Supplementary Material. Table S1. Summary of mapping results
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1 Supplementary Material Table S1. Summary of mapping results Sample Total reads Mapped (%) HNE (81.0%) HNE (81.7%) HNE (81.1%) HNE (81.7%) HNE (81.6%) HNE (81.4%) HNE (81.8%) HNE (82.2%) HNE (81.2%) HNE (81.8%) HNE (81.5%) HNE (81.6%) Unique Mapped (%) (74.9%) (75.5%) (75.5%) (75.7%) (75.5%) (75.4%) (75.2%) (75.4%) (74.8%) (74.4%) (75.4%) (74.4%) Properly mapped (%) (58.6%) (58.1%) (59.30%) (59.2%) (58.1%) (56.4%) (58.4%) (61.8%) (57.3%) (62.7%) (56.2%) (60.9%) mapped to exon 87.3% 88.0% 88.3% 87.8% 87.3% 87.1% 86.3% 86.8% 87.7% 86.9% 85.9% 87.5% Table S2. Differentially expressed genes detected from RNA-seq and microarray under 45 μm HNE treatment with the criteria abs(log 2 FC)>1 & FDR<0.01. RNA-seq Microarray Gene log 2 FC FDR Criteria log 2 FC FDR Criteria PAK1IP NO YES POLR1B NO E-05 YES RPP NO E-05 YES SLC39A NO YES TOB NO E-05 YES TXNRD NO E-09 YES DOT1L NO E-08 YES URB NO E-05 YES GCLM NO E-07 YES
2 EGR NO E-06 YES PPRC NO E-05 YES SEPT YES NO AKAP YES E-07 NO ALDH1B YES NO AURKB YES E-05 NO AXL E-11 YES NO BCL2A E-09 YES E-05 NO C3orf YES NO CCDC E-06 YES NO CCNB YES NO CLU YES E-06 NO CSE1L E-06 YES NO CYR E-07 YES NO DEPDC1B E-06 YES NO ERCC YES E-06 NO FERMT YES NO GINS YES E-05 NO KIAA YES NO KIF YES NO KLF YES E-06 NO LMNB YES NO MAST E-06 YES E-07 NO MYBBP1A E-05 YES NO MYBL YES E-05 NO NCAPH YES E-05 NO NTSR E-06 YES E-05 NO NXT YES NO PHLDA E-05 YES E-05 NO PHLDA E-06 YES NO PLAUR E-07 YES E-06 NO RCC E-05 YES E-05 NO RMRP E-08 YES NO RUNX YES E-06 NO S100A E-08 YES E-06 NO SERPINE E-07 YES E-05 NO SH2B YES E-07 NO SH3GL YES E-05 NO SLC35F E-05 YES E-06 NO SLC7A YES E-05 NO SMS E-05 YES NO SNORD3A YES NO SNRPA YES NO
3 SPOCD YES NO SRPRB YES NO TAGLN YES NO TGIF E-05 YES NO TM4SF E-06 YES E-05 NO TOP2A E-07 YES E-05 NO TPX E-10 YES E-06 NO TYMS YES NO UBE2C E-06 YES NO UHRF E-05 YES NO URB E-06 YES NO VASP YES NO WDR YES E-06 NO ZFP36L E-12 YES E-06 NO ZNF E-11 YES E-06 NO ANGPTL YES E-13 YES BHLHE YES E-07 YES CDCA YES E-07 YES CDCP E-13 YES E-09 YES DUSP YES E-09 YES FHL E-13 YES E-10 YES G0S YES E-11 YES GEM E-07 YES E-09 YES GFPT E-12 YES E-08 YES HMOX YES E-13 YES IER YES E-08 YES JUN YES E-09 YES KIF YES E-06 YES LAMB E-13 YES E-09 YES MYC YES E-08 YES PDCD1LG E-05 YES E-05 YES PIR YES E-07 YES RGS YES YES SEMA7A YES E-08 YES SLC16A E-05 YES E-06 YES SLC43A YES E-07 YES SLC44A E-11 YES E-12 YES TNFSF E-07 YES E-07 YES TRIB E-09 YES E-07 YES *YES: satisfy the criteria abs(log 2 FC)>1 & FDR<0.01; NO: don t satisfy the criteria.
4 Table S3. Overrepresented pathways at the gene level and the combined level under 15μM HNE treatment (FDR<0.05). Combined level Gene level Num. of genes FDR Num. of genes FDR Metabolic pathways MAPK signaling pathway *(2) *(0.06) Pyrimidine metabolism * * Cell cycle * * Glutathione metabolism * * *: not significant (FDR>0.05). Table S4. Overrepresented pathways at the gene level and the combined level under 30 μm HNE treatment (FDR<0.05). Combined level Gene level Num. of genes FDR Num. of genes FDR programmed cell death * * Nucleotide excision repair * * Base excision repair * * p53 signaling pathway * * DNA replication * * spindle organization * * ATP binding * * Purine metabolism * * Cysteine and methionine metabolism splicesome Endocytosis Focal adhesion Metabolic pathways MAPK signaling pathway Pyrimidine metabolism E-05 * * Cell cyle Glutathione metabolism * *
5 Table S5. Overrepresented pathways at the gene level and the combined level under 45μM HNE treatment (FDR<0.05). Combined level Gene level Num. of genes FDR Num. of genes FDR Ubiquitin mediated proteolysis DNA repair E microtubule-based process E E-09 Cell division E E-08 organelle organization E E-10 RNA transport Mismatch repair E E-06 programmed cell death Nucleotide excision repair E E-06 Base excision repair p53 signaling pathway E DNA replication E E-06 spindle organization E E-07 ATP binding E Purine metabolism Cysteine and methionine metabolism Spliceosome E E-06 Endocytosis Focal adhesion * * Metabolic pathways E MAPK signaling pathway E Pyrimidine metabolism E Cell cycle E E-08 Glutathione metabolism * * Table S6. Potential mirnas that target NEDD4 mir-144 mir-30a,b,c,d,e mir-27a,b mir-9 Potential binding sites (relative to 3 UTR start site) PhastCons score MirSVR score DIANAmT miranda mirdb mirwalk RNAhybrid 1 1,0(30a,e) 1 1 PICTAR4 0 1,0(30a,e) 1(27b),0(27a) 1 PICTAR PITA 0 1,0(30a,d,e) 0 1 RNA Targetscan
6 isoform, CDS or promoter switching We examined the 195 differentially expressions detected at the CDS level but not at the gene level to determine whether they were caused by isoform, CDS or promoter switching. Based on the results from Cuffdiff, ARHGEF2, IVNS1ABP, SEPT6, STK4, and XPO7 seemed to show differential splicing, whereas CNOT10, RND3 and TBRG4 appeared to have promoter switching. Further checking the transcript expression of each gene, we found only SEPT6 had true isoform switching between the transcript corresponding to the differentially expressed CDS and the other transcript. The CDS expression was significantly down-regulated, whereas the other transcript expression was significantly up-regulated, which led to the unchanged expression at the gene level (Figure S1). However, for IVNS1ABP and XPO7, the CDS expression was significantly down-regulated, whereas the other transcript abundance was not changed at all, which suggested differentially expressions detected at the CDS level but not at the gene level are mostly due to the noise added by the non-changed transcripts but not isoform switching (Figures S2 and S3). For ARHGEF2 and STK4, differentially expressed CDS and reported differential splicing were not from the same TSS group and the reported differential splicing occurred between two transcripts with very low expression value (FPKM<1), which was highly possible to be false positives. We checked the expression of each TSS group for CNOT10, RND3 and TBRG4, which were reported to have promoter switching between different TSS groups. We found the expression of TSS group containing the differentially expressed CDS was changed, whereas other TSS groups didn t change at all, which suggested differentially expression detected at the CDS level but not at the gene level are mostly due to the noise added by the non-changed TSS group but not promoter switching (Figure S4, S5 and S6). In summary, among 195 genes whose differential expression were detected only at the CDS level, only SEPT6 was found to have isoform switching between the differentially expressed CDS and other transcripts (Figure S1). Figure S1. The expression change of transcripts for SEPT6 across conditions. Isoform switching occurs between the transcript corresponding to the differentially expressed CDS and the other transcript. Figure S2. The expression change of transcripts for STK4 across conditions. Figure S3. The expression change of transcripts for IVNS1ABP across conditions. Figure S4. The expression change of TSS groups for CNOT10 across conditions. Figure S5. The expression change of TSS groups for RND3 across conditions. Figure S6. The expression change of TSS groups for TBRG4 across conditions.
7 SEPT6 transcript corresponding to DE CDS other transcript C0 C45
8 STK4 transcript corresponding to DE CDS other transcript 1 other transcript 2 C0 C45
9 transcript corresponding to DE CDS other transcript IVNS1ABP C0 C45
10 TSS containing DE CDS other TSS CNOT10 C0 C45
11 TSS containing DE CDS other TSS RND3 C0 C45
12 TBRG4 TSS containing DE CDS other TSS 1 other TSS 2 C0 C45
13 Detected#at#higher#dose# 15#μM## 30#μM## CDS# 30(23)# 91(78)# 77%# 86%# DEXSeq# 2(1)# 23(18)# 50%# 78%# MISO# 3(40)# 8%# 33(6)# 18%#
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