Fig 2: KLF5 upregulates AXL expression via binding to the promoter of AXL.A, B DCC-2036 downregulated the protein level and mRNA level of AXL. After the MDA-MB-231 cells were treated with DCC-2036 at 10 µM for 48 h, the protein and mRNA levels of AXL were measured by Western Blotting and qPCR. C, D Knockdown of KLF5 decreased the protein level and mRNA level of AXL. MDA-MB-231 cells were transfected with siKLF5 or control siRNA (siNC) for 48 h, followed by WB and qPCR. E Schematic representation of AXL promoter. I–V represented the putative site in the AXL promoter predicted by the Jasper database. One to three represented the qPCR products (fragments) by different AXL primers. F The information of the five highest-scoring putative sites (I–V). G Chromatin immunoprecipitation (ChIP) assay indicated that KLF5 is directly bound to AXL promoter mainly at fragment 2 in MDA-MB-231 cells. The results were shown as relative enrichment compared with IgG. H, I overexpression of KLF5 in MDA-MB-231 cells partially but significantly rescued the DCC-2036-induced reduction of KLF5 binding to fragment 2. MDA-MB-231 cells were transfected with KLF5 or vector control plasmid, followed by treating with 10 µM DCC-2036 for 48 h before WB and ChIP qPCR assay. J Regulation of AXL promoter activity by KLF5 in MDA-MB-231 cells. Wild-type (-2000 to +50 bp) or mutant promoter (site V was deleted) was cotransfected with siNC or siKLF5, and luciferase activity was measured with pRL-TK as an internal control. Data represent the mean of three independent experiments ± s.d. K Regulation of AXL promoter activity by DCC-2036 and overexpression of KLF5 in MDA-MB-231 cells. Wild-type or mutant promoter was cotransfected with KLF5 or vector control plasmid, followed by treating with 10 µM DCC-2036 for 48 h, and then luciferase activity was measured.

Fig 3: Vincristine increases sensitivity to RSL3 via targeting SUFU-YAP-ACSL4 axis(A) Immunoblot (IB) analysis of SUFU abundance in HCC1954, MCF-7, and MDA-MB-231 cells upon treatment with indicated compounds for 24 h. GAPDH was used as a loading control.(B) IB analysis of SUFU, AXL, ACSL4, and P-YAP127 levels in HCC1954, MCF-7, and MDA-MB-231 cells upon treatment with increasing doses of vincristine for 24h.(C) Confocal analysis of YAP nuclear localization in HCC1954 (lower panels) and MDA-MB-231 (upper panels) cells following the vincristine treatment for 24h; scale bar, 50µm.(D) IB analysis showing the increase in the levels of AXL and ACSL4 in HCC1954 and MDA-MB-231 cells upon a 24 h exposure to vincristine was mitigated by siRNA-mediated knockdown of YAP.(E) Viability (%) of MCF-7 and T47D cells treated with vehicle, vincristine, RSL3, or the combination of vincristine and RSL3.(F) Tumor growth measurement in mouse xenograft derived from MCF-7. An intra-tumor injection was performed with vehicle, RSL3 (40 mg/kg), RSL3 (100 mg/kg) vincristine (30 µg/kg), or the combination of RSL3 (40 mg/kg) and vincristine (30 µg/kg), n = 5 in each group.(G) Hematoxylin and eosin (H&E), Ki67, SUFU, and ACSL4-stained images of tumor tissues from tumors treated as in f; scale bar, 100µm. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test or Unpaired t test).

Fig 4: Activated AXL protects against hepatic ischemia-reperfusion injury by upregulating SOCS-1 expression. Activated AXL exerts a protective effect against hepatic I/R injury through SOCS-1-TLR4/MYD88/NF-?B signaling pathway. AXL, AXL receptor tyrosine kinase; Gas6, growth arrest-specific protein 6; I/R, ischemia/reperfusion; MYD88, myeloid differentiation factor-88; NF-?B, nuclear factor kappa-B; SOCS, suppressor of cytokine signaling protein; TLR4, toll-like receptor 4; TRAF6, tumor necrosis factor receptor–associated factor 6.

Fig 5: Activated AXL protects the liver from I/R injury. A, Serum ALT and AST levels (n = 4–6) after hepatic I/R injury in the control group and the treatment group (as shown in the figure). B, Representative histological H&E-stained images showing the area of liver necrosis after hepatic I/R injury in the control group and the treatment group (n = 4–6). ***P < 0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; AXL, AXL receptor tyrosine kinase; H&E, hematoxylin and eosin; I/R, ischemia-reperfusion; rmGas6, recombinant growth arrest-specific protein 6.