phosphorylated stat3  (Novus Biologicals)

Journal: International Journal of Oncology

Article Title: Smoking promotes colorectal cancer via the CKAP2L/AREG axis

doi: 10.3892/ijo.2026.5872

Figure Lengend Snippet: CKAP2L promotes proliferation and migration of colorectal cancer cells through the STAT3/AREG/EGFR axis. (A) Expression levels of STAT3 and p-STAT3 proteins measured by western blotting. (B) Calculation of IC 50 in HCT116 cells treated with Stattic for 24 h. (C) Levels of AREG were measured by reverse transcription-quantitative PCR and enzyme-linked immunosorbent assay. (D) Migration of cells was detected by Transwell assay (×100 magnification). (E) Proliferation of cells was measured by Cell Counting Kit 8. (F) Expression levels of proteins measured by western blotting. (G) Binding peak of STAT3 on the promoter region of AREG was detected by Cistrome Data Browser, the binding motif was predicted using the JASPAR database, and the binding of STAT3 to the AREG promoter was evaluated by chromatin immunoprecipitation assay. Data were analyzed using (A and G) Unpaired Student's t-test, and (C-F) two-way ANOVA followed by Tukey test. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. AREG, amphiregulin; CKAP2L, cytoskeleton-associated protein 2-like; EGFR, epidermal growth factor receptor; NC, negative control; p-, phosphorylated; sh, short hairpin; STAT3, signal transducer and activator of transcription 3; TSS, transcription start site.

Article Snippet: The signal transducer and activator of transcription 3 (STAT3) phosphorylation inhibitor Stattic (cat. no. HY-13818; MedChemExpress) was dissolved in DMSO for cell culture.

Techniques: Migration, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transwell Assay, Cell Counting, Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: International Journal of Oncology

Article Title: Smoking promotes colorectal cancer via the CKAP2L/AREG axis

doi: 10.3892/ijo.2026.5872

Figure Lengend Snippet: Smoking may promote colorectal cancer progression through the CKAP2L/STAT3/AREG/EGFR axis. This figure was drawn by Figdraw ( https://www.figdraw.com/ ). AREG, amphiregulin; CKAP2L, cytoskeleton-associated protein 2-like; EGFR, epidermal growth factor receptor; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The signal transducer and activator of transcription 3 (STAT3) phosphorylation inhibitor Stattic (cat. no. HY-13818; MedChemExpress) was dissolved in DMSO for cell culture.

Techniques:

Journal: Oncology Reports

Article Title: Knockdown of CCT2 inhibits the malignant progression of hepatocellular carcinoma cells by impairing STAT3 activation

doi: 10.3892/or.2026.9086

Figure Lengend Snippet: Knockdown of CCT2 inhibits STAT3 signaling activation in hepatocellular carcinoma cells. The protein levels of STAT3, p-STAT3, MCL1, MMP2 and SOX2 in (A) Huh-7 and (B) HCCLM3 cells were measured by western blotting. The protein levels of (C) p-STAT3, (D) MCL1, (E) MMP2 and (F) SOX2 in subcutaneous tumor tissue were detected by immunohistochemical staining. *P<0.05, **P<0.01 vs. sh-NC. CCT2, chaperonin containing TCP1 subunit 2; sh, short hairpin; NC, negative control; p, phosphorylated; MCL1, myeloid cell leukemia sequence 1; SOX2, SRY-box transcription factor 2.

Article Snippet: The primary antibodies were as follows: CCT2 (cat. no. 24896-1-AP), β-actin (cat. no. 66009-1-Ig), MMP2 (cat. no. 10373-2-AP), myeloid cell leukemia sequence 1 (MCL1; cat. no. 16225-1-AP) and SRY-box transcription factor 2 (SOX2; cat. no. 11064-1-AP; all Proteintech Group, Inc.) and STAT3 (cat. no. 4904) and phosphorylated (p-)STAT3 (Tyr705; cat. no. 4113; both Cell Signaling Technology, Inc.) The membranes were washed three times in TBST (0.1% Tween-20) for 5 min each at room temperature.

Techniques: Knockdown, Activation Assay, Western Blot, Immunohistochemical staining, Staining, Negative Control, Sequencing

Journal: Oncology Reports

Article Title: Knockdown of CCT2 inhibits the malignant progression of hepatocellular carcinoma cells by impairing STAT3 activation

doi: 10.3892/or.2026.9086

Figure Lengend Snippet: IL-6 abolishes the effects of CCT2 knockdown on the proliferation and invasion of hepatocellular carcinoma cells. The protein levels of STAT3 and p-STAT3 in (A) Huh-7 and (B) HCCLM3 cells were measured by western blotting. The proliferation of (C) Huh-7 and (D) HCCLM3 cells following CCT2 knockdown and IL-6 treatment was detected using EdU incorporation assay. The invasion of (E) Huh-7 and (F) HCCLM3 cells was assessed using Transwell assay. *P<0.05, **P<0.01 vs. PBS. CCT2, chaperonin containing TCP1 subunit 2; sh, short hairpin; p-, phosphorylated.

Article Snippet: The primary antibodies were as follows: CCT2 (cat. no. 24896-1-AP), β-actin (cat. no. 66009-1-Ig), MMP2 (cat. no. 10373-2-AP), myeloid cell leukemia sequence 1 (MCL1; cat. no. 16225-1-AP) and SRY-box transcription factor 2 (SOX2; cat. no. 11064-1-AP; all Proteintech Group, Inc.) and STAT3 (cat. no. 4904) and phosphorylated (p-)STAT3 (Tyr705; cat. no. 4113; both Cell Signaling Technology, Inc.) The membranes were washed three times in TBST (0.1% Tween-20) for 5 min each at room temperature.

Techniques: Knockdown, Western Blot, Transwell Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Top pathways enriched in SAVI P5 vs. P1 (iso-SAVI_SAVI cohort) by Ingenuity Pathway Analysis (IPA). Positive z-scores indicate pathway activation. Additional HC vs. SAVI analysis is shown in Figure S3A–B. B . EndMT signature heatmap in parental SAVI and isogenic iECs, with box-and-whisker plots of representative endothelial, mesenchymal, and previously reported EndMT-associated genes. Asterisks mark genes upregulated under TGFβ-induced EndMT but downregulated in SAVI iECs. C. IPA-derived transcription factor network of significant upstream regulators in the iso-SAVI_SAVI cohort (orange = activated; blue = inhibited). D . Heatmap of activation z-scores for the same transcription factors in panel C, showing similar patterns in SAVI vs. HC iECs and SAVI vs. HC lung tissue. E . Constitutive STAT3 nuclear translocation in SAVI iECs. STAT3 immunofluorescence (red) with DAPI (blue) at P5 from one SAVI and matched isogenic control line; white arrows indicate nuclear STAT3 (scale bar: 20 µm). Quantification reflects STAT3⁺ nuclei per total DAPI⁺ cells across 7–11 fields from 3–4 wells (mean ± SEM; *p < 0.05, two-tailed unpaired t-test). F . STAT3 activation in SAVI lung biopsies. Lung sections from 4 HC and 3 SAVI patients were stained for p-STAT3 Y705 (red) and CD31 (green) with DAPI (blue). Representative images (scale bar: 20 µm) and quantification of p-STAT3 Y705 /DAPI ratios (≥5 images per patient) show increased STAT3 activation in SAVI (mean ± SEM; ***p < 0.001, two-tailed unpaired t-test).

Article Snippet: Primary antibodies for CD31 (1:50, DAKO Cat#M0823), VE-cadherin (VEcad) (1:100, Santa Cruz Cat# sc-9989), or Phosphorylated STAT3 (P-STAT3 Y705 ) (1:100 Cell Signaling Technology Cat#9145S) were applied overnight at 4°C.

Techniques: Activation Assay, Whisker Assay, Derivative Assay, Translocation Assay, Immunofluorescence, Control, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Diagram of the Canonical and Non-canonical STING signaling (created with BioRender.com ). B. Constitutive STAT3 and STAT1 activation in SAVI iECs. Phospho-STAT3 Y705 and phospho-STAT1 Y701 (normalized to total protein) were measured in iECs from 3 HC and 3 SAVI donors across passages 1, 3, and 5. SAVI iECs showed increased baseline STAT3 activation (mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05, unpaired t-test). C. STING/TBK1 inhibition blocks acute cGAMP-induced STAT3 activation in HC iECs. iECs from 3 HC donors were stimulated with 2’3’-cGAMP (20µg/ml) for 30 min–24 h. STING inhibitor IFM35883 (STINGi, 2.5 μM) and TBK1 inhibitor MRT67307 (TBK1i, 5 μM) were pre-treated one hour before cGAMP. STAT3 activation at 4h was prevented by STING/TBK1 inhibition (mean ±SEM, ***p < 0.001, **p < 0.01, Mann-Whitney test). D. STING/TBK1 inhibition reduces constitutive STAT3 activation in SAVI iECs. SAVI iECs were treated with IFM35883 (2.5 μM) from P2–P5 or with TBK1i (5 μM) for 48 h. Quantification is from four experiments in iEC line from patient SAVI1 (mean ±SEM, ***p < 0.001, unpaired t-test). E. STAT3 shRNA knockdown (KD), not STAT1 KD preserves CD144 (VE-cadherin) in SAVI iECs. iECs from 3 individual SAVI patients infected with control, STAT3, or STAT1 shRNA at P2 were analyzed by flow cytometry at P5 (mean ±SEM, **p < 0.01, paired t-test). Representative flow cytometry profiles are shown in Figure S4C. F. Constitutive protein expression of SLUG/ SNAI2 is elevated in SAVI iECs and suppressed by STING inhibition. Protein from 3 HC, 3 SAVI, and 3 iso-SAVI iECs at P5 showed increased SLUG (normalized by GAPDH) in SAVI; IFM35883 (2.5 μM) treatment from P2–P5 prevented SLUG upregulation in SAVI iECs (SAVI 1) (mean ±SEM, ***p < 0.001, **p < 0.01, unpaired t-test). See also Figure S4E. G. STAT3 shRNA knockdown reduces SLUG mRNA expression (normalized by internal control 18S) in SAVI iECs at P3. Data are summarized from 3 individual SAVI patients iEC lines. *p < 0.01 as determined by two-tailed unpaired t-test.

Article Snippet: Primary antibodies for CD31 (1:50, DAKO Cat#M0823), VE-cadherin (VEcad) (1:100, Santa Cruz Cat# sc-9989), or Phosphorylated STAT3 (P-STAT3 Y705 ) (1:100 Cell Signaling Technology Cat#9145S) were applied overnight at 4°C.

Techniques: Activation Assay, Inhibition, MANN-WHITNEY, shRNA, Knockdown, Infection, Control, Flow Cytometry, Expressing, Two Tailed Test

Journal: ImmunoTargets and Therapy

Article Title: Kasugamycin Inhibits Melanoma Lung Metastasis and CHI3L1-Driven M2-Like Tumor-Associated Macrophage Differentiation

doi: 10.2147/ITT.S563951

Figure Lengend Snippet: KSM inhibits CHI3L1-stimulated M2 macrophage activation via EGFR. Bulk RNA-seq sequencing analysis on the differentiated macrophages from THP-1 cells was used to evaluate mRNA expression in the macrophage differentiation regulated by CHI3L1 and KSM. ( A ). Volcano plots showing differentially expressed genes regulated by CHI3L1. ( B ) Representative plots of the top 20 genes that are upregulated (> 2-fold) by CHI3L1 stimulation but downregulated (< 2-fold) by KSM treatment. ( C ) Representative mRNA expression of EGFR in differentiated macrophages with CHI3L1 and KSM treatment was detected by real-time qRT-PCR. ( D ) Representative immunoblots showing p-EGFR (Tyr1068) and total EGFR expression in differentiated macrophages with CHI3L1 and KSM. Right panel, densitometric quantitation on the blots of EGFR and p-EGFR. ( E ) Effect of EGFR inhibition on the expression of p-STAT3 and total-STAT3 in CHI3L1-stimulated, differentiated macrophages by gefitinib (Tocris Bioscience, #3000) treatment (1 μM, 72 hours). Right panel, densitometric quantitation on the blots of p-STAT3 and total STAT3. ( F ) Effects of EGFR inhibition on the expression of CD163 and CD206 in CHI3L1-stimulated, differentiated macrophages by gefitinib treatment (0.1 and 1 μM, 72 hours). Right panel, densitometric quantitation on the blots of CD163 and CD206. The values in ( C – F ) are the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001 (One-Way ANOVA, multiple comparisons).

Article Snippet: Protein lysates (25 μg) from lung tissues or cells were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with primary antibodies against CD163 (#PA5-109327, Thermo Fisher Scientific), CD206 (E6T5J, #24595S, Cell Signaling Technology), phosphorylated AKT (p-Akt) (193H12, #4058S, Cell Signaling Technology), and total Akt (11E7, #4685S, Cell Signaling Technology), phosphorylated EGFR (Tyr1068) (p-EGFR) (#44-788G, Thermo Fisher Scientific), EGFR (D38B1, #4267S, Cell Signaling Technology), phosphorylated Erk (p-Erk) (#9101S, Cell Signaling Technology), total Erk (#9102S, Cell Signaling Technology), phosphorylated STAT3 (Tyr705) (p-STAT3) (D3A7, #9145, Cell Signaling Technology), total STAT3 (79D7, #4904, Cell Signaling Technology) and b-actin (C4, #sc-47778 HRP, Santa Cruz Biotechnology).

Techniques: Activation Assay, RNA Sequencing, Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Inhibition