Journal: Carcinogenesis

Article Title: Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer

doi: 10.1093/carcin/bgw088

Figure Lengend Snippet: CPI-7c efficiently degrades MDM2 but leads to stabilization of p53 and induces global ubiquitination response. In ( A ), MCF-7 cells were treated with 5, 10, 15 and 20 µM CPI-7c and Nutlin-3, and expression levels of MDM2, ubiquitin and p53 were assessed by using immunoblotting and shown in the image. In panel ( B ), MCF-7 cells were grown in coverslips and treated with either vehicle or 10 and 20 µM of CPI-7c and Nutlin-3, respectively, for 24h and subjected to immunofluorescence staining and analyzed by confocal microscope. Merged confocal photographs represent the superimposition of green (p53) and red (MDM2) images, and the magnified area of the box was shown in inset pictures. Scale bar, 20 µm. Representative of three independent experiments. In panel ( C ), RT–PCR analysis shows the mRNA expression MDM2 and GAPDH genes in response to 10 and 20 µM doses of CPI-7c and Nutlin-3 treatment.

Article Snippet: Nutlin-3 and ChIP grade antibody for ubiquitin, and HDM2 (catalytic RING domain) peptide were purchased from Enzo Life Sciences.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Microscopy, Reverse Transcription Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer

doi: 10.1093/carcin/bgw088

Figure Lengend Snippet: CPI-7c physically binds to RING and N-terminal domains of MDM2 and selectively induces ubiquitination of MDM2 as well as interferes at p53–MDM2 interaction. MCF-7 cells were treated with 10 µM dose of CPI-7c and Nutlin-3 for 24h, and protein lysates were prepared for immunoprecipitation using human anti-ubiquitin. Ubiqutinated MDM2 level was measured using western blotting of anti-MDM2 antibody with proper input control and shown in panel ( A ). Similarly, ( B ) represents the level of associated p53 when lysate was immunoprecipitated using anti-MDM2 antibody. Panel ( C ) represents standard scatchard exponential hyperbolic binding curve of direct physical binding of CPI-7c and Nutlin-3 with N-terminal peptide and RING domain peptide of MDM2 in left and right panels, respectively, along with respective dissociation equilibrium constant ( Kd ) values.

Article Snippet: Nutlin-3 and ChIP grade antibody for ubiquitin, and HDM2 (catalytic RING domain) peptide were purchased from Enzo Life Sciences.

Techniques: Immunoprecipitation, Western Blot, Binding Assay

Journal: Biochemical and biophysical research communications

Article Title: Connexin43 Regulates Osteoprotegerin Expression via ERK1/2 -dependent Recruitment of Sp1

doi: 10.1016/j.bbrc.2018.12.173

Figure Lengend Snippet: (A) Diagram of the OPG proximal promoter. Numbers represent the number of nucleotides relative to the transcriptional start site (+1). Four Sp1 sites located in the region from −1486 to −1286 are shown, as are the two Runx2 binding elements located at −994 to −990 and −309 to −303, respectively. (B) Luciferase reporter assay performed in UMR106 osteoblasts co-transfected with −1486/+133 OPG Luc or −1286/+133 OPG Luc plasmid and either an empty vector (EV) control, wild type Cx43 expression vector (Cx43), or mutant Cx43 plasmids bearing the G138R or R76S mutation. ***, p-value < 0.001 relative to empty vector (EV) control sample by ANOVA.

Article Snippet: The chromatin immunoprecipitation (ChIP) grade Sp1 antibody (#39058) was from Active Motif, and the anti-Runx2 antibody (#D130–3) from MBL International.

Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Control, Expressing, Mutagenesis

Journal: Biochemical and biophysical research communications

Article Title: Connexin43 Regulates Osteoprotegerin Expression via ERK1/2 -dependent Recruitment of Sp1

doi: 10.1016/j.bbrc.2018.12.173

Figure Lengend Snippet: (A) ChIP performed in UMR106 osteoblasts transfected with an empty vector (EV) control, wild type Cx43 expression vector (Cx43), or mutant Cx43 plasmids bearing the hypomorphic G138R or R76S mutation and precipitated using Sp1 antibodies. Data show result of qPCR on bead fractions amplified by the primers to the OPG proximal promoter (n=4/group). Data are normalized to inputs. The empty vector (EV) control is set to a value of 1. ***, p-value < 0.001 relative to empty vector (EV) control sample by ANOVA. (B) (A) ChIP performed in UMR106 using Runx2 antibodies (n=4/group). (C) Model of Cx43-dependent regulation of Tnfrsf11b/OPG gene expression and inhibition of osteoclastogenesis.

Article Snippet: The chromatin immunoprecipitation (ChIP) grade Sp1 antibody (#39058) was from Active Motif, and the anti-Runx2 antibody (#D130–3) from MBL International.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Mutagenesis, Amplification, Gene Expression, Inhibition

Journal: Scientific Reports

Article Title: The dual function of PRMT1 in modulating epithelial-mesenchymal transition and cellular senescence in breast cancer cells through regulation of ZEB1

doi: 10.1038/srep19874

Figure Lengend Snippet: ( a , b ) Expression of known EMT inducers was assessed by qRT-PCR ( a ) and western blotting ( b ) in MCF10A-PRMT1 and in control vector cells. ( c , d ) Real-time RT-PCR and western blotting confirmation of ZEB1 knockdown efficiency in MDA-MB-231 cells. ( e , f ) Quantitative-ChIP experiments using anti-H4R3me2as and anti-PRMT1 to measure the levels of H4R3me2as and PRMT1 at the promoter of ZEB1 gene in MCF10A-PRMT1 cells ( e ) and MDA-MB-231-shPRMT7#1/#2 cells ( f ). All experiments were repeated at least three times. Error bars, mean ± SD, ns: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The ChIP Grade antibody against H4R3me2as was purchased from Active Motif (Cat No. 39705).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation, Knockdown

Journal: bioRxiv

Article Title: Heterochromatin and RNAi act independently to ensure genome stability in Mucorales human fungal pathogens

doi: 10.1101/2022.12.01.518749

Figure Lengend Snippet: (A) H3K9me2 (light purple) and -me3 (dark purple) and RNA polymerase II (RNA pol II, gold) enriched distribution is shown across scaffold 9. Enrichment coverage is computed as the ratio of immuno p recipitated (IP) DNA/Input DNA reads, previously normalized as log 2 c ounts p er m illion (CPM). Transcripts (mRNA, green) and sRNA (red) coverages correspond to log 2 CPM. Genes (light red) and repeated sequences (light blue) including pericentric regions (bright blue) are displayed, as well as GC content coverage as a percentage. (B) Dot plot and Pearson’s correlation coefficient (R, dark line) of H3K9me2 (x-axis) and -me3 (y-axis) average log 2 CPM enrichment values are plotted as 10-kb regions representing the whole genome. Overlapping dots are color-coded as a density scale. (C) Proportion of gene, repeated, and intergenic sequences are shown color-coded as percentage of total base pairs in the genome (inner pie chart). The proportion of H3K9-methylated base pairs in the same categories is highlighted (darker shade, outer pie chart) from the remaining non-methylated base pairs (lighter shade, outer pie chart). (D) The percentage of small RNA aligned reads according to length and 5’-end nucleotide distribution (color-coded) is depicted. (E) Schematic view of the six most abundant TE families identified in the genome, showing total length (nt) of the elements (solid black lines), open reading frames (gray rectangular arrows), and predicted protein domains (color-coded rectangles) abbreviated as follows: Zinc finger, CCHC-type (ZnF-CCHC); Endonuclease/exonuclease/phosphatase superfamily (APE); Reverse transcriptase domain (RT); Reverse transcriptase zinc-binding domain (RT-ZnB); Retrotransposon GAG domain (GAG); Aspartic peptidase domain superfamily (PR); Integrase zinc-binding domain and catalytic core (INT); Chromodomain (CD); Homeobox-like domain superfamily (HOX); Transposase, Tc1-like (Tc1-Tpn) and DDE domain (DDE); Harbinger transposase-derived nuclease domain (Harbinger-Tpn); Helitron helicase-like domain (Helitron); DNA helicase Pif1-like (Pif1). (F) Genomic distribution of the six most abundant TE families shown as color-coded rectangular arrows to indicate transcription direction (element length is scaled to the rectangle width, not including the arrow point). Centromeric ( CEN ) and telomeric (*) regions are designated in the plot. The first eleven out of twenty-four scaffolds (> 94% of the genome) containing nine centromeres and their length (Mb) are shown for reference.

Article Snippet: ChIP-grade antibodies α-H3K9me2 (ab1220, Abcam), α-H3K9me3 (ab8898, Abcam), α-RNA pol II (39497, Active Motif) were utilized to immunoprecipitate chromatin-bound DNA, following a previously-established procedure ( ).

Techniques: Methylation, Reverse Transcription, Binding Assay, Derivative Assay