Fig. 3

Givinostat acts as a specific direct inhibitor of rCGGBP1-DNA interaction in vitro. A and B: DBID using anti-FLAG (a) or anti-CGGBP1 antibodies (b) against rCGGBP1 pre-incubated with compounds as indicated show a direct inhibition by Givinostat. The multiple dot blots per compound are technical replicates. c: Quantification of the signals obtained from the dot blots of secondary screening (b) are plotted. D: Densitometric signals of DBID assay for CGGBP1 using lower concentrations of Givinostat show no inhibition at concentrations lower than 100 μM. e: Pre-incubation of rCGGBP1 with Givinostat reduces the amount of Alu DNA co-precipitating with FLAG-antibody by 70-80%. f: An input-normalized δδCt relative quantification of CGGBP1 ChIP DNA from HEK293T cells treated with 2 μM Givinostat or DMSO for the indicated durations shows inhibition of CGGBP1 occupancy at Alu elements by Givinostat. g: The DBID assay for acid-extracted nuclear proteins using total histone H3 antibody shows no inhibition of DNA-binding of histone H3 by Givinostat. h: DBID signals (background-subtracted and normalized for IgG signals) for the H3K4me3, H3K9me3 and total histone H3 show no significant change due to 100 μM Givinostat. i and j: qPCRs on CTCF and H3K4me3 ChIP-DNA show that H3K4me3 is significantly increased at Alu elements (i) whereas CTCF occupancy at a CGGBP1-regulated CTCF-binding site is significantly decreased (j) by Givinostat treatment. * indicates Mann-Whitney p-value < 0.05; ns represents p-value > 0.05. k: Nuclear-cytoplasmic fraction analysis of mock- or Givinostat-treated HEK293T cells showed that the nuclear levels of CGGBP1 and histone H3 remain unchanged upon Givinostat treatment. GAPDH serves as a loading control for the cytoplasmic fraction. l: A comparison of Givinostat or DNaseI treated genomic DNA shows Givinostat does not cause the degradation of DNA while DNaseI digests DNA into low molecular weight fragments and increases the low molecular weight smear