Fig 2: MiR-92a elevated the expression of p110d and promoted TiPs-induced inflammation by targeting KLF4.A Potential microRNAs interacting with KLF4 were sorted out from online platform The Encyclopedia of RNA Interactomes (ENCORI) and microRNAs were categorized by Venn chart. B Expressions of potential microRNAs upon 8 h TiPs stimulation were detected by qPCR assay. C, D mRNA (C) and protein levels (D) of KLF4 were respectively examined upon the transfection of mimics-miR-92a (Pre-92a) or inhibitor-miR-92a (Anti-92a). E Potential binding region of miR-92a and 3'UTR of KLF4, and relative sequence of KLF4 with mutant 3'UTR. F Dual luciferase activity was detected after 48 h transfection of wild-type KLF4 3'UTR (KLF4 3'UTR-WT) or mutant-type KLF4 3'UTR (KLF4 3'UTR-MT) with mimics-miR-92a (Pre-92a) or mimics-NC (Pre-NC). G, H mRNA level (G) and protein levels (H) of p110d were respectively examined upon the transfection of mimics-miR-92a (Pre-92a) with or without KLF4-overexpressed plasmids (KLF4+). I Total p65, total I?Ba, phosphorylated p65, and phosphorylated I?Ba were detected by WB assay to determine the activation of NF-?B signaling pathway upon 1 h TiPs stimulation in mimics-miR-92a (Pre-92a)-transfected macrophages or inhibitor-miR-92a (Anti-92a)-transfected macrophages. J Supernatant levels of TNFa and IL-6 from miR-92a-increased or miR-92a-inhibited macrophages were analysed by ELISA assay after 8 h TiPs stimulation. All data were concluded from at least three independent assays. Statistic data were displayed as mean ± SEM and were conducted unpaired t test analysis or one-way ANOVA analysis to determine significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the negative group.

Fig 3: Knockdown KLF4 exacerbated TiPs-induced osteolysis which was attenuated by knockdown p110d.A Noticeable bioluminescence signal from calvarias was detected 7 days after surgery. B 3D construction was implemented and representative images of calvarias from each group were listed after micro-CT imaging. C, D Bone mineral density (BMD) and the ratio of bone volume against total volume (BV/TV) from each group were calculated after 3D construction. E Sections loading calvarias from each group were performed H&E staining, TRAP staining and IHC staining of TNFa and IL-6. The black bar, 200 µm. Statistic data were displayed as mean ± SEM and were conducted one-way ANOVA analysis to determine significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the negative group.

Fig 4: Pik3cd transcription was mediated by KLF4 in TiPs-stimulated macrophages.A Predicted sequence logo of KLF4 binding site was acquired by JASPAR database. B mRNA expression of p110d from KLF4-knockdown RAW264.7 or KLF4-overexpressed RAW264.7 was detected. C Protein level of p110 from KLF4-knockdown RAW264.7 or KLF4-overexpressed RAW264.7 was detected. D Structure of plasmids containing mutant-type of Pik3cd promoter for luciferase reporter assay. E Relative luciferase activity upon different supplement of KLF4-overexpressed (KLF4+) plasmids. F Relative luciferase activity was analysed after transfecting plasmids containing wild-type (Pik3cd-WT) or mutant-type (Pik3cd-MT) of Pik3cd promoter with or without KLF4+ plasmids. All data were concluded from at least three independent assays. Statistic data were displayed as mean ± SEM and were conducted unpaired t test analysis or one-way ANOVA analysis to determine significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the negative group.

Fig 5: MET and AXL signaling involved in Galectin-1-mediated sorafenib resistance and ferroptosis in HCC. (A) Galectin-1-knocked-down Huh-7/SR (Huh-7/SR/shGal#23 and #24) and control (Huh-7/SR/shCtrl) cells were analyzed for RTK expression (EGFR, MET, AXL, and insulin receptor) through Western blotting. Huh-7/SR cells were treated with an MET inhibitor and AXL inhibitor R428 for 48 hr. Analysis of Galectin-1, AXL, and phospho-AXL expression (B upper panel) and MET and phospho-MET expression (C upper panel) were performed using Western blotting. Cell viability of Huh-7/SR cells cotreated with 20 µM of sorafenib and AXL (B lower panel) or an MET inhibitor (C lower panel) for 48 h. Galectin-1 overexpression after treatment with the MET inhibitor and AXL inhibitor R428 for 48 h. Analysis of AXL and phospho-AXL expression (D upper panel) and MET and phospho-MET expression (E upper panel) by using Western blotting. Cell viability of Galectin-1- overexpressing cells cotreated with 20 µM of sorafenib and AXL (D lower panel) or a MET inhibitor (E lower panel) for 48 h. (F) Oct-4, Nanog, SOX-2, and KLF4 mRNA expression determined using qRT-PCR in Galectin-1-overexpressing cells treated with an MET inhibitor and an AXL inhibitor R428. Western blot analysis was used to detect AXL, phospho-AXL, GPX4, and ferritin heavy chain 1 expression in cells cotreated with 20 µM of sorafenib and an (G) AXL inhibitor or (H) MET inhibitor. Data are presented as means ± standard deviations. **P < 0.01, and ***P < 0.001 (Student’s t test).