Fig 1: Cell proliferation/migration and inflammatory response during unilateral ischemia/reperfusion injury (IRI). (A, B) mRNA expression of Ccna 1 and matrix metalloproteinase 9 (Mmp9) in the IRI kidney assessed by qRT-PCR. (C, E) Immunohistochemistry analysis for Ki67 (a marker of cell proliferation) and Mmp9 in IRI kidneys or sham. Bar = 50 µm. (D, F) Scatter diagram presentation of the percent of the Ki67 positive cells and Mmp9 positive areas at different time points after IRI. #p<0.05 versus IRI 2 days (Bonferroni correction; two comparisons were made).(G) qRT-PCR analysis of inflammatory factors (Mcp-1, Tnfa, and Il-1ß) in the kidney. (H) Representative F4/80 stained kidney sections from IRI or sham mice. Bar = 50 µm.(I) Scatter diagram presentation of the numbers of the F4/80 positive cells at different time points after IRI. #p<0.05 versus IRI 3 days (Bonferroni correction; two comparisons were made). (J) The correlation statistical analysis between Six1 mRNA expression and related molecular markers (Ki67, p = 0.0133; Mmp9, p = 0.0012; F4/80, p = 0.166) expression in IRI kidneys. Data are mean ± SD for groups of six mice. **p<0.01, ***p<0.001, ****p<0.0001 versus sham (t-test). IRI 1d, 1 day after IRI; IRI 2d, 2 days after IRI; IRI 3d, 3 days after IRI.
Fig 2: A structural and functional role for enhancer hubs (EnHs) in forming P–En networks during myogenic differentiation.a Identification of P–En networks (NWK) in ESCs (n = 261), +Dox (n = 790), and –Dox (n = 880) iPax7 cells. (Top) Schematic of a P–En network connected through an EnH (in red). (Bottom) Quantification of P–En interactions involved in P–En networks in each population. b P–En networks of the Six1/4 locus in +Dox and -Dox iPax7 cells graphed with Cytoscape. No network was detectable at these loci in ESCs. Each node represents a pCHi-C captured region, and promoters were labeled with their corresponding gene names, while enhancers were left unlabeled. Each gray line between nodes represents a high-confidence pCHi-C interaction, and the thickness of the line indicates the strength of the interaction measured by the CHiCAGO score. Node degree from low to high reflects the relative frequency of connections from one node to other nodes within the motif. c Overlap between EnHs in +Dox and -Dox iPax7 cells. d Corresponding transcriptional changes during iPax7 cell differentiation for target genes in each EnH group from c. Only differentially expressed genes (padj < 0.05 from paired DESeq2) are included (n = 446, 430, and 547 for group ‘+Dox unique’, ‘Common’, and ‘-Dox unique’, respectively). All differentially expressed genes in iPax7 cells (n = 2831) are used as a control. The boxes denote the 25th and 75th percentile (bottom and top of box), and median value (horizontal band inside box). The whiskers indicate the values observed within up to 1.5 times the interquartile range above and below the box. Statistical significance tested with two-tailed Student’s t-test.
Fig 3: miR-106b-5p and SIX1 expression in asthmatic mice and TGF-ß1-induced BEAS-2B cells. (A) Treatment schedule of asthmatic mice model and control group. (B) Lung tissues in (a) normal and (b) asthmatic mice model (scale bar, 500 and 100 µm). Relative expression levels of (C) miR-106b-5p and (D) SIX1 in asthmatic (n=5) and normal mice (n=5) were measured by RT-qPCR. (E) Pearson's correlation analysis between miR-106b-5p and SIX1 expression. (F) SIX1 protein expression in asthmatic and normal mice. (G) SIX1 expression in the lung tissues of asthmatic and normal mice was evaluated via IHC (scale bar, 50 µm). (H) After TGF-ß1 (10 ng/ml) treatment for 6, 12, 24 and 48 h, miR-106b-5p expression in BEAS-2B cells was analyzed by RT-qPCR. (I) Western blotting and (J) RT-qPCR were performed to detect the protein and mRNA expression levels of SIX1, respectively, in BEAS-2B cells after treatment with or without TGF-ß1 (10 ng/ml) for 24 h. (K) Quantification of SIX1 expression according to IHC. Data are presented as the mean ± SD of three independent experiments. **P<0.01; ***P<0.001. miR, microRNA; SIX1, SIX1, sine oculis homeobox homolog 1; RT-qPCR, reverse transcription-quantitative PCR; IHC, immunohistochemistry.
Fig 4: O-GlcNAcylation stabilizes SIX1 via CDH1. A The mRNA levels of SIX1 were analyzed in HCC cell lines as indicated. B Levels of SIX1 were determined by western blotting in BEL7402 cells treated with CHX (10 µg/ml) for the indicated times. C Ubiquitination of SIX1 was examined in BEL7402 cells after OGA inhibitor treatment. MG132 and NEM were used to inhibit proteasome and deubiquitination, respectively. D CDH1 was detected in purified SIX1 immunoprecipitation samples from BEL7402 treated with DMSO or TMG. E SIX1 expression was examined in BEL7402 CDH1-overexpressing cells with an increasing amount of TMG. (The data from right panel of A and E were analyzed by one-way ANOVA, data from B were analyzed by two-way ANOVA, and the rest of the statistics were analyzed by Student's t-test. ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001).
Fig 5: O-GlcNAcylation has reverse effect on SIX1 expression in HCC. A Relationship between O-GlcNAcylation and SIX1 expression levels in fifty HCC samples. BEL7402 and BEL7404 were treated with OGA inhibitors or transduced with OGT lentivirus (overexpression or knockdown) as indicated. B SIX1 expression levels were analyzed by western blotting in established HCC cell lines with different manipulation of O-GlcNAcylation. C SIX1 expression in BEL7402 cells treated with an OGA inhibitor (TMG) at the indicated times. D The direct target genes of SIX1 were analyzed in established HCC cell lines. E Immunoprecipitation with anti-OGT antibody followed by western blotting with anti-SIX1 antibody. Same experiment was performed when SIX1 was immunoprecipitated and immunoblotted with anti-OGT antibody. F O-GlcNAcylation modified protein immunoprecipitated from cell extracts was analyzed by immunoblotting for anti-SIX1.
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