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PRMT5 potentiates <t>STAT3</t> activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

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1) Product Images from "PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7"

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

Journal: Advanced Science

doi: 10.1002/advs.202003047

PRMT5 potentiates STAT3 activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

Figure Legend Snippet: PRMT5 potentiates STAT3 activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

Techniques Used: Activation Assay, Stable Transfection, Expressing, Control, Western Blot, Knockdown, Phospho-proteomics, Transfection, Inhibition, Methylation, Transduction

Figure Legend Snippet: PRMT5 methylates Smad7 on R57. A) PRMT5 methylates Smad7. HEK293T cells were transfected with expression plasmids carrying MYC‐PRMT5/MEP50 and an SFB‐tagged construct, including gp130, JAK2, STAT3, Smad7, SHP2, and SOCS3. Cell lysates were harvested and precipitated with streptavidin beads. The retrieved complexes and input were analyzed by Western blotting with indicated antibodies. B) PRMT5 methylates the R57 residue on Smad7. HEK293T cells were transfected with MYC‐PRMT5/MEP50 and an SFB‐tagged Smad7 construct, i.e., wildtype Smad7 (WT) or a R‐to‐K substitution of Smad7 as indicated above the blots. Cell lysate was precipitated with streptavidin beads. Arginine di‐methylation of Smad7 was detected by Western blotting analysis. C) Mass spectrum of Smad7 Arg‐57 dimethylated peptide. Mass spectrometry identified Arg‐57 dimethylation of Smad7 in HEK293T cells expressing MYC‐PRMT5/MEP50 and SFB‐Smad7. Mass spectrometry profile of Smad7 sequence covering residue 47–64 is shown, and the dimethylated arginine side chains are indicated. D) PRMT5/MEP50 methylate Smad7, but not the R57K mutant. HEK293T cells were transfected MYC‐PRMT5/MEP50 and SFB‐Smad7 or Smad7 R57K mutant for 36 h. Cell lysates were harvested and immunoprecipitated with SYM10 antibody. The immunocomplexes and inputs were analyzed by Western blotting with indicated antibodies. E) PRMT5 methylates Smad7 in vitro. MYC‐PRMT5 or MYC‐G367A/R368A together with MYC‐MEP50 were immunopurified using anti‐MYC antibody from transfected HEK293T cells. Purified recombinant GST‐Smad7, GST‐Smad7 R57K mutant, and GST‐Smad4 were produced in E. coli . GST proteins and MYC‐PRMT5/MEP50 proteins were incubated in the presence of S‐adenosyl‐methionine to allow methylation reaction. Dimethylated Smad7 on R57 was detected by using Western blotting analysis.

Techniques Used: Transfection, Expressing, Construct, Western Blot, Residue, Methylation, Mass Spectrometry, Sequencing, Mutagenesis, Immunoprecipitation, In Vitro, Purification, Recombinant, Produced, Incubation

Arg methylation enhances Smad7 binding to gp130. A) Smad7 methylation increases its association with gp130. HEK293T cells were transfected with SFB‐Smad7 or Smad7 R57K mutant and HA‐gp130, together with MYC‐PRMT5/MEP50. Cell lysates were harvested and immunoprecipitated with Streptavidin beads. Western blotting analysis was done with appropriate antibodies. B) PRMT5 depletion blocks Smad7 methylation and its interaction with endogenous gp130. A549 tet‐on cells expressing SFB‐Smad7 were cultured with or without 1 µg mL −1 Dox for 3 d and then transfected with 40 × 10 −12 m siPRMT5. Cell lysates were harvested and immunoprecipitated with streptavidin beads. Endogenous gp130 was detected from the immunoprecipitates by using Western blotting analysis. C) Methylated Smad7 binds more tightly to gp130. HEK293T cells were transfected with indicated expression plasmids for MYC‐PRMT5, MYC‐G367A/R368A, and MYC‐MEP50 as well as SFB‐Smad7 or SFB‐R57K. Dimethylated Smad7 was immunopurified using SYM10 antibody, while total Smad7 was retrieved using an‐FLAG antibody. Bacterially expressed GST‐gp130‐ICD was purified using glutathione‐sepharose and eluted with elution buffer (10 × 10 −3 m glutathione, pH 8.0). In the in vitro binding experiments for evaluating the Smad7‐gp130 interaction, recombinant GST‐gp130‐ICD was added to the immunopurified Smad7. gp130‐ICD binding to immobilized Smad7 was analyzed by using Western blotting. D) Unmethylatable Smad7 R57K mutant loses its ability to potentiate STAT3 activation. MCF10A tet‐on cells stably expressing SFB‐Smad7 or Smad7 R57K were induced with 10 ng mL −1 Dox for 3 d, and treated with indicated concentrations of IL‐6. Cell lysates were collected and subject to Western blotting analysis.

Figure Legend Snippet: Arg methylation enhances Smad7 binding to gp130. A) Smad7 methylation increases its association with gp130. HEK293T cells were transfected with SFB‐Smad7 or Smad7 R57K mutant and HA‐gp130, together with MYC‐PRMT5/MEP50. Cell lysates were harvested and immunoprecipitated with Streptavidin beads. Western blotting analysis was done with appropriate antibodies. B) PRMT5 depletion blocks Smad7 methylation and its interaction with endogenous gp130. A549 tet‐on cells expressing SFB‐Smad7 were cultured with or without 1 µg mL −1 Dox for 3 d and then transfected with 40 × 10 −12 m siPRMT5. Cell lysates were harvested and immunoprecipitated with streptavidin beads. Endogenous gp130 was detected from the immunoprecipitates by using Western blotting analysis. C) Methylated Smad7 binds more tightly to gp130. HEK293T cells were transfected with indicated expression plasmids for MYC‐PRMT5, MYC‐G367A/R368A, and MYC‐MEP50 as well as SFB‐Smad7 or SFB‐R57K. Dimethylated Smad7 was immunopurified using SYM10 antibody, while total Smad7 was retrieved using an‐FLAG antibody. Bacterially expressed GST‐gp130‐ICD was purified using glutathione‐sepharose and eluted with elution buffer (10 × 10 −3 m glutathione, pH 8.0). In the in vitro binding experiments for evaluating the Smad7‐gp130 interaction, recombinant GST‐gp130‐ICD was added to the immunopurified Smad7. gp130‐ICD binding to immobilized Smad7 was analyzed by using Western blotting. D) Unmethylatable Smad7 R57K mutant loses its ability to potentiate STAT3 activation. MCF10A tet‐on cells stably expressing SFB‐Smad7 or Smad7 R57K were induced with 10 ng mL −1 Dox for 3 d, and treated with indicated concentrations of IL‐6. Cell lysates were collected and subject to Western blotting analysis.

Techniques Used: Methylation, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Cell Culture, Purification, In Vitro, Recombinant, Activation Assay, Stable Transfection

PRMT5 promotes STAT3 transcriptional and growth‐promoting responses. A) PRMT5 inhibition attenuates CDC25C expression in A549 cells. EPZ015666 or GSK591 (20 × 10 −6 m ) were added to A549 cells for 48 h. Cell lysates were harvested and analyzed by using qRT‐PCR to examine CDC25C mRNA levels. Data are shown as mean ± SD; n = 3. *** P < 0.001. B) PRMT5 inhibition attenuates CCNB1 expression in A549 cells. Cell treatment, harvest, and qRT‐PCR analysis were done as described in Panel A. Data are shown as mean ± SD; n = 3. *** P < 0.001. C) PRMT5 deficiency disables IL‐6/STAT3 responsiveness. GSEA showed that downregulated genes in PRMT5‐depleted A549 cells (shPRMT5‐2) were highly enriched in the IL‐6/STAT3 signaling gene set. Red, upregulated genes; blue, downregulated genes. NES = ‐1.73, FDR q value = 0.002. D) Heatmap showing expression levels (log 2 FPKM; left) and relative expression changes (log 2 (shPRMT5‐2/shCtrl); right) of the IL‐6/STAT3 signaling genes. E) Depletion of PRMT5 reduces DNA synthesis. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were subject to EdU staining to determine DNA incorporating rate (RiboBio),20x. F) Statistic analysis of the result in panel E. Data are shown as mean ± SD; n = 3. 0.01 < * P < 0.05. G) Inhibition of PRMT5 attenuates invasiveness in A549 cells. A549 cells were treated with PRMT5 inhibitors EPZ015666 or GSK591 (20 × 10 −6 m ) for 2 d, starved overnight in FBS‐free medium. 1 × 10 5 cells were plated in a transwell chamber and stained with crystal violet after 12 h. Purple color indicates crystal violet staining of the invaded cell population. H) PRMT5 depletion blocks colony formation. A549 stable cells were subject to crystal violet staining and photography.

Figure Legend Snippet: PRMT5 promotes STAT3 transcriptional and growth‐promoting responses. A) PRMT5 inhibition attenuates CDC25C expression in A549 cells. EPZ015666 or GSK591 (20 × 10 −6 m ) were added to A549 cells for 48 h. Cell lysates were harvested and analyzed by using qRT‐PCR to examine CDC25C mRNA levels. Data are shown as mean ± SD; n = 3. *** P < 0.001. B) PRMT5 inhibition attenuates CCNB1 expression in A549 cells. Cell treatment, harvest, and qRT‐PCR analysis were done as described in Panel A. Data are shown as mean ± SD; n = 3. *** P < 0.001. C) PRMT5 deficiency disables IL‐6/STAT3 responsiveness. GSEA showed that downregulated genes in PRMT5‐depleted A549 cells (shPRMT5‐2) were highly enriched in the IL‐6/STAT3 signaling gene set. Red, upregulated genes; blue, downregulated genes. NES = ‐1.73, FDR q value = 0.002. D) Heatmap showing expression levels (log 2 FPKM; left) and relative expression changes (log 2 (shPRMT5‐2/shCtrl); right) of the IL‐6/STAT3 signaling genes. E) Depletion of PRMT5 reduces DNA synthesis. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were subject to EdU staining to determine DNA incorporating rate (RiboBio),20x. F) Statistic analysis of the result in panel E. Data are shown as mean ± SD; n = 3. 0.01 < * P < 0.05. G) Inhibition of PRMT5 attenuates invasiveness in A549 cells. A549 cells were treated with PRMT5 inhibitors EPZ015666 or GSK591 (20 × 10 −6 m ) for 2 d, starved overnight in FBS‐free medium. 1 × 10 5 cells were plated in a transwell chamber and stained with crystal violet after 12 h. Purple color indicates crystal violet staining of the invaded cell population. H) PRMT5 depletion blocks colony formation. A549 stable cells were subject to crystal violet staining and photography.

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, DNA Synthesis, Stable Transfection, Control, Staining

PRMT5 promotes lung tumorigenesis. A) Depletion of PRMT5 attenuates tumorigenesis. LLC cells stably expressing shControl or mouse sh‐mPRMT5‐1 or sh‐mPRMT5‐2 were subcutaneously injected into female nude mice. Ten days after implantation, tumors were dissected and photographed. B) Measurement of tumor weight in Panel A. Data are shown as mean ± SD; n = 5 for each group. 0.01 < * P < 0.05. C) PRMT5 depletion impairs STAT3 signaling in tumors. Tumor samples were analyzed by Western blotting to examine phosphorylated STAT3 (p‐STAT3) and STAT3 target gene products such as c‐Myc and Survivin. D) PRMT5 is highly expressed in nonsmall cell lung cancer tissues (NSCLC). NSCLC tissue microarray (Alenabio) was subject to immunohistochemistry (Servicebio) using PRMT5 antibody. E) Statistic analysis of IHC score in Panel D. Statistical analysis was performed using a two‐tailed Student's t ‐test. Data are shown as mean ± SD. Lung cancer samples = 45. Normal lung tissue samples = 55. *** P < 0.001. F) A working model for PRMT5‐mediated STAT3 activation.

Figure Legend Snippet: PRMT5 promotes lung tumorigenesis. A) Depletion of PRMT5 attenuates tumorigenesis. LLC cells stably expressing shControl or mouse sh‐mPRMT5‐1 or sh‐mPRMT5‐2 were subcutaneously injected into female nude mice. Ten days after implantation, tumors were dissected and photographed. B) Measurement of tumor weight in Panel A. Data are shown as mean ± SD; n = 5 for each group. 0.01 < * P < 0.05. C) PRMT5 depletion impairs STAT3 signaling in tumors. Tumor samples were analyzed by Western blotting to examine phosphorylated STAT3 (p‐STAT3) and STAT3 target gene products such as c‐Myc and Survivin. D) PRMT5 is highly expressed in nonsmall cell lung cancer tissues (NSCLC). NSCLC tissue microarray (Alenabio) was subject to immunohistochemistry (Servicebio) using PRMT5 antibody. E) Statistic analysis of IHC score in Panel D. Statistical analysis was performed using a two‐tailed Student's t ‐test. Data are shown as mean ± SD. Lung cancer samples = 45. Normal lung tissue samples = 55. *** P < 0.001. F) A working model for PRMT5‐mediated STAT3 activation.

Techniques Used: Stable Transfection, Expressing, Injection, Western Blot, Microarray, Immunohistochemistry, Two Tailed Test, Activation Assay

Incubation:

Article Title: Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling
Article Snippet: A total of 10 μg protein per lane was separated via SDS-PAGE using 10% Tris-Gly SDS-PAGE precast gels (Beyotime Biotechnology; cat. no. P0052A), followed by transfer to PVDF membranes (MilliporeSigma) at 220 mA. .. Membranes were blocked with 5% skimmed milk in 0.05% Tween-20 at room temperature for 1 h, then incubated overnight at 4°C with one of the following primary antibodies: anti-PD-L1 (1:1,000; Shanghai Abways Biotechnology Co., Ltd.; cat. no. CY5980); anti-VEGF (1:2,000, Affinity Biosciences, Ltd.; cat. no. AF5131); anti-p-STAT3 (1:1,000, Cell Signaling Technology, Inc.; cat. no. 9145); anti-STAT3 (1:1,000, Cell Signaling Technology, Inc.; cat. no. 9139); anti-Bcl-2 (1:2,000, Shanghai Abways Biotechnology Co., Ltd.; cat. no. CY6717); anti-Bax (1:2,000, Shanghai Abways Biotechnology Co., Ltd.; cat. no. CY5059); anti-MMP2 (1:1,000, Cell Signaling Technology, Inc.; cat. no. 40994); anti-cyclin D1 (1:2,000, Santa Cruz Biotechnology, Inc.; cat. no. sc-8396); anti-cleaved-caspase 3 (1:2,000, Cell Signaling Technology; cat. no. 9664); anti-caspase 3 (1:2,000, Cell Signaling Technology; cat. no. 9662); phospho-JAK2 (1:1,000; UpingBio technology Co, Ltd.; cat. no. YP-Ab-14423); JAK2 (1:1,000; UpingBio technology Co, Ltd.; cat. no. YP-Ab-14800); SHP-1 (1:1,000; UpingBio technology Co, Ltd.; cat. no. YP-mAb-14236); anti-Tubulin (1:4,000, MilliporeSigma; cat. no. T9026); and anti-GAPDH (1:4,000, MilliporeSigma; cat. no. G8795) according to the manufacturer's protocol. .. Following washing, membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000; OriGene Technologies, Inc.; cat. nos.

Article Title: KLF4 Protects Against Sepsis-Induced Coagulopathy via Inhibition of the AKT/mTOR/STAT3 Signaling Pathway
Article Snippet: .. The PVDF membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by incubation with thrombin (ab314480, 1:1000, Abcam), PAI-1 (ab222754, 1:1000, Abcam), TF (DF6400, 1:2000, affinity), GAPDH (AF7021, 1:5000, affinity), Tubulin (AF7011,1:1000, affinity), p-AKT (4060S, 1:2000, CST), AKT (4691S, 1:1000, CST), p-mTOR (5536S, 1:2000, CST), mTOR (2983S, 1:1000, CST), KLF4 (4038T, 1:1000, CST), p-STAT3 (9145T, 1:2000, CST) and STAT3 (9139T, 1:1000, CST) were incubated at 4°C overnight. .. The next day, the membranes were incubated with HRPlabeled affinity-pure goat anti-rabbit IgG (H+L) (SA00001-2, 1:15000, Proteintech) or HRP-labeled affinity-pure goat anti-mouse IgG (H+L) (SA00001-1, 1:15000, Proteintech) for 1 h at room temperature.

Article Title: CircHIPK3 promotes the progression of B-cell acute lymphoblastic leukemia in children by binding to STAT3
Article Snippet: .. For co-localization studies, the slides were incubated with an anti-STAT3 antibody (1:1,000, CST, #9139) followed by an FITC-conjugated secondary antibody. ..

other:

Article Title: Adapalene, an RAR agonist, exerts anti-inflammatory effects by regulating macrophage polarization through RAR -mediated signaling pathways
Article Snippet: t-STAT3 , Cell Signaling , 9139 , Mouse , 1:1000.

Immunofluorescence:

Article Title: Identification of Prognostic Factors and Regulatory Pathways in Porto‐Sinusoidal Vascular Disorder
Article Snippet: Antigen retrieval was performed using proteinase K (P2308; Sigma‐Aldrich, USA). .. The primary antibodies used for immunofluorescence staining were mouse anti‐IL‐6 (MA‐45069; Invitrogen, USA), mouse anti‐STAT3 (9139s, Cell Signalling, USA), and rabbit anti‐CD36 (14347s; Cell Signalling, USA). ..

Staining:

Image Search Results

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.

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

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).

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

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

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

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.

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

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.

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

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.

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

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.

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

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1) Product Images from "PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7"

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