Fig 1: A multiplexed RNAi screen identifies MBD3 as a sensitizer to cPt treatment and suppressor of cPt resistance acquisition in A2780 EOC cells. (a) Schematic representation of the multiplexed RNAi screening strategy using the indicated chromatin-focused shRNA library. (b) Scatter plot showing the relative depletion (day 14/pool) of all genes in the library under cPt treatment compared to the vehicle control. The top-four depleted genes are highlighted. Geometric mean, n = 3. (c) String network analysis of the top four differential hits. Node radius indicates level of differential depletion. Red node color indicates a differential top hit, and light red indicates genes present in the library. Edges between nodes indicate both functional and physical protein associations, line thickness indicates the strength of the underlying data. (d) Competitive proliferation assays of A2780 EOC cells expressing the indicated shRNAs. Shown is the relative fraction of GFP+/shRNA+ cells relative to the initial measurement. After 7 days, cells were treated with vehicle or cPt (1 µM), as indicated by the red arrow, and analyzed for additional 9 days. Mean ± SEM, n = 3. (e) Competitive proliferation assays of A2780 EOC cells expressing the indicated shRNAs to determine the long-term effects of suppression of MBD3 or LSD1 expression under cPt treatment. A2780 cells expressing the indicated shRNAs were analyzed for the absolute cell number (count/mL) and the fraction of GFP+/shRNA+ cells relative to the initial measurement. Treatment with cPt (1 µM) was started, as indicated by the red arrow. Mean with range (shaded area) and individual data points are shown, n = 3.
Fig 2: An example in UCSC genome browser format. MBD3 binding pattern at VCAN gene is shown (two biological replicates; MBD3 rep1 and MBD3 rep2).
Fig 3: MBD3 suppression modulates H3K27ac in differential regions associated with resistance. (a) Scatter plot showing the fold change in H3K27ac signal of all regions with inverse change in H3K27ac signal between MBD3 suppression and cPt-resistant A2780cis cells. Fold change is shown for regions with increased acetylation in resistant cells and decreased acetylation in sensitive A2780 cells (left) and regions which display decreased H3K27ac in resistant cells and increased H3K27ac in sensitive A2780 cells (right). Respective shRNAs are indicated. Median and interquartile range are shown. (b) Number of genomic regions associated with the differentially regulated regions identified in (a). (c) Representative ChIP-seq tracks of H3K27ac signal in A2780 and A2780cis cells and A2780 cells expressing the indicated shRNAs. (d) Gene-set-enrichment analysis (ChIP-Enrich) for all genes associated to the differentially regulated regions identified in (a). All identified pathways with a p-value ≤ 0.05 were categorized as indicated.
Fig 4: Suppression of MBD3 is associated with alterations of H3K27ac within selected regulatory regions. (a,b) Profile plot of the average H3K27ac ChIP-seq signal in A2780 cells expressing control or MBD3 shRNAs for all H3K27ac peaks (a) or regions that show an increase or decrease of H3K27ac upon MBD3 suppression (b). Regions with differential H3K27ac ChIP-seq signal were identified by differential peak filtering. Average H3K27ac signals are plotted with a 10 kb window around the peak center. (c) Representative ChIP-seq tracks of H3K27ac intensity for regions with increased or decreased H3K27ac signal in MBD3 KD A2780 cells from (b). (d) Number of genomic regions associated with increased or decreased H3K27ac signal upon MBD3 suppression in A2780 cells. (e) Distribution of the H3K27ac peak distance to the nearest TSS identified by ChIP-Enrich.
Fig 5: MBD3 localizes in a cell-type specific manner by ChIP-seq.A. Exemplar loci are depicted in genome browser format (hg19, http://genome.ucsc.edu, [53]). Individual sequencing tracks are indicated to the left of the browser view. Genomic intervals and scale bar are indicated above the tracks. B. The column graph depicts the total number of peaks defined from the current study in each cell type. The colors depict peaks that overlap in the two cell types by at least one base as well as those with no overlap (cell-type specific in the figure).
Supplier Page from Abcam for Anti-MBD3 antibody