Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Sanguinarine mediated apoptosis in Non-Small Cell Lung Cancer via generation of reactive oxygen species and suppression of JAK/STAT pathway.

doi: 10.1016/j.biopha.2021.112358

Figure Lengend Snippet: Fig. 4. SNG downregulates constitutively activated JAK/STAT3 signaling pathway and IL6 mediated STAT3 activation in NCI-H-1975 and HCC-827 cellS (A). After 24 h of SNG treatment, NCI-H-1975 and HCC-827 cells were lysed, and western blotting was performed for antibodies against p-Jak2, Jak2, GAPDH, p-Src, Src, p- STAT3 (Try705 and ser727), and STAT3. (B) siRNA knockdown of STAT3. NCI-H-1975 were transfected with control (100 pM) and STAT3 siRNA (50 and 100 pM). Cells were lysed and separated, transferred on the PVDF membrane, and immunoblotted with antibodies against STAT3, Bcl-xL, caspase-3, cleaved caspase-3 and HSP60. (C) SNG inhibits IL 6 secretion in NCI-H-1975 and HCC-827 cells. The cells mentioned above were exposed to SNG for 24 h, as indicated. Using supernatant from the treated and untreated group, levels of IL 6 were determined using multiplex biometric ELISA-based immunoassay. (D) SNG inhibits IL6-induced STAT3 activation. NCI-H-1975 and HCC-827 cells were pretreated with 2.0 μM SNG for 1 h and then stimulated with IL6 (100 µg/ml). At the end of the treatment, cells were lysed and immunoblotted against p-STAT3 (Try705) and STAT3(ser727).

Article Snippet: Antibodies against caspase-9 (Cat. No. 9508), Bcl2 (Cat. No. 15071), phospho-STAT3 Tyr705- Cat. No. 9145, phospho-STAT3 Ser727- Cat. No. 9134, STAT3 Cat. No.12640, cleaved caspase-3 Cat. No. 9661, caspase-3 Cat. No. 9662, Bax (Cat. No.5023), Cytochrome c (Cat. No.12963), Tubulin (Cat. No.2144), GAPDH (Cat. No.5174), PARP ( Cat. No.9542) CLEAVED CASPASE 8 (Cat. No.9496), phospho-jak2 (Cat. No.3776), JAK2(Cat. No.3230), PH2AX(Cat. No.9718), phsopho-Src Tyr416 (Cat. No. 6943) and Src (Cat. No. 2108) were purchased from Cell Signaling Technologies (Beverly, USA).

Techniques: Activation Assay, Western Blot, Knockdown, Transfection, Control, Membrane, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Sanguinarine mediated apoptosis in Non-Small Cell Lung Cancer via generation of reactive oxygen species and suppression of JAK/STAT pathway.

doi: 10.1016/j.biopha.2021.112358

Figure Lengend Snippet: Fig. 7. SNG inhibits NCI-H-1975 xenograft tumor growth in ICR-SCID mice. Subcutaneous tumor was grown in donor mouse and then transplanted into ICR-SCID female mice and divided into treated and control groups as dis cussed under Materials and Methods. At the end of the study, the mice were sacrificed, and (A) excised tumors in each group were weighed and analyzed using graph pad prism software. The graph displays the mean ± SD of four inde pendent experiments (* P < 0.05). (B) Excised tumors were photographed (C) lysates obtained from excised tumors were immunoblotted against p-STAT3, STAT3, and actin antibodies. (D) Densitometric analysis of p-STAT3/STAT3 expression in control and SNG treated tumors. The graph displays the mean ± SD of four in dependent experiments (* P < 0.05).

Article Snippet: Antibodies against caspase-9 (Cat. No. 9508), Bcl2 (Cat. No. 15071), phospho-STAT3 Tyr705- Cat. No. 9145, phospho-STAT3 Ser727- Cat. No. 9134, STAT3 Cat. No.12640, cleaved caspase-3 Cat. No. 9661, caspase-3 Cat. No. 9662, Bax (Cat. No.5023), Cytochrome c (Cat. No.12963), Tubulin (Cat. No.2144), GAPDH (Cat. No.5174), PARP ( Cat. No.9542) CLEAVED CASPASE 8 (Cat. No.9496), phospho-jak2 (Cat. No.3776), JAK2(Cat. No.3230), PH2AX(Cat. No.9718), phsopho-Src Tyr416 (Cat. No. 6943) and Src (Cat. No. 2108) were purchased from Cell Signaling Technologies (Beverly, USA).

Techniques: Control, Software, Expressing

Journal: International journal of oral science

Article Title: Parathyroid hormone increases alveolar bone homoeostasis during orthodontic tooth movement in rats with periodontitis via crosstalk between STAT3 and β-catenin.

doi: 10.1038/s41368-020-00104-2

Figure Lengend Snippet: Fig. 3 Daily systemic injection of PTH activated STAT3 in the alveolar bone, which could be reversed by local stattic injection. Representative images of immunohistochemical staining of STAT3 (a) and p-STAT3 (Tyr705) (b) in the OTM + PD, OTM + PD + PTH and OTM + PD + PTH + S groups, and relative quantitative analysis at days 7 and 14. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1 by one-way ANOVA with Tukey’s post hoc test

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA and incubated with primary antibodies against β-actin (Huabio, China), STAT3 (Huabio, China), phospho-STAT3 (Tyr705) (Cell Signaling Technology, USA), RUNX2 (Huabio, China), OSX (Abcam, UK), ALP (Huabio, China), OPN (Huabio, China), β-catenin (Huabio, China), RANKL (Abcam, UK) and OPG (Huabio, China) at 4 °C overnight, and rabbit and mouse secondary antibodies (Signalway Antibody, USA).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: International journal of oral science

Article Title: Parathyroid hormone increases alveolar bone homoeostasis during orthodontic tooth movement in rats with periodontitis via crosstalk between STAT3 and β-catenin.

doi: 10.1038/s41368-020-00104-2

Figure Lengend Snippet: Fig. 6 IPTH promoted osteogenesis in vitro, which could be reduced by stattic. a Alp, Ibsp, Opn, Sp7, Pth1r, Cbfa1, Col1a1 and Bglap expression in each group. b Protein levels of RANKL and OPG. c RANKL and OPG mRNA levels and the RANKL/OPG ratio. d Protein levels of representative osteoblastic markers and p-STAT3 (Tyr705) and STAT3. e Alizarin red S staining and ALP staining. f Quantitative analysis of Alizarin red S staining. g Quantitative analysis of ALP. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1 by one-way ANOVA with Tukey’s post hoc test

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA and incubated with primary antibodies against β-actin (Huabio, China), STAT3 (Huabio, China), phospho-STAT3 (Tyr705) (Cell Signaling Technology, USA), RUNX2 (Huabio, China), OSX (Abcam, UK), ALP (Huabio, China), OPN (Huabio, China), β-catenin (Huabio, China), RANKL (Abcam, UK) and OPG (Huabio, China) at 4 °C overnight, and rabbit and mouse secondary antibodies (Signalway Antibody, USA).

Techniques: In Vitro, Expressing, Staining

Journal: International journal of oral science

Article Title: Parathyroid hormone increases alveolar bone homoeostasis during orthodontic tooth movement in rats with periodontitis via crosstalk between STAT3 and β-catenin.

doi: 10.1038/s41368-020-00104-2

Figure Lengend Snippet: Fig. 7 IPTH activated Wnt/β-catenin signalling, while inhibition of STAT3 activation suppressed this effect. a Colocalization of β-catenin and STAT3 by immunofluorescence. b β-catenin levels in nuclear fractions (active form) and whole cells. c Axin2 expression level. d Ctnnb1 expression level. e Sost expression level. f A diagram showing the hypothesis that PTH regulates STAT3 and Wnt/β-catenin signalling. *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s post hoc test

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA and incubated with primary antibodies against β-actin (Huabio, China), STAT3 (Huabio, China), phospho-STAT3 (Tyr705) (Cell Signaling Technology, USA), RUNX2 (Huabio, China), OSX (Abcam, UK), ALP (Huabio, China), OPN (Huabio, China), β-catenin (Huabio, China), RANKL (Abcam, UK) and OPG (Huabio, China) at 4 °C overnight, and rabbit and mouse secondary antibodies (Signalway Antibody, USA).

Techniques: Inhibition, Activation Assay, Expressing

Journal: Communications biology

Article Title: PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth.

doi: 10.1038/s42003-024-06290-7

Figure Lengend Snippet: Fig. 1 | PRMT5 expression levels are increased in lung cancer cells. a, b PRMT5 expression is elevated in a lung adenocarcinoma and b lung squamous cell carci- noma. RNA-seq data from The Cancer Genome Atlas (TCGA) were analysed on the UCSC Xena browser. c Correlation between STAT3 and PRMT5 protein levels in 12 NSCLC cell lines (NCI-H23, LU65, CORL105, CHAGOK1, NCI-H3255, NCI-

Article Snippet: STAT3 activation was monitored by western blotting using an anti-phosphorylated STAT3 (Y705) antibody (CST; 9138).

Techniques: Expressing, RNA Sequencing

Journal: International immunopharmacology

Article Title: 3,6-Anhydro-L-galactose suppresses mouse lymphocyte proliferation by attenuating JAK-STAT growth factor signal transduction and G 1 -S cell cycle progression.

doi: 10.1016/j.intimp.2024.113998

Figure Lengend Snippet: Fig. 10. Kinetic analysis of the inhibitory effect of L-AHG on the JAK-STAT signaling in activated T and activated B cells. (A, C) Following stimulation of G0 T cells (1.5 × 106/mL) and G0 B cells (2.5 × 106/mL) by immobilized anti-CD3/anti-CD28 and immobilized anti-CD40 + soluble anti-IgM + IL-4, respectively, for the indicated time periods in the absence or presence of L-AHG, cells were harvested and subjected to western blot analysis of the status of the JAK-STAT pathway components including p-JAK1 (Y1034/1035), JAK1, p-STAT1 (Y701), STAT1, p-STAT3 (Y705), STAT3 and GAPDH in activated T cells and activated B cells. A representative result is shown; two additional experiments yielded similar results. (B, D) The statistical data of western blot analysis have been drawn as bar graphs based on relative intensity of each interesting protein band. * p < 0.05, ** p < 0.01, *** p < 0.005, compared with the control. # p < 0.05, compared with selected groups.

Article Snippet: The antip-CDK4 (T172) antibody was purchased from ABclonal Technology (Woburn, MA, USA) and anti-cyclin A2, anti-cyclin E2, and anti-STAT3 antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Western Blot, Control

Journal: Molecular medicine reports

Article Title: A small GTPase‑like protein fragment of Mycoplasma promotes tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

doi: 10.3892/mmr.2013.1766

Figure Lengend Snippet: Figure 2. SGLP interacts with Rac1 and Stat3. (A) GST‑tagged SGLP (~10 µg) was used to pull‑down endogenous Rac1 and Stat3 from IL‑6 stimulated HeLa cell lysates (2 mg). The bait protein, GST‑SGLP and control GST were also immunoblotted with a GST antibody. (B) HeLa cell lysates expressing GFP‑SGLP were immunoprecipitated with a GFP antibody and then probed with anti‑Stat3 and anti‑Rac1 antibodies. The cell lysates were also immunoprecipitated with a Rac1 antibody and then probed with anti‑Stat3 and anti‑GFP antibodies. The white asterisk indicates the location of the heavy chain. The right panel shows the cell lysates of input probed with indicated antibodies. (C) HeLa cells expressing FLAG‑SGLP were fixed, permeabilized and immunostained with anti‑Stat3 (rabbit) and anti‑FLAG (mouse) antibodies in conjugation with Alexa Fluor 488 and 594 secondary antibodies, respectively. The confocal images were analyzed for colocalization using an ImageJ plugin. The boxed region is enlarged. Scale bar, 10 µm. (D) HeLa cells coexpressing GFP‑SGLP and DsRed‑Rac1 were fixed in 4% paraformaldehyde and observed by confocal microscopy. As DsRed‑Rac1 was homogenously expressed, the FRET between GFP‑SGLP and DsRed‑Rac1 was measured, and the FRET indices of GFP‑SGLP and GFP control were plotted as pseudocolor images. Scale bar, 10 µm. (E) HeLa cells, as in (D), were observed by confocal microscopy following photobleaching of DsRed‑Rac1. Images were recorded for DsRed‑Rac1 (red) and GFP‑SGLP (green) every 30 sec during and following photobleaching, and the relative fluorescence intensities were plotted. The black arrows indicate the duration of full‑lamp excitation. The negative GFP control did not exhibit such a recovery following photobleaching of DsRed‑Rac1 (data not shown). Bar represents ± SD of nine cells from three independent experiments. (F) A plot of the linear correlation of donor recovery vs. acceptor photobleaching from (E). F0 represents the original fluorescence intensity. SGLP, small GTPase‑like protein fragment; GST, glutathione S‑transferase; IL, interleukin; GFP, green fluorescent protein.

Article Snippet: The primary antibodies used were: p-Stat3 (9145) from Cell Signaling Technology, Inc. (Beverly, MA, USA); glutathione S-transferase (GST; sc-33614) and Rac1 (sc-217) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); FLAG (T510‐2) and green fluorescent protein (GFP; T508‐2) from Signalway Antibody (Baltimore, MD, USA); Myc-Tag (AM1007a) from Abgent (San Diego, CA, USA); anti-bromodeoxyuridine (BrdU; 560810) from BD Biosciences (San Jose, CA, USA) and Stat3 (51076-2-AP) from Proteintech Group (Chicago, IL, USA).

Techniques: Control, Expressing, Immunoprecipitation, Conjugation Assay, Confocal Microscopy, Fluorescence

Journal: Molecular medicine reports

Article Title: A small GTPase‑like protein fragment of Mycoplasma promotes tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

doi: 10.3892/mmr.2013.1766

Figure Lengend Snippet: Figure 3. The WxxxE motif of SGLP is required for activation of Rac1 and Stat3. The WVLGE sequence of the wt SGLP was mutated to AVLGA to generate a SGLP mutant. HeLa cells stably expressing FLAG‑tagged wt SGLP, SGLP mutant or vector control were studied in addition to the parental cells (normal). (A) A GST‑Pak1 pull‑down assay was used to determine the relative levels of Rac1 activation. (B) Western blot analysis of phosphorylated Stat3 (tyrosine‑705; p‑Stat3) and total Stat3. The densitometric ratio of (C) active‑Rac1/total‑Rac1 and (D) p‑Stat3/total‑Stat3 from three independent experiments are plotted and normalized to that of the parental cells (normal), which was set to 1. The error bar represents ± SD; t‑test, n=3; *P<0.05 and **P<0.01, compared with vector and mutant groups, separately. wt, wild‑type; SGLP, small GTPase‑like protein fragment.

Article Snippet: The primary antibodies used were: p-Stat3 (9145) from Cell Signaling Technology, Inc. (Beverly, MA, USA); glutathione S-transferase (GST; sc-33614) and Rac1 (sc-217) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); FLAG (T510‐2) and green fluorescent protein (GFP; T508‐2) from Signalway Antibody (Baltimore, MD, USA); Myc-Tag (AM1007a) from Abgent (San Diego, CA, USA); anti-bromodeoxyuridine (BrdU; 560810) from BD Biosciences (San Jose, CA, USA) and Stat3 (51076-2-AP) from Proteintech Group (Chicago, IL, USA).

Techniques: Activation Assay, Sequencing, Mutagenesis, Stable Transfection, Expressing, Plasmid Preparation, Control, Pull Down Assay, Western Blot

Journal: Molecular medicine reports

Article Title: A small GTPase‑like protein fragment of Mycoplasma promotes tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

doi: 10.3892/mmr.2013.1766

Figure Lengend Snippet: Figure 4. SGLP‑induced Stat3 activation is dependent on Rac1 activity. HeLa cells stably expressing FLAG‑tagged wt SGLP or SGLP mutant were transfected with either Myc‑tagged (A) DN‑Rac1 or (B) CA‑Rac1 plasmids for 48 h and the cells transfected with p‑RK5 vector plasmid were used as a control (mock). Cell lysates were probed with the indicated antibodies. In the lower panels, the densitometric ratios of p‑Stat3/total‑Stat3 from three independent experiments were plotted. The ratio of p‑Stat3/total‑Stat3 in wt SGLP‑transduced HeLa cells from mock group was set to 1. The error bar represents ± SD. SGLP, small GTPase‑like protein fragment; DN‑Rac1, dominant negative Rac1; CA‑RAC1 constitutively active Rac1; wt, wild‑type.

Article Snippet: The primary antibodies used were: p-Stat3 (9145) from Cell Signaling Technology, Inc. (Beverly, MA, USA); glutathione S-transferase (GST; sc-33614) and Rac1 (sc-217) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); FLAG (T510‐2) and green fluorescent protein (GFP; T508‐2) from Signalway Antibody (Baltimore, MD, USA); Myc-Tag (AM1007a) from Abgent (San Diego, CA, USA); anti-bromodeoxyuridine (BrdU; 560810) from BD Biosciences (San Jose, CA, USA) and Stat3 (51076-2-AP) from Proteintech Group (Chicago, IL, USA).

Techniques: Activation Assay, Activity Assay, Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Dominant Negative Mutation