Fig 1: Melatonin alleviated bone loss and senescence in DM1 C57BL/6 mice. A–D One week after intraperitoneal injection of STZ, C57BL/6 mice were injected with 1 g/kg glucose after overnight fasting. A, B Blood samples were collected from the tail vein at 0, 10, 20, 30, 60, 90, and 120 min. respectively, and blood glucose was measured by a OneTouch glucometer (Lifescan). C, D Blood samples were collected from the tail vein at 0, 10, and 30 min respectively, and blood insulin levels were measured using radioimmunoassay (Millipore). E–H C57BL/6 mice were intraperitoneally injected with STZ (160 mg/kg), then treated with melatonin (60 mg/kg/day) for 2 months. E Total proteins were extracted by Invent kit from femurs of DM1 mice and DM1 mice treated with melatonin. Total proteins were blotted with anti-p16, anti-γ-H2AX, anti-p21, anti-Bcl2, anti-CDK2, anti-CDK4, anti-cyclinD1, anti-Bmi1, anti-MT1, anti-MT2, anti-Rnf2, anti-p53, anti-Runx2, anti-Ezh2, anti-Suz12, anti-Sirt1 and anti-beta actin antibodies. Data of F–H were reconstructed by NRecon-1.7.3.1. Data of A–D were presented with mean ± SEM and analyzed by Graph Pad Prism 8.2.0 (San Diego, CA, USA) with two-tailed unpaired Student’s t test. Statistical significance was determined as p < 0.05
Fig 2: Activation of GPR17 inhibited glioma formation.A U87MG and U251 cells treated with vehicle or MDL29951 (300 μM) for 48 h, and CCK-8 assay was performed to examine viable cell numbers. Data represent the means ± SEM from three independent experiments. B–D BALB/c nude mice with subcutaneously xenotransplanted tumor were intraperitoneally injected with 10 mg/kg MDL29951 for 16 days, and then tumor samples were collected for the measurements of tumor sizes (B), volumes (C), and weights (D), (n = 7). E H&E staining of sections from subcutaneously xenotransplanted tumors. Scale bar 10 μm. F, G Immunofluorescent staining against Ki67 (F) and TUNEL (G) of the tumor sections, scale bar 50 μm. H IHC staining against p-PKA or RNF2 of the subcutaneously xenotransplanted tumor. Scale bar 50 μm. I A diagram depicting the working model of the inhibitory effect of GPR17 on glioma tumorigenesis. For all panels, *p < 0.05, ***p < 0.001, Student’s t-test.
Fig 3: KLF9 was a downstream target for GPR17 and RNF2 in glioma cells.A Control and U87-GPR17 cells were subjected to RNA-Seq analysis. Volcano plot depicted gene expression changes between control and U87-GPR17 cells. B Control and U87-shGPR17 cells were subjected to RNF2 ChIP-Seq analysis. Density heatmap showed the RNF2 recruitment within ±2 kb around the RNF2 peak center. C Venn diagram showing the overlapped genes in ChIP-Seq and RNA-Seq analyses. D Gene list ranked by RNA-Seq p value with RNF2 binding on promoter-TSS region. E Diagram showing RNF2 enrichment on KLF9 promoter-TSS region. F–H U87-shScr/shGPR17 (f), U87-Vec/GPR17 (G), or U87MG cells treated with Vehicle/MDL29951 (300 μM) for 48 h (H) were subjected to RNF2 and H2AK119ub ChIP assay; real-time PCR analysis was then performed to assess the relative enrichment of RNF2 and H2AK119ub in promoter-TSS region of KLF9. I Correlation plot of RNF2 and KLF9 mRNA expression in glioma, grades II–IV, from the TCGA dataset (n = 667). J, K Control, U87/U251-GPR17, or U87/U251-shGPR17 cells were prepared as in Fig. 2. Cells were harvested for real-time PCR analysis to assess the mRNA levels of RNF2 and KLF9. L Control or U87/U251-GPR17 cells were transfected with control or RNF2-overexpressing vectors for 48 h, and then subjected to real-time PCR analysis to assess the mRNA levels of KLF9. For all panels, data represent the means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.
Fig 4: GPR17 suppressed the expression of RNF2 through cAMP/PKA/p65 axis.A Correlation plot of the mRNA levels of GPR17 and RNF2 in glioma from 667 patient samples in TCGA dataset. B Control, U87/U251-GPR17, or U87/U251-shGPR17 cells were subjected to western blot to assess the protein level of RNF2. C cAMP levels were measured in control and U87/U251-GPR17 cells. D Control, U87/U251-GPR17, or U87/U251-shGPR17 cells were subjected to western blot to detect the indicated protein levels. Densitometric quantification of p-PKA/PKA, or p-p65/p65 ratio from at least three independent assays was indicated on top of each band, respectively. E The protein levels of RNF2 or p-PKA of brain sections with U87-Vec and U87-GPR17 tumors were evaluated by IHC analysis. Scale bar 50 μm. F, G U87MG or U251 cells were co-transfected with wild type (WT) or mutant RNF2 promoter (2.0 kb)-firefly luciferase, Renilla luciferase, and p65-overexpressing vectors for 24 h (F); or co-transfected with WT RNF2 promoter (2.0 kb)-firefly luciferase, Renilla luciferase and p65-overexpressing vectors for 24 h, and then treated with or without 10 μM H89 treatment for 48 h (G). Cells were then harvested for dual-luciferase reporter assay following the manufacturer’s instructions. H, I Control or U87/U251-GPR17 cells were transfected with control or RNF2-overexpressing vectors for 48 h, and then CCK-8 assays were performed to measure viable cell numbers (H), flow cytometry analyses were performed to assess mitochondrial ROS levels (I). J Control or U87/U251-GPR17 cells were transfected with RNF2 wild type/I53A mutant-overexpressing vectors for 48 h. Cells were then harvested for western blot against cleaved-caspase3. Densitometric quantification of cleaved-caspase3/caspase3 ratio from at least three independent assays was indicated on top of each band, respectively. For all panels, data represent the means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.
Fig 5: PHC1 is required for the pluripotency maintenance of hESCs.a, b Morphology and colony-forming capacity of shScr-, shPHC1.1-, and shPHC1.2-infected hESCs. Scale bars, 600 μm. Cells were stained for alkaline phosphatase activity 14 or 18 days after plating. Bar plot shows mean colony-formation efficiencies normalized to the shScr. Mean ± s.d. of n = 3 independent experiments. Two-tailed unpaired t-tests were used (**p = 0.0022, *p = 0.0458). c Comparison of tumor sizes about 1 month after injection of shScr-, and shPHC1-infected hESCs into NOD/SCID mice. Data are presented as mean ± s.e.m. In each group, 4 NOD/SCID mice were injected. Two-tailed unpaired t-tests were used (*p = 0.0211). d WB analysis of PHC1, NANOG, OCT4, SOX2, RING1B, H2AK119ub1, and ACTIN protein levels in shScr and shPHC1-infected hESCs. e WB analysis of PHC1 and NANOG protein levels in hESCs infected with control and CRISPR-CAS9 vector targeting human PHC1 gene. f Immunofluorescent co-staining of PHC1 with NANOG, OCT4, or SOX2 in shScr- and shPHC1-infected hESCs. Arrowheads showed cells with low PHC1 and NANOG signals. Scale bars, 20 μm. g The ratios of cells exhibiting PHC1highNANOGlow and PHClowNANOGlow signals to the total number of stained cells in (f) were quantified. Mean ± s.d. of n = 3 independent counts for shPHC1.1 and shPHC1.2. Two-tailed unpaired t-tests were used (p = 0.0065 for shPHC1.1, p = 0.0048 for shPHC1.2). Source data are provided as a Source Data file.
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