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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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: After blocking endogenous peroxidase activity with 3% H 2 O 2 and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.).

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

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Western blot analysis of CLIP1::ROS1-fusion expression and SHP2/MAPK and STAT3 signaling in CLIP1::ROS1-fusion iNHA; ΔTUB: abrogated microtubule interaction domain; GAPDH: loading control, p-ROS1 (Tyr2274) antibody used to validate fusion transgene activity, phospho-SHP2 (Tyr580), and p-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity and p-STAT3 (Tyr705), and p-STAT1 (Tyr701) for STAT activation. (B) Violin plots highlighting track mean speed from (C) for CCDC88A::ALK (left) and CLIP1::ROS1 (right); dots represent mean of biological replicates, significance calculated on mean values, significance calculated using unpaired two-tailed Student’s t-test, *: p-value ≤0.05. (C) Illustrative images of live cell tracking; inverted nuclear fluorescence, colored lines visualize tracks of individual cells within 12 hours; scalebar: 200µm. (D) Illustrative images of SIA assays; scalebar: 500µm. (E) SIA quantification of invading ALK- and ROS1-fusion iNHAs; left graph: number of invading cells, right graph: mean distance of invasion; one-way ANOVA (normally distributed) or Kruskal Wallis test (not normally distributed), post-hoc Dunn-Bonferroni test, *: p-value≤0.05, **: p-value <0.01,***:p-value<0.001, ****: p-value <0.0001. (F) SIA quantification of invading CCDC88A::ALK (first two graphs) or CLIP1::ROS1 (last two graphs) iNHAs treated with indicated STAT3i (Stattic) concentrations; first and third graph: number of invading cells, second and fourth graph: mean distance of invasion; one-way ANOVA (normally distributed) or Kruskal Wallis test (not normally distributed), post-hoc Dunn-Bonferroni test, *: p-value≤0.05, **: p-value <0.01, ****: p-value <0.0001.

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: Western Blot, Expressing, Control, Activity Assay, Activation Assay, Two Tailed Test, Cell Tracking Assay, Fluorescence

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Affinity purification MS/MS identifying direct interactors of ALK- and ROS1-fusions used in this study; size: −log 10 BFDR, color gradient: log 2 EFC high (red) to low (grey). (B) Immunoprecipitation validating SHC1/3 as direct interactors of ALK-fusion (top two blot) and SHP2 as direct interactor of ROS1-fusion (bottom three blots),respectively; GAPDH: loading control, p-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, p-SHC1 (Tyr239/240), and p-SHP2 (Tyr580) antibodies used to validate activity of interactors; dashed lines: marker lane. (C) Western blot analysis of MAPK signaling in CCDC88::ALK and CLIP1::ROS1 models. GAPDH: loading control, p-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, p-SHP2 (Tyr580), p-GAB1 (Tyr642), p-MEK1/2 (Ser217/221), and p-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity, p-STAT3 (Tyr705), and p-STAT1 (Tyr701) used to validate STAT activation. (D) Western blot analysis of RNAi effect in PPP1CB::ALK models. GAPDH: loading control, p-ALK (Tyr1507) antibody used to validate retained ALK activity, p-SHP2 (Tyr580) and p-GAB1 (Tyr642), used to validate shPTPN11 , p-STAT3 (Tyr705), used to validate shSTAT3 . (E) Kaplan-Meier survival curves showing tumor induced mortality upon orthotopic intracranial injection of shRNA inhibited PPP1CB::ALK cells in NSG mice, groups are represented by individual curves, with a n=8 mice per group,; grey: PPP1CB::ALK shCtrl , dark petrol: PPP1CB::ALK shSTAT3 , light petrol: PPP1CB::ALK shPTPN11 ; statistical significance determined by log-rank test, **: p-value<0.01, *:p-value<0.05.

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: Affinity Purification, Tandem Mass Spectroscopy, Immunoprecipitation, Control, Activity Assay, Marker, Western Blot, Activation Assay, Injection, shRNA

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) In vitro kinase assay validating SHP2 and STAT3 as substrates of ALK- and ROS1-fusions; GAPDH: loading control, phospho-ALK,-ROS1 antibody used to validate KD mutants, phospho-SHP2 (Tyr580) or phospho-STAT3 (Tyr705) validate ALK- and ROS1-fusion kinase specificity towards SHP2 or STAT3, respectively. (B) Western blot analysis of MAPK signaling in ALK- and ROS1-fusion models. GAPDH: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, phospho-SHP2 (Tyr580), phospho-GAB1 (Tyr642), phospho-MEK1/2 (Ser217/221), and phospho-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity, phospho-STAT3 (Tyr705), and phospho-STAT1 (Tyr701) used to validate STAT activation. (C) Subcellular fractionation of CLIP1::ROS1 samples validating increased STAT3 activity; phospho-ROS1 (Tyr2274) antibody used to validate KD mutant and phospho-STAT3 (Tyr705) used to validate pathway activity, β-TUB: cytoplasmic marker, H3: nuclear marker. (D) Western blots analyzing the effect of RTK inhibition (Entrectinib 500nM, 4hours) on MAPK and STAT signaling in ALK- and ROS1-fusion models. GAPDH: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate inhibition, phospho-SHP2 (Tyr580), phospho-GAB1 (Tyr642), phospho-MEK1/2 (Ser217/221), and phospho-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway inhibition and phospho-STAT3 (Tyr705), and phospho-STAT1 (Tyr701) for STAT inhibition.

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: In Vitro, Kinase Assay, Control, Western Blot, Activity Assay, Activation Assay, Fractionation, Mutagenesis, Marker, Inhibition

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Western blot analysis of ALK- and ROS1-fusion expression and Shp2/Mapk and Stat3 signaling in ALK- and ROS1-fusion IUE models. β-Actin: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate fusion transgene activity, phospho-Shp2 (Tyr580), phospho-Gab1 (Tyr642), phospho-Mek1/2 (Ser217/221), and phospho-Erk1/2 (Thr202/Tyr204) used to validate Mapk pathway activity and phospho-Stat3 (Tyr705) for Stat3 activation. (B) Western blots analyzing the effect of RTK inhibition (Entrectinib 100nM, 24hours) on Mapk and Stat3 signaling CLIP1::ROS1-fusion IUE models. β-Tub: loading control, phospho-ROS1 (Tyr2274) antibody used to validate inhibition, phospho-Shp2 (Tyr580), phospho-Gab1 (Tyr642), phospho-Mek1/2 (Ser217/221), and phospho-Erk1/2 (Thr202/Tyr204) used to validate Mapk pathway inhibition and phospho-Stat3 (Tyr705) for Stat3 inhibition. (C) Drug titration curves highlighting dose dependent effects of 72h treatment with Entrectinib (left), or Stattic (right) on IUE models, dark green: CCDC88A::ALK #235, light green: CCDC88A::ALK #236, berry: CLIP1::ROS1 #187; light berry: CLIP1::ROS1 #192, y-axis: linear drug concentrations, y-axis: survival normalized to DMSO control; dashed line: 50% survival; error bars: SD of 3 biological replicates. ( D) Unsupervised clustering, Euclidian distance with complete linkage heatmap of most variable transcripts, color gradient: z-score high (red) to low (blue), samples indicated at the top. (E) Enriched GO:terms for DEG in CCDC88A::ALK (upper) or GOPC::ROS1 (lower) samples. x-axis: −log 10 p-value significance established by ReViGo; left: GO:terms enriched in Entrectinib treated samples, right: GO:terms enriched in DMSO control samples

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: Western Blot, Expressing, Control, Activity Assay, Activation Assay, Inhibition, Titration