Fig. 3: IMPDH2 increases on chromatin in a DNA damage-dependent manner.
From: Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity
A Western blot of MDA-MB-231 cells cytosolic (cyt) and chromatin (chr) fractions in the control condition (DMSO) and after 3 h of etoposide treatment and 24 h of release. As cytosolic and nuclear markers, Vinculin and H3 were used, respectively (n = 3). B Quantification of nuclear IMPDH2 signal intensity in MDA-MB-231 cells treated with DMSO or etoposide in the presence (+G) or absence of guanosine supplementation (50 μM); 1 h post-supplementation with guanosine cells were exposed to 10 µM etoposide for 3 h followed by a 24 h recovery period (DMSO: nDMSO = 3011, nGuanosine = 6849; Etoposide: nDMSO = 3431, nGuanosine = 4259, outliers removed, 3 SD; unpaired two-tailed Wilcoxon test). Immunofluorescence of nuclear IMPDH2 and mean intensity quantification normalized by DMSO in MDA-MB-231 cells (DMSO, n0h = 7700; n2h = 6999; n4h = 7372; n24h = 13846; 1 μM, n0h = 7018; n2h = 6816; n4h = 7362; n24h = 10647; 2.5 μM, n0h = 6520; n2h = 7310; n4h = 8409; n24h = 9746; 5 μM, n0h = 6397; n2h = 7029; n4h = 7685; n24h = 8412; 10 μM, n0h = 6635; n2h = 7019; n4h = 6864; n24h = 8770) treated with increasing concentrations of etoposide after treatment or 2, 4 and 24h post-release (n = 3 biological replicates, unpaired two-tailed Wilcoxon test) (C), representative pictures of nuclear IMPDH2 (green) and DAPI (blue) in DMSO control condition and 10 μM etoposide treatment after 24 h of release; non-confocal mode, scale bar 15μm (D). Immunofluorescence of nuclear ɣH2AX, representative pictures (orange) and DAPI (blue) in DMSO control condition and 10 μM etoposide treatment after 24 h of release; non-confocal mode, scale bar 15 μm (E) and foci quantification in MDA-MB-231 cells (DMSO, n0h = 884; n2h = 1070; n4h = 1323; n24h = 2174; 1 μM, n0h = 642; n2h = 1322; n4h = 1270; n24h = 1927; 2.5 μM, n0h = 815; n2h = 1322; n4h = 1532; n24h = 1222; 5 μM, n0h = 651; n2h = 1382; n4h = 2212; n24h = 966; 10 μM, n0h = 657; n2h = 1140; n4h = 1182; n24h = 1226) treated with increasing concentrations of etoposide after treatment or 2, 4 and 24h post-release (n = 3 biological replicates, unpaired two-tailed Wilcoxon test) (F). All box plots indicate the median value (central line), interquartile range IQR (box boundaries), and up to 1.5*IQR beyond the box boundaries (whiskers). Source data are provided as a Source Data file.
