Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

doi: 10.1186/s13046-019-1277-x

Figure Lengend Snippet: High EREG expression promotes NF-CAF transition through the JAK2-STAT3 pathway. a & b , Representative images and quantitative analysis of western blotting showing that EREG overexpression in NFs and EREG interference in CAFs were successful. Moreover, the expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression and downregulated after EREG interference. GAPDH was used as a loading control. c & d & e , Both phosphorylated forms of JAK2 and STAT3 and the p-JAK2/JAK2 and p-STAT3/STAT3 ratios were significantly augmented after EREG overexpression. The change in expression of major participants of other pathways after EREG overexpression was not as significant. GAPDH was used as a loading control. f & g & h , Representative images and quantification of western blotting showing that expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression but was reduced after treatment with the JAK2 inhibitor AG490, indicating that AG490 antagonizes EREG-mediated NF activation. CAF markers, as well as (p-)JAK2 and (p-)STAT3 expression and p-JAK2/JAK2 and p-STAT3/STAT3 ratios decreased after EREG interference in CAFs but were restored by IL-6 treatment. GAPDH was used as a loading control. i , ELISA showing the IL-6 level in the CM of NFs/CAFs after the indicated treatment. J&K, Representative images and quantification of immunohistochemical staining in clinical samples revealed that high EREG expression was correlated with high phospho-JAK2 and phospho-STAT3 expression, while low EREG expression was correlated with low phospho-JAK2 and phospho-STAT3 expression. Magnification: 200×. *: p < 0.05, **: p < 0.01, ***: p < 0.001

Article Snippet: Western blots were performed using an SDS–PAGE electrophoresis system as described previously [ ], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies.

Techniques: Expressing, Western Blot, Over Expression, Control, Activation Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

doi: 10.1186/s13046-019-1277-x

Figure Lengend Snippet: High EREG expression in fibroblasts promotes tumor growth in vivo. a , Photographs of tumor formation in nude mice and tumor xenografts 4 weeks after inoculation. b & c , Tumor growth curves measured after injection of HSC3 cells or conditioned fibroblasts as indicated. The tumor volume was calculated every 7 days until termination. d - g , RT-PCR analysis of Ereg, Jak2, Stat3 and Il6 expression in tissues of resected tumors, revealing successful overexpression/interference of Ereg and associated upregulation or downregulation of Jak2, Stat3 and Il6 . h , Immunohistochemical staining showed that tumors that developed from EREG-overexpressing NFs had a higher level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors that developed from control NFs. Tumors that developed from siEREG-transfected CAFs showed a lower level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors developed from siNC-transfected CAFs. S: stroma, T: tumor. Magnification: 200×. i & j , Western blotting showed that CAFs significantly promote EMT in vivo compared with NFs, with decreased E-cadherin expression and increased N-cadherin and vimentin expression. This EMT-promoting ability was attenuated after EREG knockdown. On the other hand, EREG overexpression in NFs gave NFs a CAF-like EMT-promoting ability. k , Increased EREG expression in NFs led to acquisition of the CAF phenotype in a JAK2-STAT3-dependent way and supported OSCC invasion through the promotion of EMT in tumor cells

Article Snippet: Western blots were performed using an SDS–PAGE electrophoresis system as described previously [ ], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies.

Techniques: Expressing, In Vivo, Injection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Immunohistochemical staining, Staining, Control, Transfection, Western Blot, Knockdown

Journal: Journal of Pain Research

Article Title: Electroacupuncture treatment upregulates α7nAChR and inhibits JAK2/STAT3 in dorsal root ganglion of rat with spared nerve injury

doi: 10.2147/JPR.S203867

Figure Lengend Snippet: Effect of 2 Hz EA on JAK2 and STAT3 protein expression in DRG of SNI rat. Notes: ( A ) Western blotted band of p-JAK2 and JAK2. ( B ) Relative level of p-JAK2/JAK2 protein expression. ( C ) Western blotted band of p-STAT3 and STAT3. ( D ) Relative level of p-STAT3/STAT3 protein expression. Samples were collected from the L4–L6 segment of DRG on day 3, 7, 14 and 21 after surgery. * P <0.05, *** P <0.001, compared with Sham group, # P <0.05, ## P <0.01, ### P <0.001, compared with SNI group. All data were expressed as the mean ± SEM, n=4–5 per group. One-way ANOVA followed by Newman–Keuls post-hoc test. Abbreviations: EA, electroacupuncture; SNI, spared nerve injury; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; DRG, dorsal root ganglion; d, day; SEM, standard error of the mean.

Article Snippet: The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 60 mins at room temperature, and subsequently the membrane respectively immuno-labelled overnight at 4°C with antibodies of rabbit anti-α7nAChR (1:200, ab10096, Abcam, UK), anti-phospho-STAT3 (Y785) XP ® Rabbit mAb (1:200, D3A7, CST, UK), anti-STAT3 mAb (1:400, 124H6, CST, UK), anti-phospho-JAK2 (Tyr1007/1008) (1:200, 3771, CST, UK) or anti-JAK2 (D2E12) XP ® Rabbit mAb (1:400, 3230, CST, UK), anti-IL-1β (1:200, ab6722, Abcam, UK), anti-IL-6 (1:200, ab6722, Abcam, UK), anti-IL-10 (1:200, SC-365858, Santa Cruz,UK), β-actin (1:1,000, Solarbio, China) or β-tubulin (1:1,000, Solarbio, China).

Techniques: Expressing, Western Blot

Journal: Annals of Translational Medicine

Article Title: Polydatin down-regulates the phosphorylation level of STAT3 and induces pyroptosis in triple-negative breast cancer mice with a high-fat diet

doi: 10.21037/atm-22-73

Figure Lengend Snippet: The impacts of polydatin treatments on JAK2 and STAT3 expression levels. (A) STAT3 and JAK2 mRNA expressions in breast cancer tissues treated with 4T1, 4T1 + polydatin, 4T1 + polydatin + fat diet, and 4T1 + fat diet. (B) STAT3, JAK2, phospho-STAT3, and phospho-JAK2 protein expressions in BC tissues treated with 4T1, 4T1 + polydatin, 4T1 + polydatin + fat diet, and 4T1 + fat diet. All blots were subjected to scanning densitometry in triplicate, and the integrated optical density of each band was standardized to that of GAPDH in the same blot. *P<0.05, **P<0.01 was considered statistically significant. mRNA, messenger RNA; BC, breast cancer.

Article Snippet: The following primary antibodies were used: Rabbit anti-STAT3 antibody (1:1,000, Abcam, Cambridge, USA), Rabbit anti-JAK2 antibody [1:1,000, Cell Signaling Technology (CST), Danvers, MA, USA], rabbit anti-Phospho-STAT3 antibody (1:500, CST, USA), rabbit anti-IL1β antibody (1:500, Abcam, USA), rabbit anti-Phospho-JAK2 antibody (1:500, CST, USA), rabbit anti-pro Caspase-1 + p10 + p12 antibody (1:500, Abcam, USA), rabbit anti-Cleaved Caspase-3 antibody (1:1,000, CST, USA), rabbit anti-NLRP3 antibody (1:800, Abcam, USA), rabbit anti-IL18 antibody (1:500, Abcam, USA), rabbit anti-Caspase-3 antibody (1:1,000, CST, USA), rabbit anti-Caspase-1 antibody (1:500, Abcam, USA), rabbit anti-GSDMD antibody (1:500, Abcam, USA), rabbit anti-GAPDH antibody (1:3,000, Abcam, USA), and mouse β-actin monoclonal antibody (1:3,000, Abcam, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PARP Inhibitor Upregulates PD-L1 Expression and Provides a New Combination Therapy in Pancreatic Cancer

doi: 10.3389/fimmu.2021.762989

Figure Lengend Snippet: Pamiparib treatment induces PD-L1 expression via JAK2/STAT3 pathway. (A) Cells were pretreated with pamiparib (100 μM, 12h) and PD-L1 expression was assessed by immunoblotting after treatment with the concentrations (20 μM) of AG490 for 24h. (B) Cells were pretreated with pamiparib (100 μM, 12h) and PD-L1 expression was assessed by protein blotting after treatment with stattic (20 μM) for 24h. (C) Protein expression of phospho-JAK2 (p-JAK2), JAK2, and PD-L1 in SW1990 cells after being treated with pamiparib (100 μM) for the indicated times. (D) Protein expression of phospho-STAT3(p-STAT3), STAT3 and PD-L1 in SW1990 cells after being treated with pamiparib (100 μM) for the indicated times. GAPDH was used as a loading control. (E) IHC staining of p-STAT3 of nude mouse xenograft tumors in control group and treated with pamiparib group. Scale bar, 100 μm. (F) IHC staining score showed the expression of p-STAT3 was significantly upregulated in nude mice pamiparib treated group. Data are mean ± SD; n = 3 samples per group. The IHC results were analyzed by Pearson χ2 test. * P < 0.05.

Article Snippet: The primary antibodies are PD-L1 (CST #13684, Cell Signaling Technology), PD-L1 (17952- 1-AP, Santa Cruz), STAT3 (CST #9139, Cell Signaling Technology), phosphorylated STAT3 (CST #9145, Cell Signaling Technology), JAK2 (17670-1-AP, Proteintech), phosphorylated JAK2 (CST #4406, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), phosphorylated AKT (66444-1-Ig, Proteintech), ERK (CST #4696, Cell Signaling Technology), phosphorylated ERK (CST #3510, Cell Signaling Technology), PARP-1 (sc-8007, Santa Cruz), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Control, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: PARP Inhibitor Upregulates PD-L1 Expression and Provides a New Combination Therapy in Pancreatic Cancer

doi: 10.3389/fimmu.2021.762989

Figure Lengend Snippet: Diagram summarizing that pamiparib treatment induces PD-L1 expression mainly via JAK2/STAT3 in pancreatic cancer (details provided in the Discussion section).

Article Snippet: The primary antibodies are PD-L1 (CST #13684, Cell Signaling Technology), PD-L1 (17952- 1-AP, Santa Cruz), STAT3 (CST #9139, Cell Signaling Technology), phosphorylated STAT3 (CST #9145, Cell Signaling Technology), JAK2 (17670-1-AP, Proteintech), phosphorylated JAK2 (CST #4406, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), phosphorylated AKT (66444-1-Ig, Proteintech), ERK (CST #4696, Cell Signaling Technology), phosphorylated ERK (CST #3510, Cell Signaling Technology), PARP-1 (sc-8007, Santa Cruz), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage neogenin deficiency exacerbates myocardial remodeling and inflammation after acute myocardial infarction through JAK1-STAT1 signaling

doi: 10.1007/s00018-023-04974-7

Figure Lengend Snippet: Neogenin (Neo1) deficiency promoted macrophage transformation to the proinflammatory phenotype through the janus kinase 1 (JAK1)-signal transduction and activator of transcription 1 (STAT1) signal pathway, not STAT6 and P65 pathways, a The protein bands of p/T-JAK1, p/T-JAK2, p/T-STAT1, p/T-P65, p/T-STAT6 and GAPDH 3 days after myocardial infarction (MI), n = 5 in each group; b-f Quantitative analysis of p-JAK1, p-JAK2, p-STAT1, p-P65 and p-STAT6 in each group 3 days after MI, n = 5, two way ANOVA followed by Tukey’s test; g The double-labelling immunofluorescence of p-STAT1 and F4/80 3 days after MI, n = 4 in each group; h The protein bands of p/T-JAK1 and p/T-STAT1 3 days after MI in Neo1F/F and Neo1F/F;Cx3cr1cre mice; i, j Quantitative analysis of p-JAK1 and p-STAT1 in each group 3 days after MI in Neo1F/F and Neo1F/F;Cx3cr1cre mice, n = 5, two way ANOVA followed by Tukey’s test; k The protein bands and quantitative analysis of p/T-JAK1 and p/T-STAT1 in bone marrow derived-macrophages (BMDMs) 12 h after stimulation with LPS + IFN-γ, n = 3 in each group, two way ANOVA followed by Tukey’s test; l The immunofluorescence of p-STAT1 in BMDMs was analyzed by a confocal laser scanning microscope, n = 3 in each group. Data are expressed as the mean ± SEM. In b-f, *p < 0.05 vs. Sham + IgG group, #p < 0.05 vs. MI + IgG group, n.s. indicating no significance; in i and j *p < 0.05 vs. Sham + Neo1F/F group, #p < 0.05 vs. MI + Neo1F/F group; in k, *p < 0.05 vs. Sham + IgG group, #p < 0.05 vs. LPS + IFN-γ + IgG group

Article Snippet: Primary antibodies included: Neo1 (R&D system), collagen I (Servicebio), Bax (Abcam), Bcl-2 (Abcam), cleaved-caspase3 (GeneTex), phospho (p)/total (T)-P38 (CST), p/T-ERK (CST), p/T-JNK (CST), p-janus kinase 1 (JAK1) (CST), T-JAK1 (Abcam), p-JAK2 (CST), T-JAK2 (Abcam), p-signal transduction and activator of transcription 1 (STAT1) (Abcam), T-STAT1 (CST), p/T-P65 (Abcam), p/T-STAT6 (CST), GAPDH (GeneTex), Chemerin receptor 23 (ChemR23) (Santa Cruz), formyl-peptide receptor-2 (FPR2) (Santa Cruz), and G protein-coupled receptor 37 (GPR37) (Santa Cruz).

Techniques: Transformation Assay, Transduction, Immunofluorescence, Derivative Assay, Laser-Scanning Microscopy