s6k1  (MedChemExpress)

Journal: Molecular Pain

Article Title: Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

doi: 10.1177/17448069251376198

Figure Lengend Snippet: The expression of p-mTOR/p-S6K1-Gli1 signaling in the spinal cord after chronic morphine injection. (a) Western blotting showed the time course of mTOR, p-mTOR, S6K1, p-S6K1 and nucleus Gli1 after chronic morphine injection in the spinal cord ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Sham group). (b) Immunofluorescence staining results showed the expression of p-mTOR, p-S6K1 and Gli1 after chronic morphine injection in the spinal cord. ( n = 6 per group, Scale bar = 100 μm). Mor: morphine; SC: spinal cord.

Article Snippet: The selective S6K1 inhibitor PF-4708671 and GANT-61 was purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, Injection, Western Blot, Immunofluorescence, Staining

Journal: Molecular Pain

Article Title: Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

doi: 10.1177/17448069251376198

Figure Lengend Snippet: The expression of p-mTOR/p-S6K1/Gli1 signaling in the DRG after chronic morphine injection. (a) Western blotting showed the time course of mTOR, p-mTOR, S6K1, p-S6K1 and nucleus Gli1 after chronic morphine injection in the DRG. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Sham group). (b) Immunofluorescence staining results showed the expression of p-mTOR, p-S6K1 and Gli1 after chronic morphine injection in the DRG. ( n = 6 per group, Scale bar = 50 μm). Mor: morphine.

Article Snippet: The selective S6K1 inhibitor PF-4708671 and GANT-61 was purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, Injection, Western Blot, Immunofluorescence, Staining

Journal: Molecular Pain

Article Title: Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

doi: 10.1177/17448069251376198

Figure Lengend Snippet: Cellular localization of p-mTOR/p-S6K1/Gli1 signaling in the spinal cord dorsal horn after chronic morphine treatment. p-mTOR/p-S6K1/Gli1 immunoreactivity in the ipsilateral dorsal horn was mostly detected in neurons (NeuN) after chronic morphine treatment. ( n = 4 per group, Scale bar = 100 μm).

Article Snippet: The selective S6K1 inhibitor PF-4708671 and GANT-61 was purchased from MedChemExpress (Shanghai, China).

Techniques:

Journal: Molecular Pain

Article Title: Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

doi: 10.1177/17448069251376198

Figure Lengend Snippet: Treatment with mTOR inhibitor significantly attenuated chronic morphine injection-induced pain behaviors. (a) Treatment with 8 mg/kg and 16 mg/kg mTOR inhibitor KU-0063794 at the early stage (from d 1 to d 3) effectively delayed the decrease of MPE%. (b) Treatment with 8 mg/kg and 16 mg/kg mTOR inhibitor KU-0063794 at the late stage (from d 4 to d 6) significantly delayed the decrease of MPE%. (c) Early treatment with 8 mg/kg and 16 mg/kg KU-0063794 at the early stage (from d 1 to d 3) effectively mitigated thermal withdrawal latency induced by repeated morphine injections. (d) Treatment with 8 mg/kg and 16 mg/kg KU-0063794 at the late stage (from d 4 to d 6) significantly mitigated thermal withdrawal latency induced by repeated morphine injections. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the sham group, # p < 0.05, ## p < 0.01 compared with the morphine group). (e) Treatment with 8mg/kg KU-0063794 significantly suppressed the expression and activation of p-mTOR, p-S6K1 and Gli1 in the spinal cord by mTOR agonist L-leucine. ( n = 6 per group, ** p < 0.01 compared with the morphine + DMSO group, # p < 0.05, ## p < 0.01 compared with the group treated with L-leucine). i.p.: intraperitoneally; KU: KU-0063794; Leu: L-leucine; Mor: morphine; SC: spinal cord.

Article Snippet: The selective S6K1 inhibitor PF-4708671 and GANT-61 was purchased from MedChemExpress (Shanghai, China).

Techniques: Injection, Expressing, Activation Assay

Journal: Molecular Pain

Article Title: Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

doi: 10.1177/17448069251376198

Figure Lengend Snippet: The expression of BDNF in the spinal cord of mice following chronic morphine injection. (a) Western blotting showed the time course of BDNF after chronic morphine injection in the spinal cord. (b) Western blotting showed the time course of BDNF after chronic morphine injection in the DRG. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Sham group). (c) Treatment with mTOR inhibitor KU-0063794 significantly reduced morphine injection-induced BDNF upregulation. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Sham group, ## p < 0.01 compared with the morphine + DMSO group). (d) Treatment with mTOR siRNA significantly reduced morphine injection-induced BDNF upregulation. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Sham group, # p < 0.05 compared with the morphine + con-si-RNA group). (e) Injection of mTOR agonist L-leucine induced an upregulation of the protein expression of BDNF. ( n = 6 per group, ** p < 0.01 compared with the Vehicle group, ## p < 0.01 compared with the L-leucine-treated group). (f) Pharmacological S6K1 inhibition by PF-4708671 significantly reversed L-leucine-induced thermal hyperalgesia. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Vehicle group, # p < 0.05, ## p < 0.01 compared with the L-leucine-treated group). (g) Pharmacological Gli1 inhibition by GANT-67 significantly reversed L-leucine-induced thermal hyperalgesia. ( n = 6 per group, * p < 0.05, ** p < 0.01 compared with the Vehicle group, # p < 0.05, ## p < 0.05 compared with the L-leucine-treated group). (h) Treatment with mTOR inhibitor KU-0063794 significantly reversed L-leucine-induced BDNF upregulation. ( n = 6 per group, ** p < 0.01 compared with the Vehicle group, ## p < 0.01 compared with the L-leucine group). (i) Treatment with BDNF inhibitor K252 significantly reduced the decline in morphine’s maximum possible analgesic effect. ( n = 6 per group, ** p < 0.01 compared with the Vehicle group). (j-k) Treatment with BDNF inhibitor K252 significantly delayed the decrease of MPE% and suppressed thermal withdrawal latency induced by mTOR agonist L-leucine. ( n = 6 per group, ** p < 0.01 compared with the morphine + DMSO group, # P < 0.05, ## p < 0.01 compared with the group treated with L-leucine). i.p.: intraperitoneally; i.t.: intrathecally; KU, KU-0063794; Leu: L-leucine; Mor: morphine; SC: spinal cord.

Article Snippet: The selective S6K1 inhibitor PF-4708671 and GANT-61 was purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, Injection, Western Blot, Inhibition

Journal: Neoplasia (New York, N.Y.)

Article Title: SCF β-TrCP targets Ajuba for degradation in a GSK3β-dependent manner in colorectal cancer

doi: 10.1016/j.neo.2025.101175

Figure Lengend Snippet: GSK3β promotes ubiquitination and degradation of Ajuba. A Western blot analysis of HCT116 cells treated with CK1 inhibitor D 4476 (50 μM), GSK3β inhibitor CHIR-99021 (50 μM) and S6K1 inhibitor PF-4708671 (50 μM) for 24 h. B HCT116, HT29 and SW480 cells were incubated with GSK3β inhibitor CHIR-99021 (50 μM) for 24 h and collected for western blot. C Western blot analysis for HCT116 cells treated with 50 μM CHIR-99021 at different time points. D HCT116 cells cultured with GSK3β inhibitor CHIR-99021 (50 μM) for 24 h were pretreated with MG132 (20 μM) for 5 h and harvested for IP. E Western blot analysis of HCT116, SW480 and RKO cells transfected with siRNAs targeting GSK3β for 72 h. F Overexpression of Myc-GSK3β and HA-Ajuba to evaluate the expression level of exogenous Ajuba. G HEK293T cells were transfected with increasing amounts of Myc-GSK3β plasmid, followed by western blot analysis. H, I Cells were transfected with the indicated plasmids for 36 h with or without CHIR-99021 (50 μM) for 24 h and exogenous interaction between GSK3β and Ajuba was analyzed with western blot. J, K Upper panel: RKO cells transfected with two GSK3β siRNAs for 72 h were treated with CHX (50 mg/mL) for the indicated times and then were subjected to western blot. Lower panel: Quantification of Ajuba band intensity. L RKO cells were transfected with GSK3β siRNAs and pretreated with MG132 (20 μM) for 5 h. The cell lysates were immunoprecipitated and then subjected to western blot to analyze the ubiquitination level of Ajuba. M GSK3β and β-TrCP siRNAs were either cotransfected or transfected alone into HCT116 cells and the protein level of Ajuba was detected by western blot.

Article Snippet: CK1 inhibitor D 4476 (S7642), GSK3β inhibitor CHIR-99021 (S1263) and S6K1 inhibitor PF-4708671 (S2163) were purchased from Selleck.

Techniques: Ubiquitin Proteomics, Western Blot, Incubation, Cell Culture, Transfection, Over Expression, Expressing, Plasmid Preparation, Immunoprecipitation