Fig 1: Localization of INO80 and histone marks in the upstream regions.The data from ChIP followed by qPCR for HOXC11(A & B) and PAX7(C & D) are shown. A & C- Line diagram indicates the upstream region analyzed by qPCR of ChIP DNA (thick black line) mapping on chromosome12 and 1 respectively for HOXC11 and PAX7, along with the genomic positions; the bent arrow indicates the direction of transcription of the gene, and unfilled box is the hINO80 binding motif present in the upstream region. HC1-HC6 and PX1-8 are the amplicons originating from the upstream region covering 1137bp and 1403bp of HOXC11 and PAX7 respectively using primers listed (S2B Table). B&D show the enrichment as % Input for each region HC1-HB6(B) and PX1-PX8 (C) of INO80 and the H3K9me3 and K3K27me3 marks as shown in the inset (*p value<0.05 and **p<0.001 in comparison with IgG for each primer set).
Fig 2: MEME-based prediction of INO80 binding motif.(A) Snapshot of MEME output for the sequences enriched after DBINO interaction, (B) Position weight matrix of the putative INO80 motif generated by MEME.
Fig 3: Effect of INO80 binding on expression of luciferase reporter gene in HEK293T cell line.(A) Diagrammatic representation of the constructs used in the luciferase reporter assays. The constructs are; pGL3 promoter vector without the INO80 binding site, pGL3INO80 BS-Up (BS-Up) where the INO80 binding motif is cloned upstream of the promoter of luciferase reporter gene and pGL3INO80 BS-Dn (BS-Dn) where binding motif is cloned down-stream of the poly(A) signal. (B) The effect of INO80 binding on the expression of reporter gene under normal and knock-down of hINO80 condition in HEK293T cells. The reporter expression is indicated as fold change with reference to expression from pGL3 vector. The firefly luciferase counts were normalized with renilla luciferase counts. (C) The effect of knock-down of PRC members on reporter expression. The constructs pGL3, pGL3 BS-up and pGL3 BS-Dn were transfected in cells treated with siRNA-YY1, siRNA-EED and siRNA-SUZ12. (D) The effect of the mutant oligos (Fig 5A) on reporter expression. The error bars represents the standard deviation (*p value<0.05 and **p<0.001).
Fig 4: Specificity of interaction of INO80.(A) Interaction of INO80 binding motif with the nuclear extracts from siRNA-INO80 transfected cells (Sigma-EHU069661) compared to untransfected and control siRNA (sc-37007) transfected cells. (B) The supershift of the INO80 bound oligo following the addition of anti-INO80 antibody. The arrow heads indicate the three retarded oligo nucleotides, while the arrow points to the heavier complex formed after addition of the antibody.
Fig 5: Ino80 knockdown activates ATR-Chk1 signaling in human colon cancer cells(A) HT29 cells were transfected with non-specific or Ino80-specific siRNAs, and whole-cell lysates were analyzed using immunoblotting with the indicated antibodies. Band intensities were quantified using Image Gauge software (left). The normalized ratios (phosphorylated/total) of CHK1 activity are shown as a graph (right). A representative result from three independent experiments is shown. (B) SW480 cells were subjected to the same experiments as in (A). Ino80 band was indicated by star mark on the gels in (A) and (B). (C) The effect of Ino80 knockdown on the replication stress-induced ATR-Chk1 signaling in HT29 cells. After transfection was performed as in (A), HT29 cells were double-stained with either anti-ATR (top) or anti-p-CHK1 (bottom) in addition to anti-γ-H2AX antibodies. Confocal images were captured, and more than 50 cells were scored per condition. The results are presented as the percent of foci-positive cells and illustrated as a graph to the right of the representative images. (D) SW480 cells were subjected to the same experiments as in (C). *p < 0.05; **p < 0.01; ***p < 0.001; Error bars, mean ± SD of three independent experiments.
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