Journal: Differentiation; research in biological diversity

Article Title: LNGFR + THY-1 + human pluripotent stem cell-derived neural crest-like cells have the potential to develop into mesenchymal stem cells.

doi: 10.1016/j.diff.2016.04.003

Figure Lengend Snippet: Fig. 3. Cell type-specific marker analysis of human iPSC-derived LNGFRþTHY-1þ cells. (A, B) Human iPSC (WD39) derived LNGFRþTHY-1þ cells expressed NCC markers (AP2α, SOX10, NESTIN and HNK-1) as determined by immunocytochemistry (A) and by flow cytometry (B). (C) LNGFRþTHY-1þ cells were negative for the pluripotent stem cell markers, NANOG and OCT3/4, by immunocytochemical analysis. (D) LNGFRþTHY-1þ cells expressed neither hematopoietic cell markers nor the epithelial cell surface marker (CD326) by flow cytometric analysis. Scale Bars: 50 mm.

Article Snippet: After one hour incubation with blocking buffer (Nacalai Tesque) at room temperature, the following primary antibodies were applied: mouse monoclonal anti-human Ki-67 (MM1; Leica, 1:200), goat polyclonal anti-human SOX10 (R&D Systems, 1:200), rabbit monoclonal anti-human AP2α (C83E10; Cell Signaling Technology, 1:100), rabbit polyclonal anti-human NESTIN (Eurofins Genomics, 1:200), rabbit polyclonal anti-human NANOG (ReproCELL, 1:200), and mouse monoclonal anti-human OCT3/4 (C-10; Santa Cruz, 1:200).

Techniques: Marker, Derivative Assay, Immunocytochemistry, Cytometry

Journal: Scientific Reports

Article Title: Convallatoxin enhance the ligand-induced mu-opioid receptor endocytosis and attenuate morphine antinociceptive tolerance in mice

doi: 10.1038/s41598-019-39555-x

Figure Lengend Snippet: Effect of convallatoxin on opioid–induced MOR endocytosis. ( A ) Representative live cell imaging of the distribution of MOR-eGFP in CHO-K1 cells before and 30 min after drug treatment using a real-time confocal microscopy. Scale bars, 10 μm. ( B ) Representative immunofluorescence images of the distribution of MOR (red) and WGA (green) in the mouse DRG 1 h after drug treatment. The localization of MOR and WGA-labeled plasma membrane was monitored by confocal microscopy. DAPI (blue) was used as a nuclear marker. Scale bar, 20 μm. ( C ) Convallatoxin attenuated morphine-induced MOR phosphorylation. HEK-MOR cells were treated as indicated for 30 min. Phosphorylation of MOR at serine 375 ( C , D ) and total MOR expression ( C , E ) were analyzed by western blotting. Protein expression was quantified using densitometry ( D , E ). ( D ) F 3,12 = 7.36, p < 0.01; ( E ) F 3,12 = 0.08, p > 0.05 (1-way ANOVA). ** p < 0.01 versus vehicle control group. # p < 0.05 versus morphine group (Newman-Keuls post hoc tests). ( F ) Concentration-response curves of convallatoxin in morphine-induced MOR endocytosis in the presence or absence of MβCD. Data are percentages of the values for morphine (0.3 μM; ~EC 10 ) alone. ( G ) Silencing of AP2 and clathrin attenuated the effect of convallatoxin on morphine-induced MOR endocytosis. U2OS-MOR cells were transiently transfected with sh-control, sh-clathrin or sh-AP2 for 24 h, prior to MOR internalization assay. All values indicate the mean ± SD. RLU, relative light units.

Article Snippet: The rabbit monoclonal anti-AP2 α antibody (GTX62588) and mouse monoclonal anti-clathrin heavy chain antibody (GTX22731) were purchased from GeneTex.

Techniques: Live Cell Imaging, Confocal Microscopy, Immunofluorescence, Labeling, Clinical Proteomics, Membrane, Marker, Phospho-proteomics, Expressing, Western Blot, Control, Concentration Assay, Transfection

Journal: Scientific Reports

Article Title: Convallatoxin enhance the ligand-induced mu-opioid receptor endocytosis and attenuate morphine antinociceptive tolerance in mice

doi: 10.1038/s41598-019-39555-x

Figure Lengend Snippet: Effects of convallatoxin on morphine-mediated membrane potential hyperpolarization in MOR-expressing AtT-20 cells. ( A ) Flowchart showing experiments testing the effects of convallatoxin on morphine activation of GIRK channels. AtT-20 Cells were transfected with myc-MOR expression plasmid for 24 h prior to all membrane potential assays. ( B ) Acute effects of each treatment in MOR-expressing AtT-20 cells were measured using membrane potential assay. AUC: F 4,15 = 23.97, p < 0.0001 (1-way ANOVA). ( C ) After 2 h of incubation, chronic effects of each treatment on MOR desensitization were determined by rechallenging cells with 10 μM morphine. AUC: F 4,15 = 84.32, p < 0.0001 (1-way ANOVA). ( D , F , H ) AtT-20 Cells were co-transfected with myc-MOR and sh-control ( D ), sh-clathrin ( F ), or sh-AP2 ( H ) for 24 h prior to membrane potential assay. Silencing of clathrin ( F ) and AP2 ( H ) did not attenuate the acute effect of convallatoxin. Immunoblot showing expression of clathrin (F) or AP2 (H) in clathrin- or AP2-knockdown AtT-20 cells (upper panel). AUC: ( D ) F 2,12 = 88.35; ( F ) F 2,12 = 323.2; ( H ) F 2,15 = 75.45; all p < 0.001 (1-way ANOVA). ( E , G , I ) Both clathrin ( I ) and AP2 ( G ) were involved in the chronic convallatoxin effect. AUC: ( E ) F 2,12 = 458.8; ( G ) F 2,12 = 287.2; ( I ) F 2,15 = 42.45; all p < 0.001 (1-way ANOVA). *** p < 0.001 versus vehicle group. ## p < 0.01 versus morphine + convallatoxin group. $$$ p < 0.001 versus morphine-alone group (Newman-Keuls post hoc tests). All values indicate the mean ± SD.

Article Snippet: The rabbit monoclonal anti-AP2 α antibody (GTX62588) and mouse monoclonal anti-clathrin heavy chain antibody (GTX22731) were purchased from GeneTex.

Techniques: Membrane, Expressing, Activation Assay, Transfection, Plasmid Preparation, Incubation, Control, Western Blot, Knockdown

Journal: Scientific Reports

Article Title: Convallatoxin enhance the ligand-induced mu-opioid receptor endocytosis and attenuate morphine antinociceptive tolerance in mice

doi: 10.1038/s41598-019-39555-x

Figure Lengend Snippet: Down-regulation of clathrin and AP2 attenuated the effect of convallatoxin in morphine antinociceptive tolerance. ( A , B ) The sh-control, sh-clathrin, or sh-AP2 was delivered into the spinal cord of wild-type (WT) mice by using direct in vivo electroporation. Three days after surgery, the acute ( A ) and chronic ( B ) antinociceptive effects of each treatment were measured using a tail-flick test. ( A ; upper panel) Treatment F 5,24 = 0.47, min F 3,72 = 168.2, interaction F 15,72 = 1.44; ( B ; upper panel) Treatment F 5,24 = 6.9, min F 3,72 = 72.56, interaction F 15,72 = 5.81; all p < 0.001 (2-way ANOVA). Quantitative results from the upper panel of ( A ) and ( B ) are presented as AUC. ( A ; lower panel) F 5,24 = 1.43, p > 0.05; ( B ; lower panel) F 5,24 = 5.64, p < 0.01 (1-way ANOVA). ( C ) Chronic convallatoxin administration still diminished naloxone-precipitated withdrawal jumping in clathrin and AP2 knockdown mice. F 5,24 = 5.29, p < 0.01 (1-way ANOVA). ( D ) Representative immunofluorescence images of MOR (red), WGA (green) for each treatment in mouse DRG neurons were visualized using immunostaining. DAPI (blue) was used as a nuclear marker. Scale bars, 20 μm. Data in B (upper paner), *** p < 0.001 versus sh-control + morphine group (Bonferroni’s post hoc tests). Data in B (lower paner), C , D , * p < 0.05, ** p < 0.01, *** p < 0.001 versus sh-control + morphine group. # p < 0.05, ## p < 0.01 versus sh-control + morphine + convallatoxin group. § p < 0.05 versus sh-clathrin + morphine group. $ p < 0.05 versus sh-AP2 + morphine group (Newman-Keuls post hoc tests). Data are presented as the mean ± s.e.m. MPE, maximum possible effect.

Article Snippet: The rabbit monoclonal anti-AP2 α antibody (GTX62588) and mouse monoclonal anti-clathrin heavy chain antibody (GTX22731) were purchased from GeneTex.

Techniques: Control, In Vivo, Electroporation, Tail Flick Test, Knockdown, Immunofluorescence, Immunostaining, Marker

Journal: Scientific Reports

Article Title: Convallatoxin enhance the ligand-induced mu-opioid receptor endocytosis and attenuate morphine antinociceptive tolerance in mice

doi: 10.1038/s41598-019-39555-x

Figure Lengend Snippet: Convallatoxin alters morphine-induced alleviation of mechanical allodynia through clathrin and AP2 in the CFA-induced arthritic mouse model. ( A ) Experimental flowchart for effects of convallatoxin on allodynia. Mice receive an intraplantar injection of saline or CFA to induce local inflammation. Allodynia, expressed in g, is evaluated in CFA- and saline-treated mice 45 min after the last drug injection on post-inoculation days (PIDs) 14, and 18. Treatment F 7,40 = 589.4, time F 1,40 = 65.54, interaction F 7,40 = 34.96; all p < 0.001 (2-way ANOVA). ( B , C ) Quantitative results from ( A ) are presented as percentages of the threshold on PID 14 for CFA + morphine ( B ) or CFA + morphine + convallatoxin ( C ). ( B , C ) all p < 0.001 (student’s t ). ( D ) Silencing clathrin or AP2 attenuates the effect of convallatoxin on morphine antinociception. The mouse spinal cord was electroporated with sh-control , sh-clathrin , or sh-AP2 on PID 7. Treatment F 5,24 = 58.27, time F 1,48 = 1.32, interaction F 5,48 = 0.58; all p < 0.001 (2-way ANOVA). Data in (A, lower panel) and (D): *** p < 0.001 versus saline + vehicle group; ### p < 0.001 versus CFA + morphine group; $$$ p < 0.001 versus sh-control +CFA + morphine group (Bonferroni’s post hoc test). Data in B and C, right panel: ΦΦΦ p < 0.001 versus threshold of each group on PID 14; $$$ p < 0.001 versus sh-control + CFA + morphine group (Newman-Keul’s post hoc test).

Article Snippet: The rabbit monoclonal anti-AP2 α antibody (GTX62588) and mouse monoclonal anti-clathrin heavy chain antibody (GTX22731) were purchased from GeneTex.

Techniques: Injection, Saline, Control

Journal: The EMBO Journal

Article Title: Compensatory endocytosis in bladder umbrella cells occurs through an integrin-regulated and RhoA- and dynamin-dependent pathway

doi: 10.1038/emboj.2010.91

Figure Lengend Snippet: Delivery of endocytosed membrane to late endosomes/lysosomes. (A) Co-localization of EEA1, Rab11a, ZO-1, giantin, or LAMP2 with FITC-WGA 10 min after experimental voiding. (B) Co-localization coefficients for FITC-WGA and the indicated marker 10 or 90 min after experimental voiding. Values are mean±s.e.m. (n⩾7). Those values significantly different from the 10-min time point (P<0.05) are indicated with an asterisk. (C) Distribution of FITC-WGA and indicated markers 90 min after voiding. (D) Left panel: Intensity of labelled WGA pulsed for 10 min after experimental voiding ± an 80-min chase in the absence of additional marker. Alternatively, WGA was pulsed for 10 min after voiding, chased for 30 min in the absence of filling, and then chased for an additional 60 min during which time the chamber was re-filled. Values are mean±s.e.m. (n⩾7) and those values significantly different than samples fixed after the pulse (P<0.05) are indicated with an asterisk. Right panel: Distribution of Alexa488-WGA that was internalized for 10 min after voiding, chased for 30 min, and then chased for 60 min during which time the mucosal hemichamber was re-filled. (E) Co-localization of Alexa488-WGA and LAMP2 in tissue that was refilled.

Article Snippet: Other antibodies used in these studies include mouse monoclonal antibodies specific for caveolin-1 and -2, dynamin-2, EEA1, and flotillin-1 (all from BD Biosciences; San Jose, CA), mouse monoclonal anti-giantin (provided by Dr Adam Linstedt, Carnegie Mellon University), and mouse monoclonal anti-AP2 α subunit antibody AP.6 and anti-clathrin heavy chain (provided by Dr Linton Traub, University of Pittsburgh).

Techniques: Marker

Journal: The EMBO Journal

Article Title: Compensatory endocytosis in bladder umbrella cells occurs through an integrin-regulated and RhoA- and dynamin-dependent pathway

doi: 10.1038/emboj.2010.91

Figure Lengend Snippet: Dependence of CE on dynamin. (A) Localization of dynamin-2 in uroepithelial tissue. The apical surface of an umbrella cell is indicated by arrows. (B) Effect of dynasore on CT during filling and voiding. Voiding was initiated at the arrow. The boxed region in the left panel is expanded in the right-hand panel. Control data are reproduced from Figure 1A. Values are mean±s.e.m. (n⩾5), and those values significantly different than control samples (P<0.5) are indicated with an asterisk. (C) Uptake of FITC-WGA and FITC-dextran in tissue treated with dynasore (250 μM). (D) Quantitation of FITC-WGA and FITC-dextran uptake in tissue treated with MβCD (10 mM), cyto-D (25 μg/ml), C3 toxin (1 μg/ml), dynasore (250 μM), or Y-27632 (25 μM). Values are mean±s.e.m. (n⩾10), and those values significantly different than control samples (P<0.05) are indicated with an asterisk. (E, F) Rat bladders were transduced in situ with adenovirus expressing GFP alone or GFP-labelled DN-dynaminK44A (DN-dynamin). The bladder was filled with Alexa647-WGA, stimulated to undergo voiding, excised, and then fixed and processed for immunofluorescence. (E) A transduced cell expressing DN-dynaminK44A (K44A) is indicated with an asterisk. (F) The uptake of Alex647-WGA was quantified in cells expressing GFP or DN-dynaminK44A (K44A) and normalized to uptake in adjacent cells that did not express exogenous protein. Data are mean±s.e.m. (n⩾70).

Article Snippet: Other antibodies used in these studies include mouse monoclonal antibodies specific for caveolin-1 and -2, dynamin-2, EEA1, and flotillin-1 (all from BD Biosciences; San Jose, CA), mouse monoclonal anti-giantin (provided by Dr Adam Linstedt, Carnegie Mellon University), and mouse monoclonal anti-AP2 α subunit antibody AP.6 and anti-clathrin heavy chain (provided by Dr Linton Traub, University of Pittsburgh).

Techniques: Quantitation Assay, In Situ, Expressing, Immunofluorescence