constitutively active stat3 expression construct stat3-c flag prc/cmv  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Both direct and indirect suppression of MCL1 synergizes with BCLXL inhibition in preclinical models of gastric cancer

doi: 10.1038/s41419-025-07481-8

Figure Lengend Snippet: A , B Role of ELK1, STAT3 and SRF in regulating the transcriptional activity of MCL1 under basal conditions and in response to HER2-targeting drugs. SNU-216 cells expressing empty vector or the indicated transcriptional factors were left untreated or treated with 100 ng/ml trastuzumab ( A ) or 200 nM lapatinib ( B ) for 72 h or 48 h, respectively. The levels of MCL1 mRNA were determined using RT-qPCR. C Effect of HER2-targeting drugs on STAT3 and SRF expression. SNU-216, NCI-N87 and GCIY were treated with 100 ng/ml trastuzumab or 200 nM lapatinib for 72 h or 48 h, respectively. The protein levels of p-STAT3, total STAT3, and SRF were determined by Western blotting. D , E Dual luciferase reporter assays to identify the binding site(s) of STAT3 and SRF in the MCL1 promoter. Left: diagram depicting the putative binding sites of STAT3 ( D ) or SRF ( E ) in the MCL1 promoter predicted by the JASPAR database. Right: Dual luciferase reporter assays conducted in HEK293T. F Reduced binding of STATS and SRF in the MCL1 promoter upon HER2-targeting drug treatments. SNU-216 cells were left untreated or treated with 100 ng/ml trastuzumab or 200 nM lapatinib for 72 h or 48 h, respectively. ChIP was conducted using specific antibodies against STAT3, SRF, and an IgG isotype control. G Co-IP analysis of the interaction of endogenous STAT3 and SRF in SNU-216 cells. H Direct interaction of exogenous STAT3 and SRF. HEK293T cells were transiently transfected with HA-tagged STAT3, Flag-tagged SRF, either individually or both. Equivalent lysates were immunoprecipitated with anti-HA or anti-Flag antibody and immunoblotted with the indicated antibodies. I Effect of overexpressing or deleting STAT3 on SRF expression. The mRNA (left) and protein (right) levels of SRF in SNU-216 cells with STAT3 overexpression or depletion were determined. J Similar experiments to those in panel I were conducted to detect the effect of manipulating SRF on STAT3 expression in SNU-216 cells. K Reduced binding of STAT3 in the SRF promoter upon HER2-targeting drug treatments. Similar experiments to those in ( F ) were conducted. L Depletion of STAT3 attenuated the binding of SRF in the MCL1 promoter. Similar experiments to those in ( F ) were performed in SNU-216 cells engineered to express sg STAT3 . ChIP was conducted at 72 h after addition of DOX to induce the deletion of STAT3 . 2 sgRNAs were tested. M , N Deleting STAT3 enhanced the sensitivity of GC lines to BCLXL inhibition. SNU-216 ( M ) and GCIY ( N ) cells inducibly expressing sg STAT3 or the sgRNA empty vector were treated with DOX alone, or in combination with indicated concentrations of BCLXLi. Cell viability was determined 48 h later. Two-way ANOVA was used for statistical significance. O STAT3 inhibitor synergized with BCLXLi in GC cell lines, regardless of their p-STAT3 levels. GC cell lines with high p-STAT3 levels (HGC-27, MKN45 and AGS) and the ones with low p-STAT3 levels (SNU-216, NCI-N87, GCIY) were treated with indicated concentrations of BCLXLi and STAT3 inhibitor and cell viability was determined 48 h later. Data in ( A ), ( B ), ( D ), ( E ), ( I , left ), ( J , left ), ( M – O ) represent the means ± SD of ≥3 independent experiments; data in panel ( C ), ( F – H ), ( I , right ), ( J , right ), ( K ) and ( L ) are representatives of 2 independent experiments.

Article Snippet: The GV492 lentiviral expression constructs of human ELK1, ELK3, ELK4, STAT3, SRF, as well as the GV366 (C-terminally HA-tagged) and GV657 (C-terminally Flag-tagged) constructs for transient expression of human STAT3 and SRF were purchased from Shanghai Genechem Co., Ltd.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Luciferase, Binding Assay, Control, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Over Expression, Inhibition

Journal: Biomedicines

Article Title: Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

doi: 10.3390/biomedicines10071608

Figure Lengend Snippet: Figure 2. Analysis of STAT3 in HL cell lines. (A) RQ-PCR analysis of STAT3 in eight HL cell lines (left). Western blot analysis of three HL cell lines for STAT3, phospho (P)-STAT3 and alpha-tubulin (TUBA) as control (right). (B) Immuno-cytological microscopy of untreated AM-HLH cells (left) stained for STAT3 (green) and the nucleus (blue). Forced expression of GFP-labeled STAT3 in transfected AM- HLH (right). White arrowheads indicate transfected nuclei demonstrating reduced STAT3 protein in the nucleus as compared to the cytoplasm. The light blue arrowhead indicates a non-transfected cell. (C) Western blot analysis of IL6-treated AM-HLH for STAT3, P-STAT3 and TUBA (left). Immuno- cytological microscopy of IL6-treated AM-HLH cells stained for STAT3 (green) and the nucleus (blue) (middle). RQ-PCR analysis of HLX in IL6-treated AM-HLH after 4 h (right). (D) Immuno-cytological microscopy of control and TSA-treated L-540 cells stained for STAT3 (green) and the nucleus (blue). (E) RQ-PCR analysis of HLX in L-540 (left) AM-HLH (right) treated with TSA. Statistical significance was assessed by t-test and derived p-values indicated by asterisks (** p < 0.01, *** p < 0.001, n.s., not significant).

Article Snippet: A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany).

Techniques: Western Blot, Control, Microscopy, Staining, Expressing, Labeling, Transfection, Derivative Assay

Journal: Biomedicines

Article Title: Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

doi: 10.3390/biomedicines10071608

Figure Lengend Snippet: Figure 4. Knockdown and stimulation studies in AM-HLH and L-540. (A) SiRNA-mediated knock- down of IRF4 in AM-HLH resulted in elevated expression of STAT3 as analyzed by RQ-PCR (left). The

Article Snippet: A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany).

Techniques: Knockdown, Expressing

Journal: Biomedicines

Article Title: Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

doi: 10.3390/biomedicines10071608

Figure Lengend Snippet: Figure 6. Summary of the results from this study showing a gene regulatory network around STAT3. Genomic amplifications targeting particular genes are highlighted by a red background. Elevated genes are indicated in red, low level expressed genes in green while medium level expressed genes are shown in black. Functional consequences including immune escape, EBV activity, proliferation and survival are highlighted by a blue background. Bold lines indicate relationships demonstrated in this study while slight lines refer to published data.

Article Snippet: A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany).

Techniques: Functional Assay, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Shikonin inhibited STAT3 signaling pathway in melanoma cells. (A) Cells were treated with indicated concentration of shikonin for 24 h, expression levels of STAT3 and p-STAT3 were determined by the Western blot analysis (upper), and relative expression levels were analyzed by Image J software (bottom). (B) Cells were treated with 2 μM shikonin for 24 h, and then incubated with DSS. Changes in STAT3 dimerization were evaluated by immunoblotting. The arrows indicated the dimer (D) and the monomer (M) STAT3. The representative results (upper) and the relative expression levels of dimer STAT3 (bottom) were shown. (C, D) Cells were treated with indicated concentration of shikonin for 24 h, and then the intracellular distribution of STAT3 was analyzed by immunofluorescence assay (C) and immunoblotting (D) . Relative expression levels of cytosolic and nuclear STAT3 were also shown (bottom). Data were shown as mean ± SD from three independent experiments, * P < 0.05 and ** P < 0.01.

Article Snippet: Constitutively active STAT3 expression construct STAT3-C Flag pRc/CMV was obtained from Addgene (USA).

Techniques: Concentration Assay, Expressing, Western Blot, Software, Incubation, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Shikonin down-regulated protein levels and inhibited enzymatic activities of STAT3-targeted molecules in melanoma cells. A375 and A2058 cells were treated with indicated concentrations of shikonin for 24 h, and then total cell lysates were collected, expression levels of (A) Mcl-1, Bcl-2, (B) MMP-2, (D) Twist, Vimentin, and N-cadherin were evaluated by Western blot analysis (left) and relative expression levels were analyzed by Image J software (right). Data were shown as mean ± SD from three independent experiments, * P < 0.05 and ** P < 0.01. (C) The enzymatic activities of MMP-2 and MMP-9 were determined by gelatinase zymography.

Article Snippet: Constitutively active STAT3 expression construct STAT3-C Flag pRc/CMV was obtained from Addgene (USA).

Techniques: Expressing, Western Blot, Software, Zymography

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Overexpression of STAT3 in human melanoma A375 cells reduced shikonin-mediated cell growth, migration, and invasion inhibition. A375 cells were transiently transfected with an empty vector or a STAT3C-expressing construct for 24 h, and then (A) the expression level of STAT3 in A375-STAT3C cells and A375-EV cells were examined by immunoblotting. (B–D) A375-EV or A375-STAT3C cells were incubated with shikonin for 48 h or 24 h, and then (B) cell proliferation, (C) cell migratory, and (D) cell invasive abilities were measured. Representative photographs of migrated cells (left) and quantification of these cells (right) were shown. Data were mean ± SD from three independent experiments, ## P < 0.01, * P < 0.05, and ** P < 0.01.

Article Snippet: Constitutively active STAT3 expression construct STAT3-C Flag pRc/CMV was obtained from Addgene (USA).

Techniques: Over Expression, Migration, Inhibition, Transfection, Plasmid Preparation, Expressing, Construct, Western Blot, Incubation