP<0.05 vs scramble ctrl, n=3). (B) After 10–40 nmol/L integrin β4 siRNA or scramble siRNA were treated for 48 h, the Fyn levels in the VECs were assessed using western blotting (cP<0.01 vs scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Immunofluorescence, Confocal Microscopy, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 5. Effects of integrin β4 knockdown on NO production in VECs. (A) VECs were cultured under normal conditions or with siRNA for 48 h and stimulated with SPC for 0, 3, 15, or 30 min, and the supernatant was used for the NO level assay using the NO Detection Kit. In the control group (ctrl), the cells were cultured in M199 medium with 0.3% (v/v) ethanol instead of SPC. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA in the presence of SPC. The bar graph shows the changes in NO levels in VECs that had been treated with SPC (bP<0.05 vs ctrl at 3 min, eP<0.05 vs ctrl at 15 min, n=5) or with siRNA in the presence of SPC (hP<0.05 vs scramble ctrl at 3 min, kP<0.05 vs scramble ctrl at 15 min, n=5). (B) The viabilities of VECs were measured by MTT, and no significant changes were observed (n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Transfection

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 6. Effects of integrin β4 knockdown in VECs on changes in ROCK levels in co-cultured VSMCs. VSMCs that had been co-cultured with VECs that were treated with or without integrin β4-specific siRNA (β4) or scramble control siRNA in the presence of SPC were immunostained for ROCK, α-SMA, or both. The bar graph shows the relative fluorescence intensity of ROCK per VSMC as determined by laser scanning confocal microscopy (bP<0.05 vs SPC-non-stimulated VSMCs; eP<0.05 vs SPC- stimulated scramble ctrl in co-cultured system, n=4).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Fluorescence, Confocal Microscopy

Journal: Nature Communications

Article Title: Priming mobilization of hair follicle stem cells triggers permanent loss of regeneration after alkylating chemotherapy

doi: 10.1038/s41467-019-11665-0

Figure Lengend Snippet: Bu-induced proliferation followed by Cy-induced apoptosis in organ-cultured HFs. a Schedule for Bu/– and Bu/Cy treatments and subsequent analysis at 2, 4, and 6 days after alkylating chemotherapy. b Hair shaft elongation and c , hair cycle score of organ-cultured human HFs after alkylating chemotherapy ( n = 38 biological replicates/timepoint, 3 independent experiments). Note that the score of the Bu/Cy group was lower than that of the control group on day 6. d Representative images of organ-cultured HFs of experimental groups. Control HFs maintained anagen morphology, and Bu-treated HFs prematurely entered catagen stage (black arrow); however, Bu/Cy-treated HFs showed completely shrunken bulb morphology (black arrowhead). Representative images and quantification of e , Ki67 + cells among K15 + HFSCs ( n = 5 biological replicates/group), f p53 + cells among integrin β4 + basal cells ( n = 6 biological replicates/group), and g , p53 + c-caspase-3 + cells ( n = 7 biological replicates/group) in the bulge after alkylating chemotherapy. HFSCs showed remarkable cellular proliferation after Bu treatment (white arrowhead) and were completely quenched with large-scale apoptosis after Bu/Cy treatment (white arrow; immunofluorescence; scale bar = 100 μm). Bu busulfan, Cy cyclophosphamide, Bu/Cy busulfan followed by cyclophosphamide, HF hair follicle, HPF high-power field, HFSC hair follicle stem cell, c-caspase-3 cleaved caspase-3. Data are mean ± SEM. Source data are provided as a Source Data file. * p < 0.05 (vs. Con); *** p < 0.001 (vs. Con, two-way analysis of variance with Dunnett’s multiple comparisons test) in b and c ; *** p < 0.001 (vs. Con, unpaired t test) in e , f , and g

Article Snippet: For immunofluorescence, frozen tissues were sectioned at a thickness of 4 μm and incubated overnight at 4 °C with the following primary antibodies diluted in antibody diluent reagent (Invitrogen): Fas (ab82419, 1:100; Abcam, MA, USA), Ki67 (SP6, 1:200; Spring Bioscience, Pleasanton, CA, USA or MIB-1, 1:200; Dako), K15 (LHK15, 1:200; Thermo Fisher), p53 (DO-7, 1:800; Novocastra, Newcastle, England or 1C12, 1:1000; Cell Signaling, Danvers, MA, USA), K14 (GTX104124, 1:1000; GeneTex, Irvine, CA, USA), cleaved caspase-3 (5A1E, 1:400; Cell Signaling), γ-H2AX (#2577, 1:800; Cell Signaling), Shh (EP1190Y, 1:500; Abcam), Lhx2 (C-20, 1:200; Santa Cruz Biotechnology), integrin β4 (H-101, 1:200; Santa Cruz Biotechnology), phospho Akt (#9275, 1:800; Cell Signaling), phospho p38 (#9211, 1:800; Cell Signaling), TYR (T311, 1:100; Thermo Fisher, Waltham, MA, USA), TRP1 (SC25543, 1:100; Santa Cruz Biotechnology), and MITF (C5, 1:100; Thermo Fisher).

Techniques: Cell Culture, Control, Immunofluorescence