Fig 1: Increased expression of MGMT in liver cells upon depletion of NIPP1.a O6-alkyl guanine levels were measured by dot-blot assays on genomic DNA isolated from livers of male 14-days-old CTR and LKO mice that had been injected intraperitoneally with 25 mg/kg DEN 48 h before sacrifice (n = 4). Genomic liver DNA was loaded onto a nitrocellulose blotting membrane and probed with an antibody against O6-alkyl guanine (Squarix, EM2-3). b Quantification of the O6-alkyl guanine levels. Hprt, as determined by qPCR, was used for normalization. The data are expressed as means ± SD (n = 6). c, d Mgmt transcript levels were measured by qRT-PCR in the livers of untreated CTR and LKO mice (n = 3) of two weeks (c) and 48 h after a DEN injection (25 mg/kg) in 14-days-old CTR and LKO mice (d; n = 6). Hprt was used for normalization. The data are expressed as means ± SD. Total RNA was isolated from snap-frozen mouse livers using the GenElute®Mammalian Total RNA Miniprep kit (Sigma). e MGMT activity in liver extracts from untreated CTR and LKO mice of two weeks old. MGMT activity was measured according to manufacturer guidelines (MD0100, Sigma-Aldrich). The data are expressed as means ± SD (n = 3). Immunoblotting with Tata-binding protein (TBP; Abcam, ab51841) was used for normalization. f Graphic display of mutations in the indicated gene fragments, 1 month after a single injection of DEN (25 mg/kg) in male CTR and LKO mice of 14 days. Proofreading PCR for Hnf4a, Ctnnb1, and Hras was performed on genomic liver DNA from 4 CTR and 4 LKO mice. PCR products were cloned into the pGEM®-T easy Vector System (Promega) and processed for sequencing. All observed mutations are indicated in the figure. g Negative correlation between PPP1R8 and MGMT mRNA expression levels in human liver samples with hepatocellular carcinoma. The graph was derived from data generated by the TCGA Research Network (http://cancergenome.nih.gov/ and www.cbioportal.org). h, i HepG2 cells were treated during 72 h with control (Ctr) siRNA or two independent NIPP1 siRNAs (NIPP1 KD, siRNA#1 or siRNA#2). Knockdown efficiency and MGMT expression were analyzed by qRT-PCR. HPRT was used for normalization. All data are means ± SD (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t-test).