Journal: Inflammation Research

Article Title: Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo

doi: 10.1007/s00011-014-0782-9

Figure Lengend Snippet: IC 50 and Ki values of JAK inhibitors in enzyme assays

Article Snippet: Recombinant kinase domains of human JAK1 (850–end) and Tyk2 (871–end) were purchased from Carna Biosciences Inc. (Kobe, Japan), and those of JAK2 (808–end) and JAK3 (781–end) were from Millipore Corporation (Billerica, MA).

Techniques:

Journal: Inflammation Research

Article Title: Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo

doi: 10.1007/s00011-014-0782-9

Figure Lengend Snippet: IC 50 values in cytokine signaling

Article Snippet: Recombinant kinase domains of human JAK1 (850–end) and Tyk2 (871–end) were purchased from Carna Biosciences Inc. (Kobe, Japan), and those of JAK2 (808–end) and JAK3 (781–end) were from Millipore Corporation (Billerica, MA).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. * P < 0.05 vs. normal group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, shRNA, Infection

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24, and 48 h during reoxygenation after OGD when compared with normal astrocytes. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, Live Cell Imaging

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on cellular swelling in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). a Astrocyte volume analysis was performed using a Live Cell Imaging System, and cellular volume was calculated by the average value of four measured diameters. Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly blocked increases in cellular volume of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation when compared with astrocytes of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b — d Effects of inhibiting HMGB1 on spinal cord astrocytic morphology and ultrastructure were evaluated using transmission electron microscopy at 6, 12, and 24 h during reoxygenation after OGD. After OGD/R, spinal cord astrocytes showed swelling at 6, 12, and 24 h during reoxygenation. The mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and an increase in the number of lysosomes (L) were concurrent with this observation. However, astrocytic swelling, mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and the increase in lysosome (L) number after OGD/R were reduced by HMGB1 inhibition using either HMGB1 shRNA or EP (× 50,000, bar equal to 1 μm, three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Cell Culture, Live Cell Imaging, shRNA, Transmission Assay, Electron Microscopy, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on HMGB1, aquaporin-4 (AQP4), and toll-like receptor-4 (TLR4) expression in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) as well as levels of HMGB1 and interleukin-6 (IL-6) release into the surrounding medium. a Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly suppressed the increased levels of HMGB1 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 significantly suppressed the increased levels of AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 significantly suppressed increased levels of TLR4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). d HMGB1, AQP4, and TLR4 immunofluorescence on spinal cord astrocytes at 24 h into the reoxygenation process after OGD showed significantly increased membrane and cytoplasmic levels of HMGB1, AQP4, and TLR4 in the OGD/R group when compared with those in the normal group. These were markedly suppressed in both the OGD/R + HMGB1 shRNA and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e , f Inhibiting HMGB1 mitigated increases in levels of HMGB1 and IL-6 in the surrounding medium when compared with levels in the OGD/R group at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of either inhibiting high mobility group box-1 (HMGB1) or toll-like receptor-4 (TLR4) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, TLR4, myeloid differentiation primary response gene 88 (MyD88), aquaporin-4 (AQP4) upregulation, and nuclear factor-kappa B (NF-κB) activation as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a Inhibiting HMGB1 (using either HMGB1 shRNA or ethyl pyruvate (EP)) or TLR4 (using CLI-095 or C34) significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those in the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 or TLR4 significantly suppressed the increased levels of TLR4, MyD88, and AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 24 h during the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 or TLR4 significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). d Immunofluorescence results showed that either inhibiting HMGB1 or TLR4 decreased membrane and cytoplasmic TLR4 and AQP4 upregulation and attenuated the increases of nuclear NF-κB when compared with the OGD/R group at 24 h during reoxygenation (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e Inhibiting HMGB1 or TLR4 reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Activation Assay, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of nuclear factor-kappa B (NF-κB) inhibition on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, NF-κB activation, and aquaporin-4 (AQP4) upregulation, as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a NF-κB inhibition (using BAY 11-7082) significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation phase after OGD. * P < 0.05 vs. OGD/R group (three replicates). b NF-κB and AQP4 immunofluorescence in spinal cord astrocytes after 24 h of the reoxygenation process after OGD showed significantly increased nuclear levels of NF-κB and membrane and cytoplasmic levels of AQP4 in the OGD/R group. Levels were markedly attenuated in the OGD/R + HMGB1 shRNA, OGD/R + BAY 11-7082, and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). c NF-κB inhibition significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). d NF-κB inhibition significantly suppressed increased AQP4 levels in both the plasma membrane and cytoplasm of spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). e NF-κB inhibition reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Inhibition, Activation Assay, Immunofluorescence, shRNA

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of recombinant HMGB1 (rHMGB1) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. Incubation of cultured spinal cord astrocytes with rHMGB1 (0, 0.1, 1, 10, and 20 ng/ml) for 24 h did not induce dose-dependent increases in the membrane and cytoplasmic AQP4 expression (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Recombinant, Expressing, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of interleukin-6 (IL-6) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. a Spinal cord astrocytes were exposed to exogenous IL-6 at 0, 0.1, 1, or 10 ng/ml. After 24 h exposure, the membrane and cytoplasmic AQP4 expression in spinal cord astrocytes were markedly increased in the IL-6 0.1 ng/ml group, IL-6 1 ng/ml group, and IL-6 10 ng/ml group. * P < 0.05 vs. 0 ng/ml group (three replicates). b IL-6 levels increased in the surrounding medium of the OGD/R group after 24 h of the reoxygenation process after OGD. In comparison, this increase was significantly reduced in the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. OGD/R group (three replicates). c The effects of astrocyte conditioned medium (ACM) on AQP4 expression in cultured spinal cord astrocytes. Twenty-four hours exposure of spinal cord astrocytes to the ACM obtained from the OGD/R group significantly increased the membrane and cytoplasmic AQP4 expression when compared with astrocytes incubated with the ACM obtained from the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates). d Western blot analysis showed that the neutralizing anti-rat-IL-6 antibody could significantly reverse the upregulation effect of exogenous IL-6 or OGD/R ACM containing increased IL-6 on AQP4 expression in cultured spinal cord astrocytes. # P < 0.05 vs. astrocytes + IL-6 group; * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Incubation, Western Blot

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, or PMA on LIF-induced activation of signal transduction and activation of transcription_(STAT3) and ERK1/2 . Western blot was performed on NHBE cells that had been preincubated with or without AG-490, PD-98059, or PMA and then stimulated with LIF. (a) LIF induced activation of tyrosine phosphorylation of STAT3, and tyrosine phosphorylation of STAT3 was inhibited by AG-490, but not by PD-98059, and not affected by PMA. (b) LIF did not enhance the expression of total-STAT3, and its expression was not affected by AG-490, PD-98059, and PMA. (c) LIF induced activation of phosphorylation of ERK1/2, and ERK1/2 activation was inhibited by PD-98059, but not by AG-490; PMA increased the expression of p-ERK1/2 in NHBE cells, but there were no significant differences between the cells stimulated with LIF and the cells stimulated with LIF in the presence of PMA. (d) and (e) LIF did not enhance the expression of total-ERK1/2, furthermore, AG-490, PD-98059, and PMA also did not affect it. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (target/GAPDH) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Activation Assay, Transduction, Western Blot, Expressing

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by immunocytochemistry_(SABC × 200). Immunocytochemistry was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF ((a) control, (b) PD-98059, (c) AG-490, (d) LIF, (e) PMA, (f) LIF + PD-95059, (g) LIF + PMA, (h) LIF + AG-490, (i) LIF + control siRNA, (j) LIF + sham plasmid, (k) LIF + siRNA-1 against STAT3). LIF induced expression of NK-1R, which was inhibited by AG-490, PD-98059, and siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (positive cells number/total cells number) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Immunocytochemistry, Plasmid Preparation

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by RT-PCR . RT-PCR was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF. (a) LIF induced expression of NK-1R mRNA, and that was inhibited by AG-490 and PD-98059; PMA increased the expression of NK-1R mRNA in NHBE cells. (b) LIF-induced expression of NK-1R mRNA was inhibited by siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio_(target/β-actin) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: Oncogene

Article Title: WASF3 provides the conduit to facilitate invasion and metastasis in breast cancer cells through HER2/HER3 signaling

doi: 10.1038/onc.2015.527

Figure Lengend Snippet: When SKBR3 cells were treated with NRG for 30 minutes, there is a significant increase in STAT3 phosphorylation at Tyrosine 705 (Y705) (a). Only a slight increase in STAT3 phosphorylation was seen in the presence of NRG for 10 minutes (a). When these NRG-treated cells are treated with the AG490 JAK2 inhibitor, Y705 activation is suppressed (b). The same suppression is seen when the cells are treated with the either the S3I-201 STAT3 inhibitor, or the Herceptin recombinant, humanized anti-HER2 antibody (b). In STAT3 knockdown SKBR3 cells, NRG does not affect WASF3 expression levels (c). Similarly, knockdown of JAK2 also suppresses NRG-induced expression of WASF3 (d). In the luciferase reporter assay for WASF3, NRG treatment leads to a significant increase in activity, which is suppressed in STAT3 knockdown cells (e). ChIP-qPCR assays show increased levels of STAT3 at the WASF3 promoter-binding site in the presence of NRG (f). ** p<0.01; Student’s t-test.

Article Snippet: The following primary antibodies were used in our study: WASF3 (#2806), HER2 (#2242), P-HER2 (Y1221/1222, #2249), HER3 (#4754), P-HER3 (Y1289, #2842), STAT3 (#4904), P-STAT3 (Y705, #4113), ERK1/2 (#4695), P-ERK1/2 (T202/Y204, #9101) and JAK2 (#3230) (Cell Signaling Technology, MA), HA (#H9658), PY20 (#P4110) and β-Actin (#A5441) (Sigma, MO).

Techniques: Phospho-proteomics, Activation Assay, Recombinant, Knockdown, Expressing, Luciferase, Reporter Assay, Activity Assay, ChIP-qPCR, Binding Assay

Journal: JOR spine

Article Title: M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway.

doi: 10.1002/jsp2.1290

Figure Lengend Snippet: FIGURE 2 Oncostatin M (OSM) can promote the ossification of ligament flavum (LF) cells by activating the JAK2/STAT3 signaling pathway. (A) Upstream regulator analysis was used to evaluate the expression degree of each signaling pathway in osteogenic differentiation of LF cells. (B) Upregulated signal molecules downstream of OSM were analyzed by upstream regulator analysis. (C–F) Upstream regulator analysis was used to analyze the significance of the top 10 factors in each category of cytokines, kinases, transcription factors, and signaling pathway inhibitors. (G) Upstream regulator analysis was used to investigate the significance of growth factors other than OSM during osteogenic differentiation of LF cells.

Article Snippet: To investigate the effect of the JAK2/STAT3 signaling pathway on osteogenic differentiation of LF cells, the JAK2 inhibitor AZD1480 (10 μM, Santa Cruz Biotechnology), STAT3 inhibitor Stattic (5 μM, Santa Cruz Biotechnology), or blank control (DMSO) was added to the osteogenic differentiation medium with 25 ng/mL recombinant OSM protein to induce LF cells.

Techniques: Expressing

Journal: JOR spine

Article Title: M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway.

doi: 10.1002/jsp2.1290

Figure Lengend Snippet: FIGURE 4 Oncostatin M (OSM) promotes osteogenic differentiation of ligamentum flavum (LF) cells through the JAK2/STAT3 signaling pathway. (A–C) Expression of OSMR, GP130, P-JAK2, P-STAT, and osteogenic genes was detected by western blot (WB) and qPCR after 7 days of OSM cytokine induction (n = 3), two-tailed Student's t-test. (D–G) Expression of OSMR and GP130 was evaluated after knockdown in LF cells (n = 3), two-tailed Student's t-test. (H, I) LF cells with knockdown of OSMR and GP130, and stimulation with OSM. WB and qPCR were performed to evaluate expression of P-JAK2, P-STAT3, and osteogenic differentiation genes (n = 3), one-way ANOVA analysis. (J, K) In the presence of AZD1480 and Stattic, expression of P-JAK2, P-STAT3, and osteogenic genes was detected by WB and qPCR after 7 days of OSM induction in LF cells (n = 3), one-way ANOVA analysis. All data were presented as means ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To investigate the effect of the JAK2/STAT3 signaling pathway on osteogenic differentiation of LF cells, the JAK2 inhibitor AZD1480 (10 μM, Santa Cruz Biotechnology), STAT3 inhibitor Stattic (5 μM, Santa Cruz Biotechnology), or blank control (DMSO) was added to the osteogenic differentiation medium with 25 ng/mL recombinant OSM protein to induce LF cells.

Techniques: Expressing, Western Blot, Two Tailed Test, Knockdown, Standard Deviation

Journal: JOR spine

Article Title: M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway.

doi: 10.1002/jsp2.1290

Figure Lengend Snippet: FIGURE 5 M1 macorphages (Mφs) can secrete oncostatin M (OSM) and induce osteogenic differentiation of ligamentum flavum (LF) cells via the JAK2/STAT3 signaling pathway. The cytokine OSM binds to the receptor OSMR/GP130, stimulating activation and phosphorylation of JAK2. Phosphorylated JAK2 subsequently stimulates phosphorylation of downstream STAT3, causing STAT3 to form a dimer that migrates to the nucleus and binds to DNA to regulate gene transcription. Cyclooxygenase-2 inhibitors reduce OSM secretion by M1 Mφs. OSM neutralizing antibody can inhibit the binding of OSM to its receptor. Knockdown of OSMR/GP130 can obstruct signal transmission. AZD1480 inhibits both expression and phosphorylation of JAK2. Stattic can block STAT3 activation and phosphorylation.

Article Snippet: To investigate the effect of the JAK2/STAT3 signaling pathway on osteogenic differentiation of LF cells, the JAK2 inhibitor AZD1480 (10 μM, Santa Cruz Biotechnology), STAT3 inhibitor Stattic (5 μM, Santa Cruz Biotechnology), or blank control (DMSO) was added to the osteogenic differentiation medium with 25 ng/mL recombinant OSM protein to induce LF cells.

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Knockdown, Transmission Assay, Expressing, Blocking Assay

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: Multiscale analysis identifies α‐Hederin as a JAK/STAT3‐targeting compound for OC. (A) 3D Principal component analysis (PCA). PCA plot was generated using the 2500 genes with the highest variance across samples. Normal tissues are represented by orange points, while OC samples are indicated by blue points. (B) Volcano plot showing differentially expressed genes between OC and normal tissues. (C) Boxplot showing increased mRNA expression of IL‐6 in OC tissues compared to normal tissues, based on TCGA and GTEx datasets. (D) GSEA indicating significant enrichment of the IL‐6/JAK/STAT3 signaling pathway in OC. (E) Uniform Manifold Approximation and Projection (UMAP) plot of 51 786 single cells from 11 epithelial ovarian cancer (EOC) patients ( GSE165897 ), color‐coded by patient identity. (F) Expression levels of IL6, IL6ST, JAK1, and STAT3 across single‐cell populations. (G) UMAP plots show the distribution of cells before and after NACT treatment. (H) JAK1 expression levels before and after NACT treatment. (I) Workflow of structure‐based virtual screening of 2908 natural compounds targeting JAK1 and JAK2, followed by cytotoxicity validation in OC and normal ovarian epithelial cells. (J) Dose‐response curves quantifying viability of OC cells upon drug treatment for 48 h. The code names of drugs are listed on the right. (K) Dose‐response curves quantifying viability of ovarian epithelial cells upon drug treatment for 48 h. (L) Binding affinity measurements of α‐Hederin and JAK1 as measured via MST thermophoresis curve analysis. (M) Binding affinity measurements of α‐Hederin and JAK2 as measured via MST thermophoresis curve analysis. (N) Schematic structures of JAK1. Molecular docking results of α‐Hederin (green) with JAK1 (blue). The docking sites of α‐Hederin on JAK1 were highlighted in magenta. (O) Schematic structures of JAK2. Molecular docking results of α‐Hederin (green) with JAK2 (blue). The docking sites of α‐Hederin on JAK2 were highlighted in magenta. (P) Venn diagram displaying α‐Hederin targets (pink) and OC‐associated genes (yellow). The overlapping regions indicate common targets. (Q) KEGG analysis highlighted the top 20 pathways with significant enrichment. Then red box indicated the JAK/STAT3 signaling pathway. (R)The schematic diagram of the drug‐target gene network was visualized using Cytoscape software.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Generated, Expressing, Biomarker Discovery, Binding Assay, Software

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: α‐Hederin directly binds to JAK1/2 and inhibits STAT3 phosphorylation and nuclear translocation. (A,B) IF staining of p‐STAT3 and statistical analysis of fluorescence intensity. Scale bar: 10 µ m . (C) Heatmap showing relative mRNA expression of STAT3 downstream targets (MYC, CCND1, BIRC5, BCL2, VEGFA, TWIST1, MMP2, and MMP9) following α‐Hederin treatment, measured by qRT‐PCR and normalized to GAPDH. (D) Western blot analysis of total and phosphorylated STAT3, JAK1, JAK2, JAK3, and SRC in SKOV‐3 cells treated with α‐Hederin (5 or 10 µ m ) or DMSO. Statistical analysis is presented. (E,F) Kinase assay to examine the effect of α‐Hederin on JAK1 (E) and JAK2 (F) kinase activity. (G,H) The competitive binding relationship between α‐Hederin and ATP was confirmed using a pull‐down assay. (I,J) In vitro kinase assays were performed using bacterial‐purified His‐STAT3 and the active JAK1 (I) and JAK2 (J) kinase. The amount of α‐Hederin in the reaction is indicated. (K) DARTS (drug affinity responsive target stability) assay showing α‐Hederin‐mediated stabilization of JAK1 and JAK2 proteins in SKOV‐3 lysates. (L) Western blot analysis of p‐STAT3 and total STAT3 in SKOV‐3 cells with sgCtrl, sgJAK1, sgJAK2, or sgJAK1+sgJAK2, treated or not with 5 µ m α‐Hederin. Bottom panel: quantification of p‐STAT3/STAT3 ratio. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was determined by unpaired two‐tailed Student's t ‐test for two‐group comparisons and one‐way ANOVA for comparisons among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Phospho-proteomics, Translocation Assay, Staining, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot, Kinase Assay, Activity Assay, Binding Assay, Pull Down Assay, In Vitro, Purification, Stability Assay, Two Tailed Test

Journal: Endocrine-Related Cancer

Article Title: Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway

doi: 10.1677/erc-06-0027

Figure Lengend Snippet: Figure 3 Leptin activates phosphorylations of JAK2, Akt, and ERK1/2 in hepatic cells. After 1-day serum deprivation, leptin was added into the serum-free medium of Hep3B for 30 min (A) or Chang liver for 15 min (B) with the increasing concentrations as indicated, and then the protein amounts of phosphorylated forms of JAK2 (p-JAK2), Akt (p-Akt), or ERK1/2 (p-ERK1/2) were detected with western blotting analyses. The same blots were stripped and reprobed with antibodies specific for total proteins of JAK2, Akt, or ERK1/2. Similar western blotting analyses were carried out with the cell lysates of (C) Hep3B and (D) Chang liver treated with 250 ng/ml leptin for the indicated time-courses. Data represent three independent experiments. The reprobed b-actin was used as an alternative internal control. Leptin acutely and dose-dependently induces activations of JAK2, Akt, and ERK1/2 in human malignant and non-malignant hepatocytes.

Article Snippet: Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-b-actin antibody (Sigma Chemical Co.), TGF-b (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and www.endocrinology-journals.org monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.

Techniques: Western Blot, Control

Journal: Endocrine-Related Cancer

Article Title: Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway

doi: 10.1677/erc-06-0027

Figure Lengend Snippet: Figure 5 Leptin triggers JAK2-linked PI3K/Akt and MEK/ERK1/2 signaling pathways. After 1-day serum deprivation and then 30-min pre-treatment of vehicle (K), 40 mM AG490 (AG), 250 nM wortmannin (Wort), or 10 mM U0126 (U0), (A) Hep3B and (B) Chang liver were treated with vehicle (K) or 250 ng/ml leptin (C) in serum-free media for 30 and 15 min respectively. Following, the protein amounts of phosphorylated forms of JAK2 (p-JAK2), Akt (p-Akt), or ERK1/2 (p-ERK1/2) were detected with western blotting analyses. The same blots were stripped and reprobed with antibodies specific for total proteins of JAK2, Akt, or ERK1/2. The reprobed b-actin was used as an alternative internal control. Alternative inhibitors 25 mM LY294002 (LY) and 20 mM PD98059 (PD) for PI3K/Akt and MEK/ERK1/2 respectively, were tested in the similar experiments in Hep3B (C). Data represent three independent experiments. Leptin activates a JAK2-initiated signaling cascade comprising PI3K/Akt and MEK/ERK1/2 in order of occurrence.

Article Snippet: Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-b-actin antibody (Sigma Chemical Co.), TGF-b (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and www.endocrinology-journals.org monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.

Techniques: Protein-Protein interactions, Western Blot, Control

Journal: Endocrine-Related Cancer

Article Title: Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway

doi: 10.1677/erc-06-0027

Figure Lengend Snippet: Figure 9 Leptin passes through a JAK2–PI3K/Akt– MEK/ERK1/2 signaling cascade to partially reverse the TGF- b1-reduced Bcl-2/Bax ratio and thus prevent HCC apoptosis. After 24 h, Hep3B cells were seeded in 100 mm Petri dish and maintained in serum-free media, cells were treated without (K) or with (C) indicated concentrations of leptin, together without (K) or with (C) 5 ng/ml TGF-b1 in combination without (K) or with (C) 40 mM AG490 (A), 250 nM wortmannin or 10 mM U0126 (B) in serum-free media for 48 h. Subsequently, their cell lysates were subjected to western blotting analyses for detecting the cleavage of PARP and the protein amounts of Bcl-2 and Bax using b-actin as an internal control. The active PARP (116 kDa) together with the larger fragment of cleaved PAPR (89 kDa) and the calculated Bcl/Bax ratio from each treatment are indicated. Activations of JAK2, PI3K/Akt, and MEK/ERK1/2 are essential for mediating leptin inhibition on TGF-b1-induced Bax expression and resulting Hep3B apoptosis.

Article Snippet: Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-b-actin antibody (Sigma Chemical Co.), TGF-b (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and www.endocrinology-journals.org monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.

Techniques: Western Blot, Control, Inhibition, Expressing

Journal: Advances in Bioscience and Biotechnology

Article Title: CXCL10 Induces Lytic Reactivation of EBV through <i>EXTL</i>1

doi: 10.4236/abb.2024.1510039

Figure Lengend Snippet: Figure 3. Activation of the JAK2/STAT3 signaling pathway in CXCL10-induced EBV lytic reactivation is inhibited by JAK2 inhib- itor ruxolitinib

Article Snippet: Recombinant human CXCL10 (IP-10) was purchased from PeproTech (Catalog #: 300-12); CXCR3 inhibitor AMG487 was acquired from MCE (Catalog #: HY-15319); antibodies against BZLF1 and glycoprotein (gp) 350 were obtained from Santa Cruz (Catalog #: sc-53904, sc-56981); antibodies against BRLF1 were sourced from Boster Bio (Catalog #: bs-4542R); antibodies against Extl1 were procured from ImmunoWay Biotechnology (Catalog #: YN5348); antibodies against phosphorylated Janus kinase 2 (p-JAK2) and phosphorylated Signal Transducer and Activator of Transcription 3 (p-STAT3) were purchased from CST (Catalog #: 4406T; Catalog #: 9145T); antibodies against JAK2 were obtained from Affinity (Catalog #: AF6002); antibodies against STAT3 were sourced from Proteintech (Catalog #: 60199-1-Ig); JAK2/STAT3 inhibitor ruxolitinib was acquired from MCE (Catalog #: HY-50856); whole protein extraction kits were purchased from KeyGen Biotech (Catalog #: KGP250); RNA extraction kits were obtained from Shanghai Yishan Biotech (Catalog #: RN001).

Techniques: Activation Assay, Inhibition