Journal: Science Advances

Article Title: Adaptive optics two-photon endomicroscopy enables deep-brain imaging at synaptic resolution over large volumes

doi: 10.1126/sciadv.abc6521

Figure Lengend Snippet: ( A ) Experimental approach to target the dendrites and soma for single neuron recording in hippocampus CA1. SP, stratum pyramidale; SR, stratum radiatum. ( B ) Quasi-simultaneous Ca 2+ imaging of spontaneous activity from the soma (Sm) and two dendrites (D1 and D2) in awake behaving mice. Images are shown as SD projection of 600 frames. Scale bar, 5 μm. ( C ) Calcium transients (∆ F / F ) of the soma and dendrites as shown in (B). ( D ) Firing events of soma (Sm) and dendrites (D1 and D2) as shown in (B). Gray and colored curves represent the individual and average events, respectively. ( E ) Relationship between activity strength of the soma-dendrite pairs and dendrite-dendrite pair.

Article Snippet: Then, the fluorescence signal was reflected by the DM and separated from the excitation laser by dichroic mirror D1 (Semrock, FF705-Di01-25×36).

Techniques: Imaging, Activity Assay

Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: IMP decreases the production of pro‐inflammatory cytokines by supressing the NF‐κB signalling pathway in LPS‐induced THP‐1 cells. (A) THP‐1 cells were pretreated with 6.25, 12.5 or 25 μM IMP for 24 h, followed by additional exposure with or without LPS (1000 ng·mL−1) for another 24 h. Pro‐inflammatory cytokines including TNF‐α, IL‐1β, IL‐6 and IL‐10 in culture supernatants were measured by elisa. Data are expressed as the mean ± SEM from five independent experiments. # P < 0.05 compared with the DMSO control group. * P < 0.05 compared with the LPS control group. (B) The total NF‐κB p65, phospho‐p65 (p‐p65), IκBα and phospho‐IκBα (p‐IκBα) protein levels in LPS‐induced THP‐1 cells were determined by Western blotting. The numbers at the bottom indicate the relative intensity of the protein bands, with the DMSO‐treated sample set as ‘1.00’. (C) NF‐κB p65 protein levels in the cytoplasm and nucleus of LPS‐stimulated THP‐1 cells were determined by Western blotting. GAPDH and PCNA were used as cytoplasmic and nuclear markers respectively. The numbers at the bottom indicate the relative intensity of the protein bands, with the DMSO‐treated sample set as ‘1.00’. (D) NF‐κB p65 localization was performed using immunofluorescence staining and observed under a fluorescence microscope (magnification: 400×) using an anti‐NF‐κB p65 antibody (1:50) followed by an Alexa Fluor 488‐conjugated detection antibody.

Article Snippet: Mouse TNF‐α and IL‐1β elisa kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: IMP suppresses LPS‐induced NF‐κB activity and down‐regulates NF‐κB‐mediated pro‐inflammatory gene expression in LS174T cells. (A) HEK293T cells were transiently transfected with pGCL4.32 (luc2P/NF‐κB‐RE/Hygro) NF‐κB reporter, pCMV6‐entry or pCMV6‐XL4‐hPXR, and pRL‐TK. After transfection overnight, cells were treated with DMSO, IMP (6.25–25 μM) or rifampicin (RIF; 10 μM) for 24 h, followed by additional incubation with or without TNF‐α (20 ng·mL−1) for another 24 h. A standard dual luciferase assay was performed on the cell lysates. Results are expressed as fold changes compared with the DMSO control. Data are expressed as mean ± SEM from five independent experiments. # P < 0.05 compared with the TNF‐α group with or without hPXR. * P < 0.05 compared with the same treatment group transfected with empty vector and the TNF‐α group transfected with the receptor expression plasmid. (B) LS174T cells were treated with DMSO, IMP (25 μM) or rifampicin (10 μM) for 48 h, followed by additional incubation with or without TNF‐α (20 ng·mL−1) for 24 h. NF‐κB p65 localization was performed using immunofluorescence staining and observed under a confocal laser scanning microscope (magnification: 630×) using an anti‐NF‐κB p65 antibody (1:50) followed by an Alexa Fluor 488‐conjugated detection antibody. (C) NF‐κB p65, phospho‐p65 (p‐p65), IκBα and phospho‐IκBα (p‐IκBα) protein levels in LS174T cells were determined by Western blotting. The numbers at the bottom indicate the relative intensity of the protein bands, with the DMSO‐treated sample set as ‘1.00’. (D) NF‐κB p65 protein levels in the cytoplasm and nucleus of LS174T cells were determined by Western blotting. GAPDH and PCNA were used as cytoplasm and nuclear markers respectively. The numbers at the bottom indicate the relative intensity of the protein bands, with the DMSO‐treated sample set as ‘1.00’. (E) The mRNA levels of IL‐1β, ICAM1, iNOS and COX‐2 were determined using qRT‐PCR. LS174T cells were treated with DMSO, IMP (6.25–25 μM) or rifampicin (10 μM) for 48 h, followed by additional exposure to LPS (2000 ng·mL−1) for 24 h. Results are expressed as fold changes compared with the DMSO control. Data are expressed as mean ± SEM from five independent experiments. # P < 0.05 compared with the DMSO control group. * P < 0.05 compared with the LPS control group.

Article Snippet: Mouse TNF‐α and IL‐1β elisa kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay, Gene Expression, Transfection, Incubation, Luciferase, Control, Plasmid Preparation, Expressing, Immunofluorescence, Staining, Laser-Scanning Microscopy, Western Blot, Quantitative RT-PCR

Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: Knockdown of hPXR attenuated IMP‐mediated suppression of pro‐inflammatory gene expression in LS174T cells. (A) Cells were transiently transfected with hPXR siRNA and control non‐silencing siRNA. After transfection for 48 h, the cells were treated with DMSO, IMP (25 μM) or rifampicin (RIF; 10 μM) for another 48 h, followed by additional exposure to LPS (2000 ng·mL−1) for 24 h. The mRNA expression levels of IL‐1β, ICAM1, iNOS and COX‐2 were determined by qRT‐PCR. Results are expressed as fold changes compared with DMSO control. Data are expressed as mean ± SEM from five independent experiments. # P < 0.05 compared with DMSO group. * P < 0.05 compared with the same treatment group. (B) LS174T cells were transiently transfected with hPXR siRNA and control non‐silencing siRNA for 24 h and then treated with DMSO, IMP (25 μM) or rifampicin (10 μM) for another 48 h, followed by an additional incubation with or without TNF‐α (20 ng·mL−1) for 24 h. NF‐κB p65 localization was performed using immunofluorescence staining and observed under a confocal laser scanning microscope (magnification: 630×) using an anti‐NF‐κB p65 antibody (1:50) followed by an Alexa Fluor 488‐conjugated detection antibody.

Article Snippet: Mouse TNF‐α and IL‐1β elisa kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Gene Expression, Transfection, Control, Expressing, Quantitative RT-PCR, Incubation, Immunofluorescence, Staining, Laser-Scanning Microscopy

Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: IMP inhibited NF‐κB pro‐inflammatory signalling by the activation of mPXR in DSS‐induced inflamed mouse colons. (A) IMP activated mPXR signalling in healthy mice. mPXR (Nr1i2), Cyp3a11 and Mdr1 mRNA expressions were analysed by qRT‐PCR in healthy mouse colon tissues after treatment with physiological saline (normal) or IMP at a dose of 100 mg·kg−1 for 7 days. Results are expressed as fold changes compared with the normal control. Data are expressed as mean ± SEM from five independent experiments. # P < 0.05 compared with the normal group. (B) mPXR, Cyp3a11 and Mdr1 mRNA expression levels were analysed by qRT‐PCR in colon tissues from DSS‐induced mice colitis after treatment with IMP or PCN for 9 days. Gene expression levels are expressed as fold changes compared with the normal control. # P < 0.05 compared with the normal group. * P < 0.05 compared with the DSS group. (C) Protein levels were analysed by Western blotting. The numbers at the bottom indicate the relative intensity of the protein bands, with the normal‐treated sample set as ‘1.00’. (D) Western blotting was performed in inflamed colon tissues to determine the activity of NF‐κB p65, phospho‐p65 (p‐p65), IκBα and phospho‐IκBα (p‐IκBα). The numbers at the bottom indicate the relative intensity of the protein bands, with the control‐treated sample set as ‘1.00’. (E) Representative images of immunohistochemical staining of mPXR and NF‐κB in mouse colon sections (magnification: 200×). (F) TNF‐α, IL‐1β and MPO concentrations in mouse colon sections. Data are expressed as mean ± SEM for each group of mice (n = 5). # P < 0.05 compared with the normal group. * P < 0.05 compared with the DSS group.

Article Snippet: Mouse TNF‐α and IL‐1β elisa kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Saline, Control, Expressing, Gene Expression, Western Blot, Activity Assay, Immunohistochemical staining, Staining

Journal: Journal of cell science

Article Title: Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1.

doi: 10.1242/jcs.100867

Figure Lengend Snippet: Fig. 2. Translocation of Red-IZUMO1 from the outer acrosomal membrane to the sperm plasma membrane. (A) Acrosome reactions were observed using a warmed chamber (Olympus, MI-IBC) with a heater-equipped water immersion lens (Olympus, UPLSAPO 60XW). Spermatozoa were trapped on an isolated zona pellucida (see Materials and Methods). The fluorescent image was taken using a spinning-disk confocal laser scanning system (Yokogawa, CSU-X1) with a highly sensitive EM- CCD camera (Andor, iXon plus; see Materials and Methods). (B) A representative time-lapse image of Red-IZUMO1 translocation at the time of acrosomal exocytosis detected by GFP fluorescence. From the time of GFP dispersal, it took less than 30 s for Red-IZUMO1 to spread across the entire head (see also supplementary material Movie 3). (C) Schematic diagram showing the estimated translocation pathway of Red- IZUMO1 from the acrosomal membrane to the sperm plasma membrane. The inset shows the hypothetical movement of IZUMO1 in this event. (D) The fluorescence intensities from mCherry (Red-IZUMO1) and GFP (acrosomal contents) during the acrosome reaction. The total amount of Red-IZUMO1 was almost the same even after the acrosome reaction.

Article Snippet: The fluorescent image was taken using a spinning-disk confocal laser scanning system (Yokogawa, CSU-X1) with a highly sensitive EMCCD camera (Andor, iXon plus; see Materials and Methods). (B) A representative time-lapse image of Red-IZUMO1 translocation at the time of acrosomal exocytosis detected by GFP fluorescence.

Techniques: Translocation Assay, Membrane, Clinical Proteomics, Isolation, Fluorescence

Journal:

Article Title: Bax-induced cell death in tobacco is similar to the hypersensitive response

doi:

Figure Lengend Snippet: Mitochondrial localization of the GFP-TM fusion protein. (A and B) Confocal laser scanning micrographs showing a highly magnified region of a single N. benthamiana leaf trichome cell infected with PVX.GFP-TM. The tissue was imaged at 488-nm excitation wavelength to detect GFP (A) and 568-nm excitation wavelength to detect the mitochondrial marker MitoTracker Red (B). (Bar = 5 μm.)

Article Snippet: Stained tissue was examined under a Bio-Rad MRC1000 confocal laser scanning microscope using excitation light at either 488 nm, to detect GFP (emission filter 522 nm), or 568 nm, to detect the mitochondrial stains (emission filter 605 nm).

Techniques: Infection, Marker