Journal:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞

doi: 10.1074/jbc.M808879200

Figure Lengend Snippet: AG490 inhibited constitutive activation of JAK2 L611S mutant. A and B, transduced BaF3 cells were treated with AG490 (AG; 30 μm) in the absence or presence of Epo (5 units/ml) for 24 h. A, cell lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody and immunoblotted (IB) with antibodies to anti-phospho (P)-Tyr1007/1008 JAK2 or anti-HA antibody (upper). B, cell lysates were immunoblotted with anti-phospho-Thr202/Tyr204 ERK antibody, anti-ERK antibody, anti-phospho-Ser473 Akt antibody, anti-Akt antibody, anti-phospho-Tyr694 Stat5 antibody, or anti-Stat5 antibody. C and D, transduced BaF3 cells with empty virus (MSCV), WT, or JAK2 mutant (L611S) were infected with GFP as a control or constitutively active mutant of Stat5A (1*6). C, cell lysates were immunoblotted with anti-Stat5 antibody or anti-β-actin antibody. D, transduced BaF3 cells were treated with AG490 (30 μm) in the absence or presence of Epo (5 units/ml) for 24 h. The viability of these cells was determined by the trypan blue exclusion method. Results represent the mean ± S.D. of three independent experiments.

Article Snippet: AG490 was purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Activation Assay, Mutagenesis, Immunoprecipitation, Virus, Infection, Control

Journal:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞

doi: 10.1074/jbc.M808879200

Figure Lengend Snippet: AG490 induced apoptosis in cells expressing JAK2 L611S mutant. Transduced BaF3 cells were treated with AG490 (AG; 30 μm) in the absence or presence of Epo (5 units/ml) for 24 h. A, cells were fixed, treated with propidium iodide, and subjected to fluorescence-activated cell sorting analysis as described under “Experimental Procedures.” B, caspase-3 activity was measured by the cleavage of substrate, Ac-DEVD-7-amino-4-methycoumarin. Results represent the mean ± S.D. of three independent experiments. C, DNA was isolated from cells and subjected to agarose gel electrophoresis. D, XIAP, cIAP, and Bcl-XL mRNAs were determined by reverse transcription-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. E, whole cell lysates were immunoblotted (IB) with anti-XIAP antibody, anti-cIAP1 antibody, anti-Bcl-XL antibody, or anti-β-actin antibody.

Article Snippet: AG490 was purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Expressing, Mutagenesis, Fluorescence, FACS, Activity Assay, Isolation, Agarose Gel Electrophoresis, Reverse Transcription, Control

Journal:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞

doi: 10.1074/jbc.M808879200

Figure Lengend Snippet: AG490 inhibited JAK2 L611S mutant-induced tumor formation in nude mice. 1 × 107 BaF3 cells infected with empty virus (MSCV) or JAK2 mutant (L611S) were subcutaneously injected into nude mice, and the mice were treated with intraperitoneal injection of DMSO or AG490 (AG; 500 μg/day) daily for 10 days. A, nude mice were photographed 14 days postinoculation with BaF3 cells. The arrows indicate tumors in nude mice. B, a total of eight nude mice were injected with transduced BaF3 cells and treated with DMSO or AG490 (500 μg/day) daily for 10 days. For 30 days after inoculation, mouse survival was monitored daily. C, 14 days postinoculation with BaF3 cells, mice were sacrificed, and the morphological changes of liver, spleen, and lymph node were photographed. D, 14 days postinoculation, tumor, liver, spleen, and lymph node were weighed and graphed. * and ** indicate significant difference p < 0.01 and p < 0.005, respectively.

Article Snippet: AG490 was purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Mutagenesis, Infection, Virus, Injection

Journal:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞

doi: 10.1074/jbc.M808879200

Figure Lengend Snippet: AG490 inhibited JAK2 L611S mutant-induced cell penetration and invasion. 1 × 107 BaF3 cells infected with empty virus (MSCV) or JAK2 mutant (L611S) were subcutaneously injected into nude mice and treated with intraperitoneal injection of DMSO or AG490 (AG; 500 μg/day) daily for 10 days. A, 14 days postinoculation, hepatocytes and splenocytes were prepared from injected nude mice. GFP-positive cells in hepatocytes and splenocytes were monitored by flow cytometry analysis. B, 14 days postinoculation with BaF3 cells, the sections of liver and spleen were stained with hematoxylin and eosin (magnification: ×100, left; ×400, right).

Article Snippet: AG490 was purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Mutagenesis, Infection, Virus, Injection, Flow Cytometry, Staining

Journal: Scientific reports

Article Title: Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells.

doi: 10.1038/s41598-017-02056-w

Figure Lengend Snippet: Figure 2. IL-22 attenuated the ALI induced by AngII. (A) The incidence of lung injury showed remarkable decrease after IL-22 treatment. (B) In the ALI model induced by AngII, obvious oedema was noticed, together with massive infiltration of neutrophils (MPO) and macrophages (CD68), which was significantly reversed after IL-22 treatment. (C) IL22 could significantly attenuate the lung injury as revealed by pathological changes, but such phenomenon was completely reserved after AG490. HE staining and immunohistochemistry images were observed under a magnification of 200× and 400×, respectively. *P < 0.05 versus control group. #P < 0.05 versus AngII group and AngII+IL-22+AG490 group.

Article Snippet: Besides, AG490 (10 mg/ Kg, sc-202046, Santa Cruz) was given to inhibit the activity of JAK2 every two days via peritoneal injection.

Techniques: Staining, Immunohistochemistry, Control

Journal: Scientific reports

Article Title: Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells.

doi: 10.1038/s41598-017-02056-w

Figure Lengend Snippet: Figure 3. IL22 inhibited the apoptosis of PMVECs induced by AngII. (A) IL22 could attenuate the apoptosis of PMVECs significantly as revealed by TUNEL assay, while such phenomenon was completely inhibited after AG490. (B) Flow cytometry indicated the apoptosis rate of the PMVECs in the AngII+IL22 group was significantly decreased compared with the AngII group. The apoptosis rate in the AngII+IL22+AG490 group was significantly elevated compared with that of AngII+IL22 group. The raw data were listed in the Supplementary file. (C) The expression of Bcl-2 in the PMVECs significantly decreased after AngII stimulation, while the expression of Bcl-2 in the AngII+IL22 group was higher than that of AngII group. *P < 0.05 versus control group. #P < 0.05 versus AngII group and AngII+IL-22+AG490 group. Immunofluorescence images were observed under a magnification of 200×.

Article Snippet: Besides, AG490 (10 mg/ Kg, sc-202046, Santa Cruz) was given to inhibit the activity of JAK2 every two days via peritoneal injection.

Techniques: TUNEL Assay, Flow Cytometry, Expressing, Control, Immunofluorescence

Journal: Scientific reports

Article Title: Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells.

doi: 10.1038/s41598-017-02056-w

Figure Lengend Snippet: Figure 5. IL22 contributed to the expression of STAT3 in the PMVECs. (A) IL22 induced up-regulation of STAT3 in a time-dependent manner, and reached the peak level at 48 h. (B) The expression of STAT3 in the AngII+IL22 group was higher than the other groups. The raw data were listed in the Supplementary file. *P < 0.05 versus control group; #P < 0.05 versus AngII group and AngII+IL-22+AG490 group.

Article Snippet: Besides, AG490 (10 mg/ Kg, sc-202046, Santa Cruz) was given to inhibit the activity of JAK2 every two days via peritoneal injection.

Techniques: Expressing, Control

Journal: Scientific reports

Article Title: Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells.

doi: 10.1038/s41598-017-02056-w

Figure Lengend Snippet: Figure 4. IL22 contributed to the expression of STAT3 in the lung tissues in mice. Immunohistochemistry (A, B) indicated IL22 induced up-regulation of STAT3 in the lung tissues, but such fact was inhibited after AG490. The raw data were listed in the Supplementary file. (C) Western blot analysis indicated the expression of STAT3 in the lung tissues in the AngII+IL22 group was higher than the other groups. *P < 0.05 versus control group; #P < 0.05 versus AngII group and AngII+IL-22+AG490 group. The images of immunohistochemistry were observed under a magnification of 400× and 1000×, respectively.

Article Snippet: Besides, AG490 (10 mg/ Kg, sc-202046, Santa Cruz) was given to inhibit the activity of JAK2 every two days via peritoneal injection.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Scientific reports

Article Title: Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells.

doi: 10.1038/s41598-017-02056-w

Figure Lengend Snippet: Figure 6. IL-22 significantly stimulated phosphorylation at tyrosine 705 of STAT3 in PMVECs. (A) The expression of pY705-STAT3 was up-regulated after IL22 interference, and reached the peak level at 30 min. (B) Immunofluorescence analysis indicated the expression of pY705-STAT3 significantly increased together with accumulation in the nucleus after IL22 interference. (C) The expression of pY705-STAT3 in the AngII+IL22 group was higher than the other groups. The raw data of Western blot were listed in the Supplementary file. *P < 0.05 versus control group; #P < 0.05 versus AngII group and AngII+IL-22+AG490 group.

Article Snippet: Besides, AG490 (10 mg/ Kg, sc-202046, Santa Cruz) was given to inhibit the activity of JAK2 every two days via peritoneal injection.

Techniques: Phospho-proteomics, Expressing, Immunofluorescence, Western Blot, Control

Journal: Scientific reports

Article Title: Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells.

doi: 10.1038/s41598-017-02056-w

Figure Lengend Snippet: Figure 7. IL-22 significantly stimulated nuclear STAT3 expression in PMVECs. (A) STAT3 was mainly localized in the cytoplasm in the control group and AngII group. The expression of STAT3 in the nucleus in the AngII+IL22 group significantly increased, while its expression in the nucleus significantly decreased in the AngII+IL22+AG490 group. (B) Western blot analysis indicated the STAT3 in the nucleus in the AngII+IL22 group was significantly higher than the other groups. The raw data were listed in the Supplementary file. *P < 0.05 versus control group; #P < 0.05 versus AngII+IL-22+AG490 group.

Article Snippet: Besides, AG490 (10 mg/ Kg, sc-202046, Santa Cruz) was given to inhibit the activity of JAK2 every two days via peritoneal injection.

Techniques: Expressing, Control, Western Blot

Journal: Frontiers in Nutrition

Article Title: Central Nervous System Inflammation Induced by Lipopolysaccharide Up-Regulates Hepatic Hepcidin Expression by Activating the IL-6/JAK2/STAT3 Pathway in Mice

doi: 10.3389/fnut.2021.649640

Figure Lengend Snippet: AG490 significantly reduced p-STAT3 protein expression and eliminated the changes in Fpn1 and Ft-L expression induced by lipopolysaccharide (LPS) in the liver. C57BL/6 wild-type male mice were pre-treated with AG490 (5 mg/kg, IP injection) in 10% dimethyl sulfoxide (DMSO) or vehicle 30 min before LPS (ICV injection, 5 μg in 2 μL of sterile saline). The expression levels of p-STAT3 (A) , Fpn1 (B) , and Ft-L proteins (C) in the liver were measured using western blot. Data are presented as means ± SEM (% control) ( n = 4). For statistical analysis, one-way ANOVA with Tukey's post hoc test was performed.

Article Snippet: The JAK2 inhibitor AG490 was purchased from Selleckchem (Houston, TX, USA); the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Injection, Sterility, Saline, Western Blot, Control

Journal: BMC Complementary Medicine and Therapies

Article Title: Potentilla reptans L. postconditioning protects reperfusion injury via the RISK/SAFE pathways in an isolated rat heart

doi: 10.1186/s12906-021-03456-2

Figure Lengend Snippet: Experimental protocols. All experimental groups were first perfused for 30 mins on the Langendorff apparatus to allow the isolated hearts to stabilize. The hearts were then divided into different groups. All groups were subjected to 30 mins of regional ischemia followed by 100 mins of reperfusion. IR; Ischemia-reperfusion, IPOST; Ischemic postconditioning (3 × 10 s ischemia and reperfusion at the onset of reperfusion period), Etpost; ethyl acetate fraction from Potentilla reptans root (2 μg/ml) was applied at the onset of reperfusion, L-NAME; NO inhibitor (50 μM), Wort; Wortmannin a PI3K/AKT inhibitor (400 nM), PD; PD98059 (2′-Amino-3′-methoxyflavone) an ERK1/2 inhibitor (400 nM), AG; tyrphostin (AG490, 400 nM) a JAK/STAT3 inhibitor, HD; 5-hydroxy decanoate (5HD,1 μM) a mitoKATP channel blocker

Article Snippet: Wortmannin (Wort; the PI3k/Akt inhibitor), PD98059 (PD; the ERK1/2 inhibitor), Tyrphostin AG490 (the JAK/STAT3 inhibitor), and 5-hydroxy decanoate (a mitoKATP channel blocker) were purchased from “Santa Cruz Biotech, Dallas, Texas, USA”.

Techniques: Isolation

Journal: BMC Complementary Medicine and Therapies

Article Title: Potentilla reptans L. postconditioning protects reperfusion injury via the RISK/SAFE pathways in an isolated rat heart

doi: 10.1186/s12906-021-03456-2

Figure Lengend Snippet: Western blot analysis. A Western blot analysis shows the relative intensity of AKT (Tyr473) phosphorylation in heart tissue of IR, Etpost (2 μg/ml) and Etpost+Wort groups that were adjusted relative to GAPDH. B Western blot analysis illustrates the relative intensity of ERK1/2 (Thr183/185) phosphorylation in heart tissue of IR, Etpost (2 μg/ml) and Etpost+PD groups that were adjusted relative to GAPDH. C Western blot analysis displays the relative intensity of STAT3 (Tyr705) phosphorylation in heart tissue of IR, Etpost (2 μg/ml) and Etpost+AG groups that were adjusted relative to GAPDH. The data were expressed as mean ± SEM. * P < 0.05 vs. IR group and # P < 0.05 vs. Etpost (2 μg/ml). Etpost: ethyl acetate fraction of P. reptans root; Wort: Wortmanin (PI3K/Akt inhibitor); PD: PD98059 (ERK inhibitor); AG: AG490 (JAK/STAT3 inhibitor)

Article Snippet: Wortmannin (Wort; the PI3k/Akt inhibitor), PD98059 (PD; the ERK1/2 inhibitor), Tyrphostin AG490 (the JAK/STAT3 inhibitor), and 5-hydroxy decanoate (a mitoKATP channel blocker) were purchased from “Santa Cruz Biotech, Dallas, Texas, USA”.

Techniques: Western Blot, Phospho-proteomics

Journal: Biochimica et biophysica acta

Article Title: Prolactin and glucocorticoid signaling induces lactation-specific tight junctions concurrent with β-casein expression in mammary epithelial cells.

doi: 10.1016/j.bbamcr.2016.04.023

Figure Lengend Snippet: Fig. 4. Prl and Dex influence the expression patterns of Cldn3 and Cldn4 in MECs (A) Immunostaining images of Cldn3 (green) and Cldn4 (green) with Ocln (red) in MECs cultured with or without Prl (0.5 U/mL) and Dex (1 μM) for 5 days. Nuclei stained with DAPI are blue. The scale bars represent 10 μm. (B–F) The results of western blotting and densitometry analysis of Cldn3 and Cldn4 with β-actin used as an internal control. MECs were cultured for 5 days with or without Prl (0.5 U/mL or 0–2.5 U/mL) and Dex (1 μM). MECs were pretreated with 10 μM AG490 (JAK inhibitor) and 5 μM RU486 (glucocorticoid receptor antagonist) 1 h before Prl (0.5 U/mL) and Dex (100 nM) treatment. The data are presented as the mean (SEM); n = 5; *p b 0.05 and **p b 0.005. (G) The expression of Cldn3 and Cldn4, determined by qRT-PCR. The data are presented as the mean (SEM); n = 6; *p b 0.05 and **p b 0.005.

Article Snippet: AG490 (JAK inhibitor; cat. no. T9969) and RU486 (GR antagonist; cat. no. M3321)were purchased from LKT Laboratories, Inc. (St. Paul, MN).

Techniques: Expressing, Immunostaining, Cell Culture, Staining, Western Blot, Control, Quantitative RT-PCR

Journal:

Article Title: Signalling events involved in interferon-?-inducible macrophage nitric oxide generation

doi: 10.1046/j.1365-2567.2003.01620.x

Figure Lengend Snippet: Effect of specific inhibitors of Janus kinase 2 (JAK2), extracellular signal-regulated kinase 1 and 2 (Erk1/Erk2) and MAPK kinase 1 and 2 (MEK1/2) on interferon-γ (IFN-γ)-induced macrophage nitric oxide (NO) generation, and inducible nitric oxide synthase (iNOS) gene and protein expression. Cells were treated with increasing doses of AG-490 (JAK2 inhibitor) (a), apigenin (Erk1/Erk2 inhibitor) (b) or PD 98059 (MEK1/2 inhibitor) (c) for 1 hr prior to stimulation with IFN-γ (100 U/ml). After 24 hr of incubation, supernatants were collected and submitted to the Greiss reaction to evaluate nitrite generation (mean ± SEM, n = 3). (d) to (f) iNOS regulation has been monitored in cells pretreated, as described above, with specific inhibitors prior to stimulation with IFN-γ for 8 hr (iNOS gene expression) or 24 hr (iNOS protein expression). After incubation, either total RNA or total proteins were extracted, and used for Northern and Western blots, respectively. These results are representative of one of three experiments performed independently. GAPDH, glyceraldehyde 3-phosphate dehydrogenase probe used to confirm equal RNA loading.

Article Snippet: The JAK2 inhibitor, AG-490, and the NF-κB inhibitors, CAPE and BAY 11–7082, were purchased from Biomol (Plymouth Meeting, PA).

Techniques: Expressing, Incubation, Gene Expression, Northern Blot, Western Blot

Journal:

Article Title: Signalling events involved in interferon-?-inducible macrophage nitric oxide generation

doi: 10.1046/j.1365-2567.2003.01620.x

Figure Lengend Snippet: Effect of specific second-messenger inhibitors on interferon-γ (IFN-γ)-induced signal transducer and activator of transcription 1α (STAT1α) binding to the murine inducible nitric oxide synthase (iNOS) promoter. J774 macrophages were treated with specific inhibitors against extracellular signal-regulated kinase 1 and 2 (Erk1/Erk2) (50 µm of apigenin, lanes 3 and 4), Janus kinase 2 (JAK2) (75 µm of AG-490, lanes 5 and 6) and MEK1/2 (50 µm of PD 98059, lanes 7 and 8) for 1 hr prior to IFN-γ stimulation for 15 min. The last lane represents nuclear extracts preincubated with unlabelled oligonucleotide (100 ×) for band specificity evaluation. This result is representative of one of three different experiments.

Article Snippet: The JAK2 inhibitor, AG-490, and the NF-κB inhibitors, CAPE and BAY 11–7082, were purchased from Biomol (Plymouth Meeting, PA).

Techniques: Binding Assay

Journal:

Article Title: Signalling events involved in interferon-?-inducible macrophage nitric oxide generation

doi: 10.1046/j.1365-2567.2003.01620.x

Figure Lengend Snippet: Time-dependent activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1α (STAT1α) and extracellular signal-regulated kinase 1 and 2 (Erk1/Erk2) by interferon-γ (IFN-γ) in J774 macrophages: effect of signalling inhibitors on their phosphorylation states. Cells were treated with IFN-γ (100 U/ml) for different time-periods (0–60 min) and cell lysates were subjected to immunoblot analysis by using antibodies specific for the phosphorylated forms of Janus kinase 2 (JAK2) (a); STAT1α (b) and (c); and Erk1/Erk2 (d). (e) To monitor IFN-γ-dependent Erk1/Erk2, JAK2 and STAT1α activation in the presence of apigenin, AG-490 or PD 98059, cells were treated with maximal doses of these specific inhibitors for 1 hr prior to stimulation with IFN-γ (for 15 min). Cell lysates were then subjected to Western blotting and incubated with the same antibodies mentioned above (for panels a–d). Equal protein loading was verified by using anti-JAK2, anti-STAT1α and anti-Erk1/Erk2 specific antibodies. Results are representative of three experiments performed independently.

Article Snippet: The JAK2 inhibitor, AG-490, and the NF-κB inhibitors, CAPE and BAY 11–7082, were purchased from Biomol (Plymouth Meeting, PA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation