Fig 1: MZF1-uPEP/YY1/MZF1 expression is associated with NB outcome. (A) Representative images of immunohistochemical staining showing the expression patterns of MZF1-uPEP, YY1, and MZF1 in tumor cells of NB specimens (arrowheads, brown). Scale bars: 50 µm. (B) Kaplan-Meier curves indicating overall survival of 42 NB patients with high or low MZF1-uPEP immunostaining in 88 (GSE16476) NB cases with low or high expression levels of YY1 (cutoff value=1003.1). (C and D) Western blot (C) and real-time qRT-PCR (D, normalized to ß-actin) assays showing the expression of YY1, MZF1, and target genes in normal dorsal root ganglia (DG), NB tissues (n=42), and NB cell lines. (E) Mining of a public microarray dataset (GSE16476) revealing the levels of YY1 in NB tissues with different status of age, death, or INSS stages. (F) The positive expression correlation of YY1 with MZF1, HK2, or PGK1 in 88 NB cases (GSE16476). (G) The mechanisms underlying MZF1-uPEP-suppressed tumor progression: as an uORF-encoded small peptide, MZF1-uPEP directly binds to YY1 to repress its transactivation, resulting in decreased transcription of MZF1 and downstream target glycolytic genes, and reduced aerobic glycolysis and tumor progression. Log-rank test for survival comparison in B. Student's t test compared the difference in D and E. Pearson's correlation coefficient analysis for gene expression in F. * P<0.05 vs. DG. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in C and D. Bars are means and whiskers (min to max) in E.
Fig 2: Cellular stability of YY1 mRNA and protein. Influence of the transcriptional inhibitor actinomycin-D (ACT-D, 5 µg/mL) (a-d) and of the translational inhibitor cycloheximide (CHX, 100 µg/mL) (e, f) on YY1 expression in the B-NHL cell lines RAMOS (a, b and e) and U2932-R2 (c, d and f). a YY1 mRNA quantification relative to GAPDH by qRT-PCR after 0 h, 30 min, 1 h, 2 h, 3 h and 6 h of ACT-D treatment of RAMOS and (c) of U2932-R2. Error bars indicate 95% confidence interval of the mean expression. MYC levels were analyzed as a positive control for a fast turnover mRNA. b Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h of ACT-D treatment in RAMOS and (d) in U2932-R2. MYC was used as a positive control for a fast turnover protein and GAPDH as loading control. The arrow indicates the caspase-cleaved YY1 form. e Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h CHX treatment of RAMOS and (f) of U2932-R2. MYC expression was determined as a positive control for a fast turnover protein and GAPDH and histone 3 (H3) served as loading controls. The arrow indicates the caspase-cleaved YY1 form. Numbers underneath the blots refer to the relative amount of YY1 normalized to GAPDH levels according to densitometric analyses of the blots. The 0 h sample was set to 1 at each time point, treatment and cell line
Fig 3: Molecular distinctions between CPAM and PPB lung biopsies. qRT-PCR expression analyses of DICER1, YY1, FGF9, FGF10, BMP4, SPRY2, ETV4, ETV5, ELF5, SHH, IRX2 and IRX5 genes in human lung biopsies from male (blue) and female (pink) control, CPAM (unknown type, Type I and Type II) and PPB (unknown type, Type I, Type II and Type III) patients, indicating a specific PPB molecular signature. Means±s.e.m. are shown. *Padj<0.05, **Padj<0.01, ***Padj<0.001 (Kruskall–Wallis, Dunn's post hoc with Bonferroni correction). CTL, control (n=14-16); CPAM, congenital pulmonary airway malformation (n=7); PPB, pleuropulmonary blastoma (n=7-12).
Fig 4: ARGLU1 enhanced the transcriptional activity of AP1 and YY1 on MLH3, MSH2, MSH3 and MSH6. (a) Venn diagram of ARGLU1 IP-MS result and transcription factors of 4 MMR genes (MLH3, MSH2, MSH3 and MSH6) predicted by JASPAR. (b) Co-IP result of ARGLU1 with YY1 and SP1. (c) ChIP-qPCR results of YY1 and SP1 in HGC27 cells with or without ARGLU1 knock-down. (d) Relative luciferase activity of MLH3 in shARGLU1, siSP1 or siYY1 treated HGC27 and (e) MGC803 cells. (f) Schematic diagram of the molecular mechanism of ARGLU1 enhancing the transcriptional activity of SP1 and YY1 on MMR genes in GC. The data are the means ± SDs of three independent experiments (two-tailed t-test, *, p < 0.05, **, p < 0.01, ***, p < 0.001, NS, not significant).
Fig 5: Mechanism of Smurf2 in cerebral ischemic injury via YY1/HIF1a/DDIT4 axis. Smurf2 promoted YY1 ubiquitination and degradation to decrease HIF1a and DDIT4 expression, decreasing neuron apoptosis, which exerted neuroprotective effects on cerebral ischemic injury. DDIT4, DNA damage–inducible transcript 4 gene; HIF1a, hypoxia-inducible factor-1 alpha; Smurf2, Smad ubiquitination regulatory factor 2; YY1, Yin Yang 1.
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