Fig 2: 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."

Fig 3: JAK/STAT pathway mediates β‐catenin activation in CXCR4 signalling. A, Representative Western blots show JAKs and p‐JAKs expression in HKC‐8 cells. HKC‐8 cells were incubated with SDF‐1α (100 ng/mL) for 0, 5, 10, 15, 30 and 60 min. B, Representative Western blots show STATs and p‐STATs expression in HKC‐8 cells. HKC‐8 cells were incubated with SDF‐1α (100 ng/mL) for 0, 15, 30, 60, 120 and 240 min. C, The relative abundance of GSK3β mRNA was assessed by quantitative real‐time PCR. HKC‐8 cells were transfected with CXCR4 or STAT3 or STAT6 expression plasmid (pFlag‐CXCR4, pFlag‐STAT3 or pHA‐STAT6) or pcDNA3 for 24 h. Total RNA was extracted and analysed by real‐time PCR. *P < .05 versus pcDNA3 group (n = 3). D, Representative micrographs show the colocalization of β‐catenin and STAT3 or STAT6 inHKC‐8 cells. HKC‐8 cells were treated with SDF‐1α (100 ng/mL) for 12 h. White arrows indicate the colocalization of β‐catenin and STAT3 or STAT6 in nuclei. E‐G, Representative (E) Western blots and graphical representations of (F) p‐GSK3β (Ser9) and (G) active β‐catenin expression in four groups. HKC‐8 cells were transfected with siRNA to STAT3 or STAT6 or negative control (NC) and then treated with SDF‐1α for 12 h.*P < .05 versus control group (n = 3 to 4); †P < .05 versus SDF‐1α plus NC group (n = 3 to 4). H, Representative micrographs show the expression of fibronectin in four groups as indicated. HKC‐8 cells were transfected with siRNA to STAT3 or STAT6 or NC and then treated with SDF‐1α for 12 h. Cells were then immunostained for fibronectin (red) and counterstained with DAPI (blue).White arrow indicates positive staining. I, Schematic presentation maps show that through binding of SDF‐1α to its receptor CXCR4, JAK2 and JAK3 are phosphorylated to trigger the activation and nuclear translocation of STAT3 and STAT6, which repress the transcription of GSK3β and the assembly of the β‐catenin degradation complex. The release of β‐catenin from degradation complex in the cytoplasm would lead to the accumulation of nuclear β‐catenin, which mediates fibrotic injury in tubular cells through binding to TCF/LEF transcription factors

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.