Journal: Autophagy

Article Title: Regulation of autophagy by DNA G-quadruplexes.

doi: 10.1080/15548627.2020.1769991

Figure Lengend Snippet: Figure 2. PDS downregulates the ATG7 protein and the expression of Atg7 in astrocytes. (A) Primary astrocytes were cultured for 3 weeks from cortical tissue isolated from rat embryos. Cultures were fixed and stained with Alexa Fluor 633 phalloidin (red), DAPI (blue), and an antibody against GFAP (green). Most cells stained with phalloidin were positive for GFAP (94 ± 6%). Over 200 cells were analyzed, and results were pooled from three independent experiments. Scale bar: 40 µm. (B) Cultured primary astrocytes were treated with a vehicle (control) or with PDS (2 μM) overnight. Astrocytes were then processed to measure mRNA levels. Expression levels of Atg7 and Tbp (housekeeping gene as control) were determined by qRT-PCR. Atg7: p = 0.017 (t-test), *p < 0.05; Tbp: p = 0.7949 (t-test), n.s., non-significant. Results were pooled from six experiments. (C) Cultured primary astrocytes were treated with a vehicle (control) or with PDS (2 μM) overnight. Astrocytes were then processed to measure the levels of ATG7 and SQSTM1. ATG7: *p = 0.019 (t-test), SQSTM1: *p = 0.018 (t-test). Results were pooled from six experiments. (D) An example of western blotting experiments in (C). (E) Dendra2-LC3 was used to measure autophagy flux. Two cohorts of astrocytes were co-transfected with Dendra2- LC3 and an empty plasmid, or with Dendra2-LC3 and untagged SPHK1 (sphingosine kinase 1, a positive control). Astrocytes co-transfected with Dendra2-LC3 and an empty plasmid were treated with a vehicle (control, cont), or with 0.5 µM PDS overnight. After treatment, cells were photoswitched and longitudinally imaged, and the decay of the red fluorescence over time was used to calculate the half-life of Dendra2-LC3. The half-life of Dendra2-LC3 is normalized to one with respect to control astrocytes. cont vs SK1: *p = 0.001, cont vs PDS: **p = 0.001, SK1 vs PDS: **p = 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons testing). Fifty cells per group were analyzed from two independent experiments. (F) An example of a photoswitching experiment with Dendra2-LC3 and SPHK1 (untagged). Astrocytes co-transfected with Dendra2-LC3 and SPHK1 were treated with a vehicle overnight. Cells were photoswitched and longitudinally imaged. Autophagosomes are depicted with white arrows. Blue arrow depicts photoswitching. Scale bar: 25 µm.

Article Snippet: Antibodies against GFAP were from Santa Cruz Biotechnology (sc-9065; 1:100).

Techniques: Expressing, Cell Culture, Isolation, Staining, Control, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Positive Control, Fluorescence

Journal: Life science alliance

Article Title: Mast cell extracellular trap formation underlies vascular and neural injury and hyperalgesia in sickle cell disease.

doi: 10.26508/lsa.202402788

Figure Lengend Snippet: Figure 1. Sickle microenvironment drives mast cell extracellular trap (MCET) release. (A) Dorsal skin of female control HbAA mice treated with the vehicle and sickle HbSS mice treated with the vehicle or GSK484 (sc, 20 mg/kg/day) was imaged with a laser scanning confocal microscope in 100-μm immunostained (see the Materials and Methods section for details) sections. Z-stacks of 1.0 μm were sequentially acquired at 60x magnification. Association of mast cells with vasculature and nerve bundles is shown: nerve bundles (cyan, anti-neurofilament H- 200) surrounded by degranulating mast cells (green, anti-c-Kit) loaded with tryptase granules (red, anti- tryptase). Mast cells are extending green nanotubes interspersing and disrupting the nerve bundles, and tryptase granules are intertwined around the nerve bundles, suggestive of MCET formation in HbSS mice. (B) Mast cells, indicated with c-Kit and tryptase, associate with CD31+ (magenta) endothelial cells in HbSS skin. White arrows indicate mast cell interactions with nf or bv. (C) Incitement of a sickle microenvironment with TNF-α (1 ng/ml), hemin (40 μM), or TNF-α/hemin caused MCET formation in mast cells cultured from skin of HbSS female mice, shown with the formation of rigid DNA fibers (stained with cell-permeable DAPI and SYTO 13 and cell-impermeable SYTOX Orange). (D) HbSS mast cells treated with TNF-α/hemin showed citrullinated histones (red) and DNA-binding fluorescent probes, DAPI (cyan), SYTO 13 (green), and SYTOX Orange (red), indicating an extranuclear DNA that overlaps with histone citrullination. (E) MCETs were elongated in HbSS and HbAA mast cells with TNF-α, hemin, or combined treatment, and to a greater extent in HbSS mast cells compared with TNF-α or hemin alone. The data are analyzed with regular two-way ANOVA with Tukey’s post hoc multiple comparisons test. Data shown are the mean ± SEM of N = 17–24 cells. (F) TNF-α/hemin-induced MCET formation was abrogated by pretreatment with scrambled siRNA (10 μM), the TLR4 inhibitor TAK242 (1 μM), siRNA silencing of FcεR1 and TLR4 (10 μM each), and PAD4 inhibition with GSK484 (10 μM). Each image represents multiple images from five different 5.0-mo-old female mice. Abbreviations: bv, blood vessel; MCET, mast cell extracellular trap; nf, nerve fiber; PAD4, peptidylarginine deiminase 4; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor alpha; trp, tryptase.

Article Snippet: Mast cells were also transfected with FcεR1 siRNA (10 μM, #sc-45268; Santa Cruz), TLR4 siRNA (10 μM, #sc-40261; Santa Cruz), or scramble siRNA negative control (10 μM, # 4390843; Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (#13778100; Thermo Fisher Scientific) per the manufacturer’s recommendation.

Techniques: Control, Microscopy, Cell Culture, Staining, Binding Assay, Inhibition

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