Journal: ACS Central Science

Article Title: Chemogenetic Approach Using Ni(II) Complex–Agonist Conjugates Allows Selective Activation of Class A G-Protein-Coupled Receptors

doi: 10.1021/acscentsci.8b00390

Figure Lengend Snippet: Selective activation of the engineered M1R in primary rat cortical astrocytes. (a) Schematic illustration of selective activation of the His 4 tag-fused M1R over endogenous mAChRs by coordination tethering. (b) Confocal microscopic images of astrocytes co-transfected with AcGFP (green) and M1R(1.18H4) immunostained by an anti-HA tag antibody (magenta). Nuclei were stained with Hoechst33258 (blue). Scale bar: 20 μm. Ca 2+ responses evoked by 30 nM (c) MAC(M1R) and (d) control-2 in astrocytes harboring the plasmids of (red) M1R(1.18H4), (blue) WT M1R, or (black) the control vector ( n = 18). Fura-2 ratiometric images of astrocytes expressing M1R(1.18H4) (e) before and (f) during perfusion of 30 nM MAC(M1R) . Red and blue represent high and low Ca 2+ concentration, respectively. Scale bar: 100 μm. (g) Average Δratio plot evoked by 5 min of treatment of 30 nM MAC(M1R) and control-2 ( n = 11–23). *** denote significant differences from the group of M1R(1.18H4)/ MAC(M1R) /inhibitor(−) (***: P < 0.001, one way analysis of variance with Dunnett’s post hoc test). F(6/117) = 23.05. Concentration–response curves of (h) M1R(1.18H4) and (i) WT M1R on (red) MAC(M1R) and (black) control-2 ( n = 20–25). The data represent the mean ± SEM. Reproducibility of all experiments was confirmed at least two times.

Article Snippet: Primary rat cortical astrocytes were obtained from P2 neonatal SD rat pups (both male and female were used) (Japan SLC) according to the protocol reported by Guaza et al .

Techniques: Activation Assay, Transfection, Staining, Control, Plasmid Preparation, Expressing, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: Differential interference contrast images of astrocytes cultured on ( A ) PLL glass, ( B ) nanofibrillar scaffolds, ( C ) Aclar, and ( D ) PLL Aclar. Note: Scale bars, 100 μm. Abbreviations: PLL Aclar, poly-L-lysine-functionalized planar Aclar; PLL glass, poly-L-lysine-functionalized planar glass.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Cell Culture

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: AFM astrocyte morphology investigation on PLL glass. ( A ) AFM high pass filtered composite height image of astrocytes by J scanner show lamellipodia formation (scale bar, 20 μm). ( B – D ) Close-ups by E scanner of arrow regions a–d shown in ( A ): stress fibers ( B ); filopodia anchors ( C ); and cell–cell interaction ( D ) (scale bars, 2 μm). ( E and F ) Cross-section profiles of cells e and f in ( A ). Note: Axis units are in μm with astrocyte soma extent identified by red arrow. Abbreviations: AFM, atomic force microscopy; PLL glass, poly-L-lysine-functionalized planar glass.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Microscopy

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: AFM astrocyte morphology investigation on nanofibrillar scaffolds. ( A ) AFM GHPF composite height image of astrocytes cultured on nanofibrillar surfaces by J scanner. Scale bar, 20 μm. ( B – D ) Cross section profiles of cells b–d in ( A ). Note: Axis units are in μm with astrocyte soma extent identified by red arrow. Abbreviations: AFM, atomic force microscopy; GHPF, Gaussian high pass filtering.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Cell Culture, Microscopy

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: Atomic force microscopy high pass filtered composite height images of astrocytes cultured on nanofibrillar scaffolds by J scanner. ( A ) Dashed box identifies the intercellular connection. ( B ) Close-up of the intercelluar connection by E scanner. Dashed box identifies a contacted area. ( C ) Close-up of a contacted area by E scanner. Note: The scale bars show 10, 2, and 0.5 μm in ( A ), ( B ), and ( C ), respectively.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Microscopy, Cell Culture

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: ( A ) AFM GHPF composite height image of astrocytes cultured on Aclar surfaces by J scanner. Scale bar 20 μm. ( B – D ) The cross sections shown by b–d in ( A ) were given in corresponding section profiles. Note: Axis units are in μm. Abbreviations: AFM, atomic force microscopy; GHPF, Gaussian high pass filtering.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Cell Culture, Microscopy

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: AFM investigation of astrocytes cultured on PLL Aclar surfaces. ( A ) Composite AFM deflection image of astrocytes by J scanner. Scale bar, 20 μm. ( B ) AFM deflection image of an astrocyte by J scanner. Scale bar, 10 μm. Dotted arrow identifies cross section. Dashed box identifies a cell edge. ( C ) Corresponding cross section profile. Axis units are in μm with astrocyte soma extent identified by red arrow. ( D ) Corresponding cell edge showing nanoscale filopodia by E scanner. Notes: Scale bar, 2 μm. Abbreviations: AFM, atomic force microscopy; PLL Aclar, poly-L-lysine-functionalized planar Aclar.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Cell Culture, Microscopy

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: Immunocytochemistry structural investigation results. Left to right: fluorescence images of astrocytes cultured on PLL glass, nanofibrillar scaffolds, Aclar, and PLL Aclar substrates. Immunnolabeling and corresponding phalloidin are represented in red and green, respectively. Astrocytes were labeled with ( A ) anti-Cdc42, ( B ) anti-Rac1, and ( C ) anti-RhoA. Note: Scale bars, 10 μm. Abbreviations: PLL Aclar, poly-L-lysine-functionalized planar Aclar; PLL glass, poly-L-lysine-functionalized planar glass.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Immunocytochemistry, Fluorescence, Cell Culture, Labeling

Journal: International Journal of Nanomedicine

Article Title: Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

doi: 10.2147/IJN.S32681

Figure Lengend Snippet: Overlaid fluorescence images of astrocytes cultured on nanofibrillar scaffolds ( A ) and PLL glass ( B ) labeled with phalloidin (F-actin) and anti-RhoA. The fluorescence was represented in green for F-actin and red for RhoA in ( A ) and ( B ). The scatter plots of F-actin and RhoA for nanofibrillar scaffolds ( C ) and PLL glass ( D ) showed partial colocalization with a mean PC of 0.66 and 0.58 respectively. Note: Scale bar in (A) 10 μm, and (B) 20 μm. Abbreviation: PLL glass, poly-L-lysine-functionalized planar glass.

Article Snippet: Briefly, rat cerebral cortical astrocytes were prepared from postnatal day P2 Sprague Dawley rats and grown to confluence in astrocyte medium in 75 cm 2 tissue culture flasks.

Techniques: Fluorescence, Cell Culture, Labeling