phosphorylated p stat3  (Cell Signaling Technology Inc)

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: 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