recombinant full length human stat3  (Creative BioMart)

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 selectively targets STAT3. (A) Structure of YY002. (B) YY002 inhibited the STAT3 luciferase reporter activity. IL-6 was used as an activator of STAT3 ( n = 2). (C) YY002 inhibited the ATP production ( n = 2). (D) YY002 inhibited the OXPHOS rate (OCR = oxygen consumption rate, n = 2). (E) Direct binding of YY002 to STAT3 SH2 or STAT3 127–722 was determined by MST experiments ( n = 3). (F) The binding affinities between YY002 and STAT3 was detected by surface plasmon resonance (SPR) assay. (G) YY002 selectively bound to STAT3 and other STAT members ( n = 3). (H) The shRNA knock-down efficiency in PANC-1 and Capan-2 was confirmed by Western Blot. (I, J) The pancreatic cancer cell lines shNC, shSTAT3-1# and shSTAT3-2# of PANC-1 (I) and Capan-2 (J) were treated with YY002, and the proliferation was determined after 72 h of treatment ( n = 3). (K) BxPC3 cells were treated with 50 nM YY002 for 24 h, and then RNA-Seq assay was performed. The results were analyzed by gene set enrichment analysis (GSEA). (L) Capan-2 cells were treated with different concentrations of STAT3 inhibitors or YY002, and the proliferation was tested by MTS ( n = 2). (M) YY002 and Stattic directly bound to STAT3 127–722 , The binding affinities were determined by MST experiments ( n = 3). Data shown as mean ± SD ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by one-way ANOVA following multiple comparison.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Luciferase, Activity Assay, Binding Assay, SPR Assay, shRNA, Knockdown, Western Blot, RNA Sequencing, Comparison

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 directly binds to STAT3 SH2 domain. (A) Computer docking model predicting that YY002 bound to the STAT3-SH2 domain. (B, C) YY002 binding to STAT3-SH2 and its indicated mutants. The binding affinities were measured by MST experiments ( n = 3). (D) PANC-1 shSTAT3-1# cells that were infected with indicated lentivirus vectors to reintroduce specific SH2 mutants were treated with indicated YY002 concentrations, and cell viability was measured ( n = 3). Data shown as mean ± SD. (E) Re-expression of STAT3 mutant in STAT3 knockdown PDAC cells. The shSTAT3-1# PANC-1 cells that were infected with the indicated lentivirus expression vectors, and the transfection efficacy was detected by Western blots. (F) In vitro enzyme inhibition assays.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Binding Assay, Infection, Expressing, Mutagenesis, Knockdown, Transfection, Western Blot, In Vitro, Enzyme Inhibition Assay

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits pancreatic cancer cell growth, STAT3 Tyr705 phosphorylation and STAT3 nuclear function. (A) YY002 inhibited the proliferation of pancreatic cancer cell lines but only had a limited effect on normal cells ( n = 2). (B) The protein expression level of STAT3, p-STAT3 Tyr705 and p-STAT3 Ser727 in different cell lines. (C) STAT3 phosphorylation and downstream gene expression were measured by Western Blot in Capan-2, HPAC and BxPC-3 cells after treatment with YY002. (D) Down-stream gene expression was measured by quantitative real-time PCR in Capan-2, HPAC and BxPC-3 cells after treatment with YY002 ( n = 2). (E) YY002 inhibited the entry of STAT3 into the nucleus of BxPC3, Capan-2 and PANC-1 cells. Scale bar, 50 μm. Data shown as mean ± sd. ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by one-way ANOVA following multiple comparison.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Phospho-proteomics, Expressing, Gene Expression, Western Blot, Real-time Polymerase Chain Reaction, Comparison

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits STAT3 Ser727 phosphorylation and mitochondrial OXPHOS. (A) Oxygen consumption rate (OCR) was evaluated by the Seahorse XF96 extracellular flux analyzer and OXPHOS was inhibited in BxPC-3 ( n = 2), CFPAC-1 ( n = 3) and Capan-2 ( n = 4) cells treated with YY002. (B) Knockdown of STAT3 in CFPAC-1 inhibited OXPHOS ( n = 3). (C) CFPAC-1 shSTAT3-1# cells were treated with YY002 or vehicle (DMSO), and the level of OXPHOS was measured ( n = 3). (D, E) The glycolysis level of BxPC-3 (D) and CFPAC-1 (E) cells treated with different concentrations of YY002 was determined ( n = 3). (F) A brief description of the mitochondrial electron transport chain. (G, H) Capan-2 cells were treated with 50 nM YY002 or vehicle control and injected with the indicated drugs 1, 2, 3, 4 sequentially, and the mitochondrial respiration was determined by Seahorse instrument assay ( n = 3). Data shown as mean ± sd.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Phospho-proteomics, Knockdown, Control, Injection

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits pancreatic cancer growth in vivo . (A, B) The PANC-1 tumor volumes of mice were recorded every 4–5 days ( n = 8). Data shown as mean ± SEM * P < 0.05, * * P < 0.01 and **** P < 0.0001 by One-way ANOVA followed multiple comparison. (C) At the end of the experiment, the tumors in each group were excised, weighed and counted. (D-E) MIA PaCa-2 cells were injected into mice which were treated with different concentrations of YY002 orally ( n = 8). (F) The tumor volume was recorded every 4 days and after 24 days of administration, the tumors were harvested. Data shown as mean ± SEM * P < 0.05, * * P < 0.01 and **** P < 0.0001 by One-way ANOVA followed multiple comparison. At the end of the experiment, the tumors in each group were excised, weighed and counted. (G) Quantification of STAT3, pSTAT3 Tyr705 , and pSTAT3 Ser727 immuno-staining and representative images of PANC-1 tumor.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: In Vivo, Comparison, Injection, Immunostaining

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits metastasis of pancreatic cancer in vivo . (A) PAN02-Luciferase cells were implanted orthotopically into the pancreas tails of male C57/BL6 mice. Different concentrations of YY002 were given orally for 4 weeks. Quantification of bioluminescence in pancreatic orthotopic xenograft model( n = 7). (B) PAN02-Luciferase cells were inoculated intravenously into male C57/BL6 mice. Quantification of bioluminescence in pancreatic cancer liver metastasis mouse model ( n = 8). (C) Overall survival rates of the additional independent orthotopic of pancreatic cancer. Log-rank (mantel-cox) test was used ( n = 9 in Control, 10 mg/kg, 20 mg/kg groups; n = 8 in 5 mg/kg group). (D) Overall survival rates of the additional independent liver metastatic models of pancreatic cancer. Log-rank (mantel-cox) test was used ( n = 8 in each group). (E) AsPC-1 cells were implanted orthotopically into the pancreas tails of mice, and then mice were treated with different concentrations of YY002 for 4 weeks, The survival time was counted continuously ( n = 9 in Control, 10 mg/kg, 20 mg/kg groups; n = 8 in 5 mg/kg group). Data shown as mean ± sd. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by One-way ANOVA followed multiple comparison. (F-J). YY002 induced the death of other STAT3-dependent tumors. (F) YY002 induced various STAT3-dependent cancer cells death. The tumor cells treated with YY002 in different concentrations, the cell viabilities were determined by MTS assays ( n = 2). (G, H) The STAT3 phosphorylation and the expression of STAT3 were measured by Western Blot in MDA-MB-231 and SUDHL-1 cells after treatment with YY002. (I) MDA-MB-231 cells were injected into mice which were treated with YY002 or BBI608 orally ( n = 10). (J) SUDHL-1 cells were injected into mice which were treated with YY002 or BBI608 orally ( n = 10).

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: In Vivo, Luciferase, Control, Comparison, Phospho-proteomics, Expressing, Western Blot, Injection