Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control

Journal: International Journal of Oncology

Article Title: Combined inhibition of IL-6 and IL-8 pathways suppresses ovarian cancer cell viability and migration and tumor growth

doi: 10.3892/ijo.2022.5340

Figure Lengend Snippet: Baze and SCH inhibit the expression of target genes downstream of the IL-6 and IL-8 pathways in human ovarian cancer cells. SKOV3 and CAOV3 cells were treated with DMSO, a single drug or their combination. The levels of p-STAT3, p-AKT, p-S6 and survivin were determined by western blot analysis. (A) CAOV3. (B) SKOV3. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 (DMSO vs. Baze, SCH or Baze + SCH). B+S, Baze + SCH; B10, BAZE 10 µ M; B5, BAZE 5 µ M; BAZE, Baze; S50, SCH 50 µ M; p-phosphorylated; SCH, SCH.

Article Snippet: The following primary antibodies were purchased from Cell Signaling Technology, Inc. and diluted at 1:1,000 in 5% milk: Phosphorylated (p)-STAT3 (Y705; rabbit mAb; cat. no. 9131S), STAT3 (rabbit mAb; cat. no. 4904S), p-S6 (rabbit mAb; cat. no. 4858S), S6 (rabbit mAb; cat. no. 2217S), p-AKT (rabbit mAb; cat. no. 4060S), AKT (rabbit mAb; cat. no. 4691S), survivin (rabbit mAb; cat. no. 2808S) and GAPDH (rabbit mAb; cat. no. 2118S).

Techniques: Expressing, Western Blot

Journal: BMJ Open Diabetes Research & Care

Article Title: Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

doi: 10.1136/bmjdrc-2020-001242

Figure Lengend Snippet: JAK/STAT, inflammatory and oxidative stress pathways in kidney tissue of BTBR ob/ob model. (A) Representative images of phosphorylated (p-)STAT3, p-p65 NF-κB and p-NRF2, and quantification of positive cells in glomerular and tubular fields of BTBR WT and ob/ob mice. Magnification ×400 and ×630. Arrows indicate positive stained cells. (B) Representative images of SOCS1/SOCS3 proteins and quantification of positive stained area per tubular field. Magnification ×200. (C) Real-time PCR analysis of JAK/STAT, inflammatory and oxidative stress genes. Values normalized by endogenous control gene 18s are expressed as n-fold of the average value from BTBR WT. Data are shown as scatter dot plots and mean±SD of each group (n=5–7 mice/group); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus BTBR WT control. (D) Schematic protein–protein interaction prediction of JAK/STAT, inflammatory and oxidative stress markers in Mus musculus according to STRING software. More information can be found at https://string-db.org/ . a.u., arbitrary units; BTBR, black and tan brachyuric; GCS, glomerular cross section; JAK/STAT, Janus kinase/signal transducers and activators of transcription; NF-κB, nuclear factor-κB; NRF2, nuclear factor erythroid 2-related factor 2; ob/ob, obese/obese; SOCS, suppressor of cytokine signaling; WT, wild type.

Article Snippet: The primary antibodies for immunodetection were sourced as follows: phosphorylated (p-) STAT3 serine 727 (Cell Signaling Technology Cat# 9134, RRID:AB_331589, dilution 1:100), p-STAT1 tyrosine 701 (Cell Signaling Technology Cat# 7649, RRID:AB_10950970, dilution 1:50), p-p65 subunit of nuclear factor-κB (NF-κB) serine 536 (Santa Cruz Biotechnology Cat# sc-33020, RRID:AB_2179018, dilution 1:100), p-nuclear factor erythroid 2-related factor 2 (NRF2) serine 40 (Abcam Cat# ab76026, RRID:AB_1524049, dilution 1:2000), SOCS1 (Abcam Cat# ab62584, RRID:AB_956316, dilution 1:1000), SOCS3 (Abcam Cat# ab16030, RRID:AB_443287, dilution 1:200), F4/80 monocytes/macrophages (Bio-Rad Cat# MCA497, RRID:AB_2098196, dilution 1:70), CD3 T lymphocytes (Agilent Cat# M7254, RRID:AB_2631163, dilution 1:100), Wilms tumor protein-1 (WT-1; Agilent Cat# M3561, RRID:AB_2304486, dilution 1:100), perilipin-1 (sc-390169, dilution 1:50, Santa Cruz Biotechnology, USA) and 4-hydroxy-2-nonenal (4-HNE; Abcam Cat# ab46545, RRID:AB_722490, dilution 1:200).

Techniques: Staining, Real-time Polymerase Chain Reaction, Control, Software

Journal: BMJ Open Diabetes Research & Care

Article Title: Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

doi: 10.1136/bmjdrc-2020-001242

Figure Lengend Snippet: MiS1 treatment inhibits kidney JAK/STAT activation and renal microinflammatory milieu in the BTBR ob/ob model. (A) Graphs and images represent the changes observed in JAK/STAT activation (p-STATs) in diabetic mice treated with active MiS1 (2 µg and 4 µg) and inactive mutated peptide (Mut 4 µg) compared with vehicle controls (Veh), quantified per number of positive cells p-STAT1+ and p-STAT3+, both at the glomerular and tubular fields. Magnification ×630. (B) Representative images of immunohistochemistry against F4/80 and CD3. Magnification ×200 and ×630. Graphs represent the quantification of average number of monocytes/macrophages F4/80+ and CD3+ T lymphocytes, both at the glomerular and interstitial fields. Arrows indicate positively stained cells. (C) Gene expression analysis of mRNA related with JAK/STAT pathway ( Stat1 , Stat3 , Socs1 and Socs3 ), inflammatory cytokines ( Tnfα and Il-12 ) and chemokines ( Mcp-1 and Rantes ), and kidney damage markers ( Kim-1 and Ngal ) were evaluated by real-time PCR, being normalized in each sample by endogenous control gene 18s and expressed as n-fold the average value obtained in the vehicle group (Veh). Data are shown as scatter dot plots and mean±SD of each group (n=6–7 mice/group); *p<0.05, **p<0.01, ****p<0.0001 versus diabetic vehicle control. BTBR, black and tan brachyuric; GCS, glomerular cross section; JAK/STAT, Janus kinase/signal transducers and activators of transcription; ob/ob, obese/obese.

Article Snippet: The primary antibodies for immunodetection were sourced as follows: phosphorylated (p-) STAT3 serine 727 (Cell Signaling Technology Cat# 9134, RRID:AB_331589, dilution 1:100), p-STAT1 tyrosine 701 (Cell Signaling Technology Cat# 7649, RRID:AB_10950970, dilution 1:50), p-p65 subunit of nuclear factor-κB (NF-κB) serine 536 (Santa Cruz Biotechnology Cat# sc-33020, RRID:AB_2179018, dilution 1:100), p-nuclear factor erythroid 2-related factor 2 (NRF2) serine 40 (Abcam Cat# ab76026, RRID:AB_1524049, dilution 1:2000), SOCS1 (Abcam Cat# ab62584, RRID:AB_956316, dilution 1:1000), SOCS3 (Abcam Cat# ab16030, RRID:AB_443287, dilution 1:200), F4/80 monocytes/macrophages (Bio-Rad Cat# MCA497, RRID:AB_2098196, dilution 1:70), CD3 T lymphocytes (Agilent Cat# M7254, RRID:AB_2631163, dilution 1:100), Wilms tumor protein-1 (WT-1; Agilent Cat# M3561, RRID:AB_2304486, dilution 1:100), perilipin-1 (sc-390169, dilution 1:50, Santa Cruz Biotechnology, USA) and 4-hydroxy-2-nonenal (4-HNE; Abcam Cat# ab46545, RRID:AB_722490, dilution 1:200).

Techniques: Activation Assay, Immunohistochemistry, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: Naunyn-Schmiedeberg's archives of pharmacology

Article Title: Modulation of AMPK by esomeprazole and canagliflozin mitigates methotrexate-induced hepatotoxicity: involvement of MAPK/JNK/ERK, JAK1/STAT3, and PI3K/Akt signaling pathways.

doi: 10.1007/s00210-025-03908-3

Figure Lengend Snippet: Fig. 7 The regulation of the p-JAK1/p-STAT3 signaling pathway by ESOM and CANA. To verify the effect of ESOM and CANA on various groups, we measured A the relative protein expression of p-JAK1 via western blot analysis and B the relative protein expres- sion of p-STAT3 using western blot analysis. Data are shown as mean ± SE (n = 6). *, **, # indicate a significant difference between the control group, MTX group, and ESOM and CANA + MTX group, respec- tively, using one-way ANOVA test at a p value < 0.01 followed by Tukey

Article Snippet: The membranes were incubated at 4 °C overnight with 1:1000 dilutions of JAK1 antibody (Santa Cruz Biotechnology, sc-376996, USA), STAT3 antibody (Abcam Co., EPR787Y, UK), JNK antibody (Santa Cruz Biotechnology, sc-7345, USA), ERK1 antibody (Santa Cruz Biotechnology, sc-271270, USA), and p38 antibody (Biorbyt, orb127559, USA), respectively, then incubated at room temperature for 1 h with 1:2000 dilutions of HRP-linked anti-rabbit antibody (Cell Signaling Technology, 7074, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: Resveratrol and AG490 Overcome Glioblastoma Cells’ Resistance to Monotherapy by Inhibiting JAK2/STAT3 Signalling Pathway

doi: 10.3390/cancers18050794

Figure Lengend Snippet: RES and AG490 suppress STAT3 signaling pathway activation in GBM cells. ( a ) Representative immunofluorescence images showing pSTAT3 (green) and nuclear staining with Hoechst (blue) in LN428 and U251 cells treated with RES, AG490, or RES + AG490. Merged images display the overlay of pSTAT3 and nuclear signals. ( b ) Representative immunocytochemistry results demonstrating pSTAT3 protein expression. ( c ) Representative Western blot results showing total STAT3 and pSTAT3 protein expression in both cells treated with RES, AG490, and RES + AG490. GAPDH serves as the loading control. ( d ) Densitometric quantification of STAT3 and pSTAT3 protein expression levels normalized to GAPDH in LN428 and U251 cells. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 100 µm.

Article Snippet: Afterword, the membranes were blocked with 5% skim milk for 2 h, and washed thrice with Tris-buffered saline (TBS-T, 8 min each), and incubated overnight at 4 °C with primary antibodies, Rabbit polyclonal anti STAT3 (1:1000, Protein Tech, Rosemont, IL, USA 10253-2-AP), Rabbit polyclonal anti pSTAT3 (1:1000, abs118973), Rabbit polyclonal anti BAX (1:1000, Protein Tech, USA 50599-2-lg), Rabbit polyclonal anti BCL-2 (1:1000, Protein Tech, USA 26593-1-AP), and Rabbit polyclonal anti- GAPDH (1:5000, Proteintech, Wuhan, China 10494-1-AP).

Techniques: Activation Assay, Immunofluorescence, Staining, Immunocytochemistry, Expressing, Western Blot, Control

Journal: Oncogene

Article Title: Stat3 contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration.

doi: 10.1038/sj.onc.1209531

Figure Lengend Snippet: Figure 1 Stat3 expression and activation are elevated in keloid skin tissues. (a) Hematoxylin–eosin stain of paraffin tissue sections of normal skin (NS1) and keloid scar (KS28). D, E and K indicate dermis, epidermis and keratin, respectively. (b) Cryosections of normal skin and keloid scar tissue were stained with either anti-rabbit immunoglobulinG (a-rabbit IgG) or polyclonal anti-Stat3 (C-20) (a- Stat3) and counterstained with 4,6-diamidino-2-phenylindole (DAPI). (c) Cryosections were probed with anti-mouse IgG (a-mouse IgG) or monoclonal anti-phosphoStat3 (pStat3) (a-pStat3) and counterstained with DAPI. Pictures were taken together with phase contrast and all scale bars represent 50 mM. (d) Equal amounts of tissue lysates from normal skin (NS) and keloid samples (KS) were subjected to Western blot analysis with anti-pY705 Stat3 antibody. The blot was stripped and reprobed with anti-pS727 Stat3, and subsequently with anti-Stat3 and anti-actin.

Article Snippet: Polyclonal anti-phosphoTyr705 Stat3, monoclonal antiphosphoSer727 Stat3, anti-pJak1 (Tyr1022/1023), anti-pJak2 (Tyr1007/1008), anti-phosphoSrc (Tyr416), anti-phosphoEGFR (Tyr845) and anti-Bcl-XL antibodies were purchased from Cell Signaling Technology, whereas monoclonal pStat3 (B7) (sc-8059), polyclonal Stat3 (C-20) (sc-482), monoclonal cyclin D1 (sc-20044), anti-mouse IgG (sc-2025) and anti-rabbit IgG (sc-2027) antibodies were acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Staining, Western Blot

Journal: Oncogene

Article Title: Stat3 contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration.

doi: 10.1038/sj.onc.1209531

Figure Lengend Snippet: Figure 2 Enhanced Stat3 phosphorylation and expression in keloid fibroblasts (KFs). (a) Western blot analyses of total cell lysates from normal fibroblasts (NFs) and KFs with equal amounts of protein, and antibodies as indicated. The graph on the upper right illustrates the pY705 Stat3 level normalized to Stat3 expression, whereas the graph on the lower right depicts an average of the normalized pY705 Stat37s.d. of the KFs and NFs presented. (b) Normal fibroblasts, NF5 and NF4, and KFs, KF48 and KF8, were cultured in serum-free media for 2 days before stimulating with 10% fetal bovine serum. Total cell lysates were harvested at day 1–5, and phosphorylations of Stat3 were analysed by Western blot after loading equal amounts of protein. (c) NF4 and KF48 fibroblasts were incubated in serum-free Dulbecco’s modified Eagles’s medium for 2 days before harvesting daily over 5 days to investigate the serum-independent phosphorylation of Stat3. Equal amounts of total cell lysates were subjected to Western blot analysis with antibodies as indicated. Arrowheads indicate the position of the protein bands.

Article Snippet: Polyclonal anti-phosphoTyr705 Stat3, monoclonal antiphosphoSer727 Stat3, anti-pJak1 (Tyr1022/1023), anti-pJak2 (Tyr1007/1008), anti-phosphoSrc (Tyr416), anti-phosphoEGFR (Tyr845) and anti-Bcl-XL antibodies were purchased from Cell Signaling Technology, whereas monoclonal pStat3 (B7) (sc-8059), polyclonal Stat3 (C-20) (sc-482), monoclonal cyclin D1 (sc-20044), anti-mouse IgG (sc-2025) and anti-rabbit IgG (sc-2027) antibodies were acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Cell Culture, Incubation