Fig. 5: PSIP1 deficiency increases the sensitivity of cancer cells to drugs that induce transcription-coupled DNA damage.
From: PSIP1/LEDGF reduces R-loops at transcription sites to maintain genome integrity
a Western blotting of S9.6, H3 and IgG immunoprecipitated (IP) from RWPE-1 cell nuclear extract with PSIP1/p75, PARP1, γ-H2AX and PCNA (negative control) antibodies. Lysate treated with RNASEH before the pulldown was used as a negative control. IgG and H3 IP were negative controls, and 5% of the nuclear extract was used as input. b Western blotting for PSIP1-IP with PSIP1, PARP1, γ-H2AX and PCNA antibodies, IgG served as a negative control. 10% of the nuclear extract was used as input. c Western blotting for eGFP-PARP1 and HA-PSIP1 co-IPs was performed using GFP-trap beads. αGFP ab was to detect eGFP-PARP1 and αHA to detect HA-PSIP1/p75 and HA-p52, along with γ-H2AX and PCNA antibodies. d TGCA expression data showing levels of PSIP1 transcripts in cancers of the bladder (BLCA; n = 408), breast (BRCA; n = 1097), cervical (CESC; n = 305), kidney chromophobe (KICH; n = 67), kidney renal clear cell (KIRC; n = 533), Kidney renal papillary cell (KIRP; n = 290), lung (LUAD; n = 515), lung squamous cell (LUSC; n = 503), pancreatic (PAAD; n = 178) and prostate (PRAD; n = 497). The data was obtained from the UALCAN online tool. The centre line indicates a median value; the boxes and whiskers indicate 25th to 75th and 10th to 90th percentiles, respectively. e Western blot showing the levels of PSIP1 in prostate normal and cancer cells and their sensitivity to olaparib as determined by CCK-8 assay (f). Mean survival ± SD is plotted (n = 3 independent experiments). g Western blot confirming the depletion of PSIP1 by shRNA in LNCaP cells. h Cell survival assay showing % survival of control and PSIP1 knockdown LNCaP cells with indicated doses of olaparib determined using the CCK-8 assay (mean ± SD; n = 3 independent experiments). i Similar to h but for indicated doses of illudin-S. j Working model showing the role of PSIP1 in reducing R-loop level at transcription sites to minimise transcription-replication conflict leading to DNA damage. Source data are provided as a Source Data file.
