Journal: Journal of enzyme inhibition and medicinal chemistry

Article Title: Dual inhibition of EGFR and IL-6-STAT3 signalling by miR-146b: a potential targeted therapy for epithelial ovarian cancer.

doi: 10.1080/14756366.2021.1963240

Figure Lengend Snippet: Figure 2. The expression patterns of EGFR and IL-6-STAT3 in ovarian cancer. (A) Analysis of of IL-6 expression in ovarian cancer samples from TCGA database at http://gepia.cancer-pku.cn/. (B) Analysis of STAT3 expression in ovarian cancer tissues from the https://www.oncomine.org database. (C, D) The mRNA and protein lev- els of EGFR in ovarian cancer cell lines as determined by qPCR and Western blotting. (E) Analyses of mRNA levels of IL-6 in ovarian cancer samples (n ¼ 24) and control samples (n ¼ 24). (F) The relative mRNA levels of IL-6 in ovarian cancer cell lines. (G) The protein level of IL-6 as determined by Western blotting in ovarian cancer cell lines. (H) The concentration of IL-6 in cell supernatants cell supernatants as determined by flow cytometry in ovarian cancer cell lines. (I) The relative mRNA levels of STAT3 in ovarian cancer cell lines. (J) The relative expression of p-STAT3 (Y705, S727) and STAT3 in ovarian cancer cell lines was examined by Western blotting, and GAPDH was used as the internal control. The data are expressed as the means ± SDs; ns: not significant; p < 0.05; p < 0.01; p < 0.001.

Article Snippet: The antibodies used for Western blotting were as follows: anti-GAPDH (#60004–1-Ig, Proteintech, Wuhan, China), anti-IL-6 (#66146-Ig, Proteintech, Wuhan, China), anti-STAT3 (#4904 CST, Danvers, MA, USA), anti-P-STAT3 (Y705) (#9145, CST, Danvers, MA, USA), anti-P-STAT3 (S727) (#49081, CST, Danvers, MA, USA), anti-EGFR (#4267, CST, Danvers, MA, USA), anti-Flag (F1804, Sigma, USA), HRP-conjugated anti-mouse IgG (#70-GAM007, MultiSciences, China), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (#70-GAR007, MultiSciences, China).

Techniques: Expressing, Western Blot, Control, Concentration Assay, Flow Cytometry

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 1 Elevated miR-762 expression is associated with gefitinib resistance in NSCLC cells. a RT-qPCR analysis of miR-762 expression in different NSCLC cells. Relative expression levels of miR-762 were obtained in each sample by normalization of the expressions of miR-762 to that of the U6 snRNA signal. For presentation of data, expression levels of miR-762 from NuLi-1 cells were taken as 100% and the others were normalized accordingly. Each value is a mean ± S.E.M. from three independent experiments. Different superscript letters denote groups that are statistically different (P < 0.05). b The IC50 value (inhibitory concentration to produce 50% cell death) following a 48-h exposure to gefitinib was determined in PC-9 and PC-9/GR cells using MTT assay. c The IC50 value following a 48-h exposure to gefitinib was determined in A549 and A549/GR cells using MTT assay. d-e Characterization of miR-762 expression in different NSCLC cells using RT-qPCR. The value indicates the relative expression levels of miR-762 in the cells (PC-9/PC-9/GR and A549/A549/GR cells) at different batches of gefitinib resistant induction

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, MTT Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 2 IL-6 serves as a potential upstream regulator of miR-762 induction in NSCLC cells. a-b Characterization of expression levels of different cytokines in different NSCLC cells using RT-qPCR. Each value is a mean ± S.E.M. from three independent experiments. c A549 cells were incubated with different cytokines, including IL-1α (5 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml) and IL-8 (50 ng/ml) for 24 h, followed by RT-qPCR analysis of miR-762 expression. d A549 cells were stimulated with different doses of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression. e A549 cells were stimulated with 10 ng/ml of IL-6 for different durations as indicated, followed by RT-qPCR analysis of miR-762 expression. f A549 cells were stimulated with 10 ng/ml of IL-6, in the presence or absence or co-treatment with 5 μM of WP1066, for 24 h, followed by RT-qPCR analysis of miR-762 expression. Inhibition of STAT3 activation was verified using immunoblotting analysis of pSTAT3 expression (upper panel). g A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA. 48 h later, knockdown of STAT3 in A549 cells was validated using immunoblotting. h 48 h after siRNA treatment, A549 cells were stimulated with 10 ng/ml of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Incubation, Inhibition, Activation Assay, Western Blot, Transfection, Knockdown

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 3 miR-762 upregulation desensitizes NSCLC cells to gefitinib treatment. a 48 h after transfection with miR-762 inhibitors or negative controls (NC), PC-9/GR and A549/GR cells were subjected to RT-qPCR analysis of miR-762 expression. b PC-9/GR and A549/GR cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). c Cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). d In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and ** P < 0.01 when comparing Inhibitors + vehicle to Inhibitors + gefitinib). e 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9 and A549 cells were subjected to RT-qPCR analysis of miR-762 expression. f PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). g PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). h In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and **P < 0.01 when comparing Mimics + vehicle to Mimics + gefitinib)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 4 ABR serves as the direct target of miR-762 in NSCLC cells. a Prediction of putative target genes of miR-762 by Target scan and miRDB programs. b PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by immunoblotting analysis of ABR expression. c PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by RT-qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). d PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. e PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by RT- qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). f Predicted miR-762 binding sites in the 3′-UTR of ABR gene. g miR-762 mimics/Mimics- NC (25 pmol/well) and pGL3-ABR 3’UTR-Luc reporter (0.25 μg/well), together with 0.001 μg of the Renilla luciferase reporter (Promega, Beijing, China), were co-transfected into subconfluent proliferating NIH/3 T3 cells for 24 h, followed by measurement of the relative luciferase activity (*P < 0.05)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 5 ABR overexpression alleviates miR-762-induced gefitinib resistance. a PC-9 and A549 cells that stably expressed the exogenous ABR was established as described in Materials and methods. PC-9/ABR and A549/ABR cells were transiently transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. b 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm. c 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell apoptosis was assayed using an ApoStrand™ ELISA Apoptosis Detection Kit at 405 nm. Different superscript letters denote groups that are statistically different (P < 0.05). d 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were subjected to a xenograft model to measure the in vivo gefitinib sensitivity, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 when comparing Mimics + gefitinib + vector to Mimics + gefitinib + pCMV3-ABR)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Over Expression, Stable Transfection, Transfection, Western Blot, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Plasmid Preparation

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