Fig 1: The expression of MEST and VCP in metastatic cancer and a schematic diagram of the action mechanism of MEST. A Two representative IHC images of MEST and VCP expression in primary lung cancer tissues and in corresponding metastatic tumor tissues. Differences in MEST and VCP expression scores between lung cancer and metastatic tumor tissues are shown (n = 30). Scale bar, 50 μm. B Working model of MEST on promotion of cancer metastasis. MEST is upregulated in highly invasive lung cancer cells, it interacts with VCP and increases VCP to capture IκBα for degradation, leading to the nuclear translocation of p65 to activate the NF-κB pathway for cancer metastasis. In addition, MEST can induce IκBα phosphorylation via the STAT3/IKK pathway, mediating NF-κB signaling activation at multiple levels
Fig 2: Inhibition of active STAT3 results in the induction of apoptotic cell death in ASCs.(A) Cells were plated in a 6-well plate at a density of 2 × 105 per well and were then treated with 2 µM Stattic or DMSO. The number of cells was counted at 24 and 48 hours after treatment. The cell proliferation assay was performed in triplicate (p < 0.001 at the time point of 24 hours, while p < 0.001 at 48 hours, one-way ANOVA). (B) Levels of cleaved caspase-3 and -7, PARP, cleaved PARP, and XIAP were determined in cells treated with 2 and 5 µM of Stattic by immunoblot assay. (C) Levels of Bcl-2 family members, Bcl-2, Bcl-X, and Mcl-1, in ADs and ASCs treated at indicated Stattic concentrations were examined by immunoblot assay. These proteins are labeled as the intrinsic pathway on the right. Levels of death receptor signaling related proteins in Stattic-treated cells were investigated by immunoblot assay. These proteins are labeled as the extrinsic pathway on the right. (D) ADs and ASCs were plated in a 6-well plate at a density of 5 × 105 per well and were treated with 2 µM Stattic for 48 hours. Harvested cells were stained with Annexin V/7-AAD for 15 minutes and subjected to flow cytometric analysis. The frame with dashed and solid lines refer to early apoptotic cells and late apoptotic cells, respectively.STAT3, signal transducer and activator of transcription 3; ASC, adapted suspension cell; AD, adherent cell; PARP, poly(ADP-ribose) polymerase.*p < 0.01, †p < 0.001, Student's t-test.
Fig 3: MAGEC2 contributes to STAT3 hyperactivation in ASCs.(A) Levels of total STAT3 and pY-STAT3 in the basal ADs and ASCs were evaluated by immunoblot assay. (B) Cells were lysed in the presence or absence of MG132 20 µM for 10 hours and immunoprecipitated with pY-STAT3. Endogenous levels of poly-ubiquitinated pY-STAT3 and pY-STAT3 were measured by immunoblotting. (C) Cells were transfected with siControl or siMAGEC2 for 48 hours and levels of STAT3, pY-STAT3, and pS-STAT3 were evaluated. (D) Protein levels of STAT3 downstream target genes, cyclin D1, MMP9, and survivin, were examined by immunoblot assays in cells treated with siControl and siMAGEC2. (E) Depletion of siMAGEC2 efficiency in ADs was evaluated by over-exposure immunoblotting.MAGEC2, melanoma-associated antigen C2; ASC, adapted suspension cell; AD, adherent cell; STAT3, signal transducer and activator of transcription 3; MMP-9; matrix metallopeptidase 9.
Fig 4: Myeloid deletion of Cdc42 enhances the M2-type differentiation of macrophages via regulating STATs signaling. THP1 cells were pretreated with PMA (100 ng/mL) and ML141 (10 μM) for differentiation and then stimulated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 1 hour for M1 induction, or IL4 and IL13 (both at 40 ng/mL) for 48 hours for M2 induction. The images (A) and quantitative results (C–F) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were detected by Western blot analysis in THP1 cells, n = 3. The images (B) and quantitative results (G–J) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were measured by Western blot analysis in BMDMs stimulated with LPS (100 ng/mL) for 3 hours or IL4 (40 ng/mL) for 48 hours, respectively, n = 3. n. s., no significance; ∗P < .05; ∗∗∗P < .001.
Fig 5: Selective Inhibition of the JAK3-Dependent Signaling Pathway by MJ04. A Assessment of cell cytotoxicity induced by MJ04 and its determination of IC50 values across different cancer cell lines. B Examination of the impact of MJ04 on the phosphorylation status of JAK1, JAK3, and STAT3. C Evaluation of the effect of Tofacitinib on the phosphorylation of JAK1, JAK3, and STAT3. D Assessment of MJ04’s impact on STAT3 phosphorylation in the presence of IL-6 of A549. GAPDH served as the loading control. Densitometry analysis results for each immunoblot are depicted in the accompanying graph to the right. All data analysis was performed using MS Excel, GraphPad Prism-V8.0 Software, and Image-J. Statistical significance was determined through one-way analysis of variance (ANOVA). Data points are presented as mean ± SD, and significance values are denoted as *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005."
from Cell Signaling Technology for Phospho-Stat Antibody Sampler Kit