anti phosphorylated stat3  (Proteintech)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 affects the STAT3 signaling. ( A ) A schematic diagram of Epac1’s role in the JAK/STAT3 signaling pathway during the OF activation process. ( B ) TAO OFs transfected with sh- Epac1 or Epacl overexpression vector, treated with TGFβ1, and determined for the protein level of p-STAT3 and STAT3 using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus NC-transfected OFs; ## P < 0.01 versus sh-NC-transfected OFs. ( C ) The protein level of p-STAT3 and STAT3 in TAO mouse OAT and OMT was determined using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy mice; # P < 0.05, ## P < 0.01 versus TAO + AAV-NC mice. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; sh, short-hairpin RNA; NC, negative control.

Article Snippet: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Virus, shRNA, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: STAT3 mediates the effects of Epac1 on TGFβ1-treated TAO OFs. ( A – G ) Under TGFβ1 treatment, TAO OFs were transfected with sh- Epac1 with or without the JAK-STAT pathway inhibitor Stattic (2 g/mL for 24 hours) and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); α-SMA and fibronectin expressions using IF staining ( D ); the content of collagen I in cell supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); the protein level of α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus TGFβ1 + sh-NC; ## P < 0.01 versus TGFβ1 + sh- Epac1 + Stattic. sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

Techniques: Transfection, CCK-8 Assay, Migration, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: International Journal of Immunopathology and Pharmacology

Article Title: Cucurbitacin B inhibits Th17 cell differentiation via the suppression of the JAK/STAT pathway and alleviates collagen-induced arthritis in mice

doi: 10.1177/03946320251348715

Figure Lengend Snippet: Screening of potential gene targets for cucurbitacin B (CuB) in RA. (a) Venn diagram showing 134 common genes associated with RA and CuB. (b) Protein-protein interaction (PPI) network of the 134 shared target genes, with different colored edges representing various interaction types. (c) The top 10 key genes identified based on the “Degree” ranking, including TNF, IL1B, STAT3, and others, were visualized for further analysis.

Article Snippet: The cell lysates were then transferred onto PVDF membranes, which were incubated overnight at 4°C with the following primary antibodies: anti-phosphorylated STAT3 (Cell Signaling, Danvers, MA, USA), anti-STAT3 (Cell Signaling, Danvers, MA, USA), anti-phosphorylated JAK2 (Abcam, Cambridge, UK), anti-JAK2 (Abcam, Cambridge, UK), and anti-GAPDH (Cell Signaling, Danvers, MA, USA).

Techniques:

Journal: International Journal of Immunopathology and Pharmacology

Article Title: Cucurbitacin B inhibits Th17 cell differentiation via the suppression of the JAK/STAT pathway and alleviates collagen-induced arthritis in mice

doi: 10.1177/03946320251348715

Figure Lengend Snippet: CuB Suppresses STAT3 Phosphorylation and Th17 Cell Differentiation. CD4+ T cells from DBA/1 mice were cultured under Th17-polarizing conditions with or without CuB (50 nM or 100 nM) for 72 hours. (a) CuB reduced IL-17 mRNA expression in a dose-dependent manner. (b) IL-17 protein levels in the culture medium decreased with increasing CuB concentration. (c) Flow cytometry showed a dose-dependent reduction in IL-17A+ Th17 cells. (d) CuB inhibited RORγt mRNA expression, a key transcription factor for Th17 differentiation, in a dose-dependent manner. (e) Western blot analysis of phosphorylated STAT3 (p-STAT3, Tyr705) and JAK2 (p-JAK2, Tyr1007/1008) in Th17-polarized CD4+ T cells treated with CuB (100 nM) for 48 and 72 hours. Vinculin was used as a loading control. Data are presented as the mean ± SD of three wells from one of three experiments. Statistical analysis for (a–d) was performed using one-way ANOVA followed by Dunnett’s post hoc test (compared to Th17-polarizing condition without CuB). Statistical significance in (e) was determined using Student’s t -test. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus Th17-polarizing condition without CuB treatment.

Article Snippet: The cell lysates were then transferred onto PVDF membranes, which were incubated overnight at 4°C with the following primary antibodies: anti-phosphorylated STAT3 (Cell Signaling, Danvers, MA, USA), anti-STAT3 (Cell Signaling, Danvers, MA, USA), anti-phosphorylated JAK2 (Abcam, Cambridge, UK), anti-JAK2 (Abcam, Cambridge, UK), and anti-GAPDH (Cell Signaling, Danvers, MA, USA).

Techniques: Phospho-proteomics, Cell Differentiation, Cell Culture, Expressing, Concentration Assay, Flow Cytometry, Western Blot, 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: 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