phosphorylated stat3  (Cell Signaling Technology Inc)

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: Primer sequences for reverse transcription-quantitative PCR.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Sequencing

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: mRNA expression analysis by RT-qPCR, and assessment of self-renewal and tumor growth in H460-derived SFCs. (A) BEP2D, H460 and A549 cells were treated with DFOG (0–20 µM) for 48 h, and cell viability was assessed using a Cell Counting Kit-8 assay. H460 and A549 cells were treated with DFOG (5 µM) for 24 h. RT-qPCR was used to evaluate the effects of DFOG (5 µM) on tumor-suppressive miRNAs, including miR-671-5p, miR-148a-3p, miR-340-5p, miR-342-3p, miR-34a-5p and miR-152-3p in (B) H460 and (C) A549 cells. (D) Comparison of miR-152-3p expression between H460 cells and H460-derived SFCs. (E) STAT3 mRNA levels and (F) p-STAT3 protein levels. Rates of (G) sphere formation and (H) colony formation (scale bar, 100 µm). Western blot analysis of (I) CD44 and CD133 expression, as well as (J) Oct4 and Sox2 expression. *P<0.05, **P<0.01 (n=3). DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR/miRNA, microRNA; p-, phosphorylated; RT-qPCR, reverse transcription-quantitative PCR; SFC, sphere-forming cell.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Cell Counting, Comparison, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: DFOG induces miR-152-3p expression, and inhibits self-renewal and tumor growth in H460-derived SFCs. At the indicated concentrations, DFOG (A) upregulated miR-152-3p expression, and (B) decreased STAT3 mRNA expression and (C) p-STAT3 protein levels in H460-derived SFCs. (D) Sphere formation and (E) colony formation were reduced (scale bar, 100 µm). Western blot analysis showed downregulation of (F) CD44 and CD133 expression, as well as (G) Oct4 and Sox2 expression. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001 (n=3). (H) Images of tumor tissue; volume quantification; weight quantification; H&E staining and immunohistochemical staining using an anti-p-STAT3 antibody (scale bar, 50 µm). Quantification of p-STAT3 protein levels and miR-152-3p levels in xenograft tumors of nude mice bearing H460-derived SFCs treated with DFOG at the indicated doses. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA; p-, phosphorylated; SFC, sphere-forming cell.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Expressing, Derivative Assay, Western Blot, Staining, Immunohistochemical staining

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: miR-152-3p mimic enhances DFOG-induced downregulation of p-STAT3 levels and inhibits self-renewal in H460-derived SFCs. Expression levels of (A) miR-152-3p and (B) STAT3 mRNA, and (C) p-STAT3 protein levels. (D) Spheres and (E) colonies formed were quantified (scale bar, 100 µm). Western blot analysis of (F) CD44 and CD133, as well as (G) Oct4 and Sox2 expression in H460-derived SFCs transfected with miR-152-3p mimic and/or treated with DFOG (5 µM). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 (n=3). DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA; NC, negative control; p-, phosphorylated; SFC, sphere-forming cell.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Negative Control

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: miR-152-3p inhibitor antagonizes DFOG-induced suppression of p-STAT3 levels and self-renewal in H460-derived SFCs. Expression levels of (A) miR-152-3p and (B) STAT3 mRNA, and (C) p-STAT3 protein levels. (D) Spheres and (E) colonies formed were quantified (scale bar, 100 µm). Western blot analysis of (F) CD44 and CD133, as well as (G) Oct4 and Sox2 expression in H460-derived SFCs transfected with miR-152-3p inhibitor and/or treated with DFOG (5 µM). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 (n=3). DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA; NC, negative control; p-, phosphorylated; SFC, sphere-forming cell.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Negative Control

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: STAT3 inhibitor enhances DFOG-induced suppression of self-renewal in H460-derived SFCs. Expression levels of (A) miR-152-3p and (B) STAT3 mRNA, and (C) p-STAT3 protein levels are shown. (D) Spheres and (E) colonies formed were quantified (scale bar, 100 µm). Western blot analysis of (F) CD44 and CD133, as well as (G) Oct4 and Sox2 expression in H460-derived SFCs treated with S3I 201 (10 µM) and/or DFOG (5 µM). *P<0.05, **P<0.01 and ***P<0.001 (n=3). DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA; p-, phosphorylated; SFC, sphere-forming cell.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Derivative Assay, Expressing, Western Blot

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: Effect of STAT3 overexpression and DFOG co-treatment on miR-152-3p expression. Expression levels of (A) miR-152-3p and (B) STAT3 mRNA, and (C) STAT3 protein levels in H460-derived sphere-forming cells transfected with pcDNA-STAT3 and/or treated with DFOG (5 µM). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 (n=3). DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Over Expression, Expressing, Derivative Assay, Transfection

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: STAT3 is a direct target of miR-152-3p ( https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid ). (A) Representation of the predicted miR-152-3p binding site in the 3′-UTR of STAT3 mRNA. (B) Luciferase activity of 3′-UTR-WT and 3′-UTR-MUT STAT3 3′-UTR reporters in H460 cells after transfection with the miR-152-3p mimic or miR-mimic-Cont. (C) Luciferase activity of 3′-UTR-WT STAT3 3′-UTR in H460 cells transfected with the miR-152-3p mimic and/or treated with DFOG (5 µM). *P<0.05, **P<0.01 (n=3). (D) Mechanism of action of DFOG regulating the miR-152-3p/STAT3 axis and suppressing self-renewal in non-small cell lung carcinoma. 3′-UTR, 3′-untranslated region; Cont, control; DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA; MUT, mutant; p-, phosphorylated; WT, wild-type.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Control, Mutagenesis

Journal: Oncology Reports

Article Title: 7‑Difluoromethoxyl‑5,4'‑di‑n‑octylygenistein targets the STAT3 pathway by upregulating microRNA‑152‑3p expression to inhibit self‑renewal and tumor growth in non‑small cell lung carcinoma

doi: 10.3892/or.2025.8899

Figure Lengend Snippet: DFOG induces miR-152-3p expression, and inhibits STAT3 activation and self-renewal in A549-derived SFCs. At the indicated concentrations, DFOG (A) upregulated miR-152-3p expression, and decreased (B) STAT3 mRNA expression and (C) p-STAT3 protein levels in A549-derived SFCs. (D) Spheres and (E) colonies formed were quantified (scale bar, 100 µm). Western blot analysis of (F) CD44 and CD133, as well as (G) Oct4 and Sox2 expression in A549-derived SFCs. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 (n=3). DFOG, 7-difluoromethoxyl-5,4′-di-n-octylygenistein; miR, microRNA; p-, phosphorylated; SFC, sphere-forming cell.

Article Snippet: Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described ( ).

Techniques: Expressing, Activation Assay, Derivative Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Solid stress compression enhances breast cancer cell migration through the upregulation of Interleukin-6

doi: 10.3389/fcell.2025.1541953

Figure Lengend Snippet: Incremental solid stress compression upregulates protein levels of IL-6 and SNAI1 as well as secretion of IL-6 in a biphasic manner (A) Representative western blots of cell lysates harvested from MDA-MB-231 cells under 16 h of incremental solid stress compression using IL-6 and SNAI1 antibodies. GAPDH was used as loading control (B) Densitometry quantification of IL-6 protein levels using Image Lab. Bar graph represents the mean relative fold changes compared to uncompressed across three independent experiments (n = 3). Error bars represent standard deviation. Statistical analysis was performed using 2-tailed student T-test (*) p ≤ 0.05, (**) p ≤ 0.01 (C) Densitometry quantification of SNAI1 protein level using Image Lab. Bar graph represents the mean relative fold changes compared to uncompressed across three independent experiments (n = 3). Error bars represent standard deviation. Statistical analysis was performed using 2-tailed student T-test, (*) p ≤ 0.05, (**) p ≤ 0.01. (D) (Top) Representative Western blot of conditioned media harvested after 16 h of incremental solid stress compression and probed with anti-IL-6 antibody. (Bottom) Representative Ponceau S Staining highlighting equal protein loading of the conditioned media. (E) Densitometry quantification of IL-6 cytokine secretion using Image Lab. Bar graph represents the mean relative fold changes compared to uncompressed across three independent experiments (n = 3). Error bars represent standard deviation. Statistical analysis was performed using 2-tailed student T-test, (*) p ≤ 0.05 and (N.S) Not Significant. (F) Representative Western blot image using anti-phospho-STAT3 (pSTAT3) at Tyr-705 antibody for MDA-MB-231 cells under 16 h of incremental solid stress compression. (G) Densitometry quantification of pSTAT3 using Image Lab. Bar graph represents the mean relative fold changes compared to uncompressed across three independent experiments (n = 3). Error bars represent standard deviation. Statistical analysis was performed using 2-tailed student T-test, (*) p ≤ 0.05 and (N.S) Not Significant.

Article Snippet: Phosphorylated STAT3 Tyrosine-705 (D3A7) XP 9145 , Cell Signaling , Rabbit , 1:1000 in 5% BSA.

Techniques: Western Blot, Control, Standard Deviation, Staining