Fig 2: YB1 regulates miR-205/200b?ZEB1 axis by inhibiting miRNA expression. A-B. The expression levels of miR-205 and miR-200b were analyzed in HCCLM3 and MHCC97H cells stably expressing YB1 shRNAs (A) as well as SMMC7721 and BEL7402 cells stably overexpressing YB1 (B) by qPCR analysis. U6 was used as an internal control for qPCR. C. A schematic representation of the luciferase reporter constructs with ZEB1 or SIP1 3'UTR (left panel). The complementary binding sites among ZEB1 3'UTR luciferase reporter, miR-205-5p, and miR-200b-3p (right panel). D. The regulation of YB1 on the miR-205/200b-ZEB1 axis was identified by luciferase reporter assays, which were performed by cotransfecting ZEB1 3'UTR luciferase reporter with pre-miR-205 or pre-miR-200b expressing plasmid, YB1 expressing plasmid, and specific miR-205 or miR-200b inhibitors into HEK293T cells. E. YB1 regulated miR-205/200b-ZEB1 axis in a dose-dependent manner. Luciferase activity assays were performed by cotransfecting ZEB1 3'UTR luciferase reporter with YB1 expressing plasmid and pre-miR-205 (left panel) or pre-miR-200b (right panel) expressing plasmid into HEK293T cells. The most representative results of three independent biological experiments were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.001

Fig 3: 5-FU induces Y-box binding protein 1 (YB-1) phosphorylation at S102 in KRAS(G13D)-mutated HCT116 cells but not in KRAS wild-type SW48 cells. (A) KRAS wild-type SW48 and KRAS(G13D)-mutated HCT116 cells were treated with increasing concentrations of 5-FU for 72 h. Thereafter, protein samples were isolated, and phosphorylation of YB-1 was analyzed by Western blotting using a phospho-specific antibody. Actin was detected as the loading control. The histogram represents the mean ratio of phosphorylated YB-1 (P-YB-1)/YB-1 from three independent experiments normalized to untreated HCT116 control cells. (B) A proliferation assay was performed following the same treatment conditions. Histograms indicate the mean number of cells after treatment with the indicated concentrations of 5-FU normalized to the control condition in each cell line (9 data points from three biologically independent experiments). Asterisks indicate a significant antiproliferative effect of 5-FU as analyzed by Student’s t-test (* p = 0.05, ** p = 0.01, *** p = 0.001, and **** p = 0.0001; n.s.: nonsignificant). (B, left part) Comparison of absolute cell counts of control conditions in SW48 and HCT116 cells.

Fig 4: a4 regulates IR Tyr phosphorylation via YBX1 and PTP1B.a Schematic diagram showing the regulation of insulin signaling in brown preadipocytes via a4. Specifically, a4 binds to the catalytic subunit of protein phosphatase 2A (PP2Ac) and is known to regulate Ser/Thr phosphorylation. For the mass spectroscopic proteomic analysis. we compared the Control (n = 1) and a4 overexpressing (n = 2, biological replicates) samples. b a4-complex containing YBX1. Flag-tagged a4 immunoprecipitation showed the interaction between YBX1 with a4 as well as that between PP2Ac and a4 in brown preadipocytes (n = 3 biologically independent cell clones/group). c Western blotting of phosphorylation of insulin signaling molecules in Control and a4KO brown preadipocytes following indicated concentrations of insulin stimulation for 10 min. d Effect of a4 on insulin signaling-based densitometric immunoblotting analysis with antibodies to a4 and phosphorylated IR, Akt, YBX1, as well as PTP1B in lysates of a4 Knockout brown preadipocytes (a4 KO) before and 10 min after insulin stimulation (see c). Data are presented as mean ± SEM (one-way ANOVA post hoc Bonferroni test, n = 3, each cell was generated from biologically independent animals from three independent experiments, Statistical significance is shown as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***)). e Luciferase reporter assay for PTP1B transcription was performed as described by Fukada and Tong45. Cells were transfected with the expression vectors of a4 and/or YBX1 in the presence of Ptp1b-Luc reporter or mock plasmid DNA in HEK293cells. Data are presented as mean ± SEM (One-way ANOVA post hoc Bonferroni test, n = 8 technical replicates per group, ***p < 0.0001). f Model of the regulation of a4 in insulin-activated IR phosphorylation. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and S6K pathways. a4 stabilizes PP2A and dephosphorylates mTORC2 with Rictor. a4 also binds to Y-box protein 1 (YBX1), which functions as a transcription factor for Tyr phosphatase PTP1B. a4 blocks YBX1 nuclear translocation and inhibits PTP1B expression, thus in feedback regulation, promoting the enhanced signaling through the insulin receptor. Source data are provided as a Source Data file.

Fig 5: The GSDME–YBX1–mucin pathway mediates tumour progression in patients with PDAC.a, The expression profile of GSDME and YBX1 from the TCGA Research Network (https://cancergenome.nih.gov/). Data are presented as box plots, where the centre line shows the median, the bounds of the box show the first and third quartiles, whiskers extend to the most extreme values within 1.5-times the interquartile range, and dots denote outliers reaching past the 1.5 interquartile range. n = 179 for PDAC tissues and n = 171 for adjacent tissues. N, normal adjacent tissue; T, tumour tissues; TPM, transcripts per million. b,c, Immunohistochemical images (left) and quantification (right) of GSDME (b) and MUC13 (c) from sections of tumour tissues and adjacent tissues of patients (P1–P3) with PDAC (n = 10). Scale bars, 50 µm. d–f, Correlation analysis between the level of GSDME methylation (d), YBX1 (e) or MUC1 (f) and overall survival of patients with PDAC (n = 177). g, Schematic of the GSDME–YBX1–mucin pathway to regulate the resistance of PDAC to digestive enzymes. P values were determined by two-tailed Mann–Whitney test (a–c) or two-sided Pearson’s correlation test (d–f). The data represent the mean ± s.d.Source data