Fig 1: PARP9/DTX3L regulates of PARP14 post-transcriptionally. A) Immunoblot analysis of a panel of cell-lines, including HNSCC (head and neck squamous cell carcinoma), GBM (glioblastoma multiforme), PRAD (Prostate adenocarcinoma) and PAAD Pancreatic adenocarcinoma. B) Correlation of PARP14 and DTX3L protein levels in cell lines, showing Pearson correlation R. C) RT-qPCR analysis of total mRNA using gene-specific primers for PARP9, PARP14 and DTX3L in cells transfected with siRNAs specific to each respective gene or NC1: negative control. The relative expression levels were quantified using the ΔΔCt method and presented as RQ. The expression values were normalised to ACTB as the reference gene. The data is presented as the mean values, with the error bars representing the standard deviation (SD). D) Immunoblot analysis of PARP14, PARP9 and DTX3L in FaDu cells following 48 hr transfection with siRNAs specific to each respective gene or NC1. Actin was used as a loading control.
Fig 2: Mapping and prediction of PARP9-DTX3L interaction. A) Depiction of GFP-flag tagged PARP9 were included in an anti-FLAG co-IP of PARP9 truncations co-expressed with DTX3L and probed with anti-DTX3L (top blot) or anti-FLAG (bottom blot). B) AF2 prediction of DTX3L KHL2-5 complexed with PARP9 truncation containing PARP9 KHL and 9cat domains. Insert of PAE plot showing high confidence in arrangements of DTX3L (KHL2-5) and PARP9 interactions pITM = 0.9. C) Superimposed competing PARP9 (pink) and PARP14 (blue) interaction with DTX3L KHL4 (green). D) Three alanine substitutions in DTX3L KHL4 were made in two regions identified in the model for potential contacts with PARP9. FL DTX3L DLS/AAA and KLF/AAA were co-expressed with PARP9 9C (top) or PARP14 14C (bottom) which was pulled down with anti-FLAG beads and blotted for DTX3L and FLAG. Lanes have been rearranged (top) from the same blot to be consistent with anti-FLAG co-IP of PARP14C.
Fig 3: PARP9 and PARP14 expression in vitro and in vivo.(a) TMT-derived 0-h-normalized protein abundance profiles for PARP9 and PARP14 from mouse RAW264.7 and human THP-1 M(IFNγ) and M(IL-4) data sets. (b) PARP9 and PARP14 gene expression at 24 h after stimulation (n=3). (c) PARP9 and PARP14 protein expression visualized by western blot. The time course in the relative protein abundances of PARP9 and PARP14 normalized to β-actin were quantified (graph, n=3). *P<0.05 and **P<0.01, respectively, by Student's t-test. Error bars indicate s.d. (d) Representative images of PARP9 and PARP14 expression in atherosclerotic plaques from the aorta of an Apoe−/− mouse (n=3) fed a high-fat diet and from the carotid artery of a human (n=5). Scale bars, 100 μm.
Fig 4: PARP14 is removed rapidly in the absence of PARP9/DTX3L but independently of the proteasomal or lysosomal degradation. A) Immunoblot analysis and quantification of PARP14 degradation in UMSCC74A after cycloheximide (100 μM) treatment. B) Immunoblot analysis and quantification of PARP14 degradation in siPARP9-transfected cells following CHX (100 μM). C) Immunoblot analysis of PARP14 in NC1, siPARP9 and siDTX3L treated UMSCC74A cells in the presence of proteasomal inhibitor, MG132 (10 μM) or DMSO control. D) Immunoblot analysis of PARP14 in NC1, siPARP9, siDTX3L and siPARP9/siDTX3L UMSCC74A cells in the presence of CQ (10 μM).
Fig 5: Haematopoietic PARP14 in mouse atheromata and PARP9–PARP14 expression in human plaques.(a) Representative image and quantification of aortic root lesion formation and CD68+ macrophage accumulation (green, Alexa 488) in high-fat and high-cholesterol diet-fed LDLR−/− mice whose bone marrow was reconstituted by PARP14−/− mice (BMTPARP14−/−→LDLR−/− mice, n=5), compared with LDLR−/− mice with PARP14+/+ bone marrow (BMTPARP14+/+→LDLR−/− mice, n=6–7). Scale bars, 100 μm. (b) mRNA expression of the aorta from a. n=6–8. (c) Immunofluorescence staining of PARP14 and PARP9 proteins (green, Alexa 488) in human carotid plaques. CD68 (red, Alexa 594). Nuclei (blue, 4,6-diamidino-2-phenylindole, DAPI). Scale bars, 100 μm; insets, 10 μm (n=5). Prevalence of PARP14+ or PARP9+ macrophages in macrophage-poor versus macrophage-rich plaques. *P<0.05 and **P<0.01, respectively, by Student's t-test. Error bars indicate s.d.
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