Journal: bioRxiv

Article Title: Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

doi: 10.1101/182360

Figure Lengend Snippet: ( A and B ) Role of Na + /K + -ATPase in the effect of CTSs. U2OS-MOR cells are transiently transfected with sh-control ( A ) or sh-Na + /K + -ATPase α1 ( B, lower panel), 24 h prior to the MOR internalization assay. ( B , upper panel) Immunoblot showing Na + /K + -ATPase α1 expression in knockdown U2OS-MOR cells. ( C ) Concentration-response curves of CTSs in morphine-induced MOR endocytosis in the presence or absence of the endocytosis inhibitor MβCD. Data are percentages of the values for morphine alone (0.3 µM; ∼EC10). ( D ) Silencing of adaptor protein 2 ( AP2) or clathrin attenuates the effect of convallatoxin on morphine-induced MOR endocytosis. ( D , lower panel) U2OS-MOR cells are transiently transfected with sh-control , sh-clathrin or sh-AP2, 24 h prior to the MOR internalization assay. ( D , upper panel) Immunoblots showing clathrin or AP2 expression in clathrin - or AP2- knockdown U2OS-MOR cells. ( E and F ) CTSs fail to modulate morphine-mediated β-arrestin-2 recruitment ( E ) or inhibition of cAMP accumulation ( F ). CHO-K1-MOR cells ( E ) and human embryonic kidney (HEK)-MOR cells ( F ) are treated with various concentrations of morphine in the absence or presence of CTSs. ( G, lower panel) Silencing of β-arrestin-2 fails to regulate the effect of convallatoxin on morphine-induced MOR endocytosis. U2OS-MOR cells are transiently transfected with sh-control or sh-β-arrestin-2 , 24 h prior to the MOR internalization assay. ( G , upper panel) Immunoblot showing β-arrestin-2 expression in knockdown U2OS-MOR cells. ( H ) Involvement of Gi/o protein in the effect of CTSs on MOR endocytosis. U2OS-MOR cells are pretreated with pertussis toxin 18 h prior to the internalization assay. ( I ) Convallatoxin and/or morphine do not attenuate MOR expression. HEK-MOR cells are treated as indicated for 30 min. Total MOR expression is analyzed by immunoblotting and quantified by densitometry. F 3,12 = 0.45; p > 0.05 (1-way ANOVA).

Article Snippet: Blots were incubated with rabbit monoclonal anti-MOR antibody (1:1000; Cat. NBP1-96656; Novus Biologicals), mouse monoclonal anti-Na + /K + -ATPase α1 subunit antibody (1:1000; Cat. ab7671; Abcam), rabbit monoclonal anti-AP2 α antibody (1:1000; Cat. GTX62588; GeneTex), mouse monoclonal anti-clathrin heavy chain antibody (1:1000; Cat. GTX22731; GeneTex), or mouse monoclonal anti-β-actin antibody (1:5000; Cat. A5316; Sigma) for 16 h. Following incubation, blots were washed and incubated in horseradish peroxidase-conjugated secondary antibody (1:1000; Cat. NA931V, NA934V; GE Healthcare) for 1 h. Signals were detected using a chemiluminescence enhancement kit (Millipore), and the density of the immunoblots was determined using ImageJ (NIH).

Techniques: Transfection, Control, Western Blot, Expressing, Knockdown, Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

doi: 10.1101/182360

Figure Lengend Snippet: ( A and B ) shRNA ( sh-control , sh-clathrin, or sh-AP2 ) is transfected into the spinal cord of B6 mice by in vivo electroporation. Seven days later, mice are sacrificed and the expression levels of clathrin ( A , green), AP2 ( B , green) and NeuN ( A and B , red) are visualized by immunostaining. DAPI (blue) is a nuclear marker. n = 5–10 per group. Scale bars, 20 µm.

Article Snippet: Blots were incubated with rabbit monoclonal anti-MOR antibody (1:1000; Cat. NBP1-96656; Novus Biologicals), mouse monoclonal anti-Na + /K + -ATPase α1 subunit antibody (1:1000; Cat. ab7671; Abcam), rabbit monoclonal anti-AP2 α antibody (1:1000; Cat. GTX62588; GeneTex), mouse monoclonal anti-clathrin heavy chain antibody (1:1000; Cat. GTX22731; GeneTex), or mouse monoclonal anti-β-actin antibody (1:5000; Cat. A5316; Sigma) for 16 h. Following incubation, blots were washed and incubated in horseradish peroxidase-conjugated secondary antibody (1:1000; Cat. NA931V, NA934V; GE Healthcare) for 1 h. Signals were detected using a chemiluminescence enhancement kit (Millipore), and the density of the immunoblots was determined using ImageJ (NIH).

Techniques: shRNA, Control, Transfection, In Vivo, Electroporation, Expressing, Immunostaining, Marker

Journal: bioRxiv

Article Title: Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

doi: 10.1101/182360

Figure Lengend Snippet: ( A and B ) The sh-control , sh-clathrin , or sh-AP2 plasmids are delivered into the spinal cord of WT mice using direct in vivo electroporation. Seven days after surgery, the antinociceptive effects after acute ( A ) and chronic ( B ) treatment with drugs are measured using the tail-flick test. ( A , left panel) Treatment F5,24 = 0.47, time F3,72 = 168.2, interaction F15,72 = 1.44; ( B , left panel) Treatment F5,24 = 6.9, time F3,72 = 72.56, interaction F15,72 = 5.81; all p < 0.001 (2-way ANOVA). Quantitative results from the left panel of ( A ) and ( B ) are presented as AUCs. ( A , right panel) F5,24 = 1.43; p > 0.05; ( B , right panel) F5,24 = 5.64; p < 0.01 (1-way ANOVA). ( C ) Representative immunofluorescence images (upper panel) and quantification (lower panel) of MOR (red), and WGA (green) for each treatment in mouse DRG neurons. DAPI (blue) is a nuclear marker. Scale bars, 20 µm. F 5,36 = 14.47; p < 0.001 (1-way ANOVA). Data in ( B , left panel): $$$, p < 0.001 versus sh-control + morphine group (Bonferroni’s post hoc test). Data in ( B , right panel), ( C , lower panel): $$, p < 0.01; $$$, p < 0.001 versus sh-control + morphine group; %, p < 0.05; %%, p < 0.01 versus sh-control + morphine + convallatoxin group (Newman-Keul’s post hoc test). MPE, maximum possible effect.

Article Snippet: Blots were incubated with rabbit monoclonal anti-MOR antibody (1:1000; Cat. NBP1-96656; Novus Biologicals), mouse monoclonal anti-Na + /K + -ATPase α1 subunit antibody (1:1000; Cat. ab7671; Abcam), rabbit monoclonal anti-AP2 α antibody (1:1000; Cat. GTX62588; GeneTex), mouse monoclonal anti-clathrin heavy chain antibody (1:1000; Cat. GTX22731; GeneTex), or mouse monoclonal anti-β-actin antibody (1:5000; Cat. A5316; Sigma) for 16 h. Following incubation, blots were washed and incubated in horseradish peroxidase-conjugated secondary antibody (1:1000; Cat. NA931V, NA934V; GE Healthcare) for 1 h. Signals were detected using a chemiluminescence enhancement kit (Millipore), and the density of the immunoblots was determined using ImageJ (NIH).

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

Journal: bioRxiv

Article Title: Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

doi: 10.1101/182360

Figure Lengend Snippet: ( 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, 16, and 18. Treatment F 7,40 = 384.2, time F 2,80 = 40.7, interaction F 14,80 = 23.48; all p < 0.001 (2-way ANOVA). ( B and 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 ) F 2,15 = 22.32; ( C ) F 2,15 = 24.7; all p < 0.001 (1-way ANOVA). ( 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 2,48 = 1.32, interaction F 10,48 = 0.58; all p < 0.001 (2-way ANOVA). ( E ) Representative immunofluorescence images (left panel) and quantification (right panel) of MOR (red) and WGA (green) for each treatment in mouse DRG neurons. DAPI (blue) is the nuclear marker. Scale bar, 20 µm. F 5,30 = 7.36; p < 0.001 (1-way ANOVA). Data in ( A , lower panel) and ( D ): ***, p < 0.001 versus saline + vehicle group; ###, p < 0.001 versus CFA + morphine group; $$$, p < 0.001 versus sh-control + CFA + morphine group (Bonferroni’s post hoc test). Data in B , C and E , right panel: Φ, p < 0.05; ΦΦΦ, p < 0.001 versus threshold of each group on PID 14; $$$, p < 0.001 versus sh-control + CFA + morphine group (Newman-Keul’s post hoc test).

Article Snippet: Blots were incubated with rabbit monoclonal anti-MOR antibody (1:1000; Cat. NBP1-96656; Novus Biologicals), mouse monoclonal anti-Na + /K + -ATPase α1 subunit antibody (1:1000; Cat. ab7671; Abcam), rabbit monoclonal anti-AP2 α antibody (1:1000; Cat. GTX62588; GeneTex), mouse monoclonal anti-clathrin heavy chain antibody (1:1000; Cat. GTX22731; GeneTex), or mouse monoclonal anti-β-actin antibody (1:5000; Cat. A5316; Sigma) for 16 h. Following incubation, blots were washed and incubated in horseradish peroxidase-conjugated secondary antibody (1:1000; Cat. NA931V, NA934V; GE Healthcare) for 1 h. Signals were detected using a chemiluminescence enhancement kit (Millipore), and the density of the immunoblots was determined using ImageJ (NIH).

Techniques: Injection, Saline, Control, Immunofluorescence, Marker