Supplementary Materials: Gene Expression Signatures in AML-12 Hepatocyte Cells Upon Dengue virus Infection and Acetaminophen Treatment Figure S1. Detection of DENV−2 in AML−12 cell cultures. AML−12 cells were grown in DMEM/Ham's F12. The presence of DENV2 into the cells was performed by immunocytochemistry, using a mouse anti−flavivirus envelope protein antibody (4G2), diluted 1:10, and Novolink™ Polymer Detection Systems. Images are shown at 100X magnification. The brown staining indicates the presence of DENV−2 in the cell cultures. Ab control: cells labeled with peroxidase−conjugated antibody. Anti−Env (4G2): cells labeled with mouse anti−Flavivirus envelope protein antibody (4G2) and peroxidase−conjugated antibody. Untreated = cells not incubated with DENV−2 and untreated with APAP (Negative controls). DENV = cells incubated with DENV−2 at a MOI = 1. APAP + DENV = cells incubated with DENV−2 at a MOI = 1 (48 h) and 1 mM APAP (24 h). Figure S2. Evaluation of DENV infection in AML−12 cells by RT−PCR. Total RNA extracted from AML−12 cells was used to run RT−PCR. The cDNA was submitted to PCR assays according to [65] modified by [66]. The PCR products were run at 1% agarose/TAE (Tris−acetate, 0,5 mM EDTA) and stained using SYBR Safe—DNA Gel Stain (Thermo Fisher Scientific). The positive control (C+) pUC− Viruses 2020, 12, 1284 2 of 180 cloning vector, containing a sequence designed for annealing with primers. (C−) PCR reaction control (no cDNA). Untreated = cultured cells (not incubated with DENV−2 and untreated with APAP. APAP = cells treated with APAP 1 mM (24 h). DENV = cells incubated with DENV−2 at a MOI = 1 (24 h). APAP+DENV = cells incubated with DENV−2 at a MOI = 1 (48 h) and treated with 1 mM APAP (24 h). APAP CC50= cells treated with APAP 20 mM (24 h). Figure S3. Multidimensional scaling plot of sequencing libraries of AML−12 cells infected by DENV−2 and/or treated with APAP. The multidimensional scaling plot based on pairwise gene expression profiling distances among AML−12 cells infected by DENV−2 and/or treated with APAP. On the x−axis, it is possible to observe the separation of DENV2−infected cells (DENV−infected and DENV−infected/APAP−treated cells) of the non−infected groups (Untreated and APAP−treated cells). DENV−infected cells: cells incubated for 48 hours with DENV−2 (MOI=1); APAP−treated cells: cells treated with 1mM APAP for 24 hours after additional 24 hours of culture; DENV−infected/APAP−treated cells: cells incubated for 24 hours with DENV−2 (MOI=1) and further treated with 1mM APAP for 24 hours; Untreated cells: untreated cells cultured for 48 hours. Viruses 2020, 12, x 3 of 180 Figure S4. Dot plot of hierarchical summaries of GO terms enriched in differentially expressed genes AML−12 hepatocytes after infection by DENV−2 and treatment with APAP in gene clusters 2 (A), 4 (B), and 5 (C). Data were obtained from the analysis of the GO terms that were significantly enriched within the Biological Process categories (p.adjust < 0.05) among DEGs from each cluster presented in Figure 2. Dots represent the enriched GO terms after summarization with the REVIGO tool. The color of the dots represents the p−adjust values following Benjamini−Hochberg (BH) significance testing. The position of the dots in the x−axis (Count) is related to the amount of DEGs associated with the GO term. The size of the dots (GeneRatio) represents the number of DEGs related to the number of genes associated with a GO term in the Mus musculus genome. Table S1. Differentially expressed genes identified in AML−12 hepatocytes cells after infection by DENV−2 and/or treatment with APAP. Differentially Expressed Genes in APAP−Treated Cells Compared to Untreated Cells Gene logFC logCPM F p Value FDR Serpinb9b −0.81 5.00 102.13 2.89 x 10−15 1.95 × 10−11 Akr1b7 −0.74 7.09 96.32 9.65 × 10−15 4.35 × 10−11 Viruses 2020Gm13857, 12, x −0.72 6.17 91.88 2.49 × 10−14 8.44 × 104−11 of 180 Azgp1 −0.66 4.62 72.99 1.97 × 10−12 5.33 × 10−09 Eno3 −0.87 5.09 102.17 8.23 × 10−12 1.86 × 10−08 Arhgap36 −0.66 4.39 65.53 1.31 × 10−11 2.52 × 10−08 Crip1 −0.80 6.01 90.22 5.10 × 10−11 7.67 × 10−08 Serpina7 −1.35 1.41 57.90 1.01 × 10−10 1.37 × 10−07 Cyp2c68 −0.70 3.67 55.16 2.18 × 10−10 2.46 × 10−07 Cyp2c55 −0.76 3.80 59.18 1.68 × 10−09 1.62 × 10−06 Gm48702 −0.63 4.78 56.36 2.40 × 10−09 2.17 × 10−06 Lamc2 −0.61 3.87 44.17 5.71 × 10−09 4.16 × 10−06 Ttc36 −0.62 4.81 53.72 7.92 × 10−09 5.36 × 10−06 Spink4 −0.96 2.04 42.81 8.74 × 10−09 5.63 × 10−06 Gm36099 −0.88 2.50 40.98 1.57 × 10−08 8.49 × 10−06 Wnk2 −1.12 1.40 40.17 2.03 × 10−08 1.02 × 10−05 Pax8 −0.60 3.54 39.18 2.81 × 10−08 1.36 × 10−05 Fabp5 −0.59 3.65 38.58 3.42 × 10−08 1.60 × 10−05 Apol7a −0.79 3.63 44.62 9.38 × 10−08 3.73 × 10−05 Asgr2 −0.67 2.97 35.08 1.10 × 10−07 3.83 × 10−05 Apobec1 −0.64 3.72 33.59 1.84 × 10−07 5.40 × 10−05 Hsf2bp −0.59 2.95 29.47 7.90 × 10−07 1.77 × 10−04 X2010204K13Rik −0.93 1.34 29.44 7.97 × 10−07 1.77 × 10−04 Ifi27l2b −1.01 1.81 27.21 1.80 × 10−06 3.21 × 10−04 Tekt4 −0.64 2.77 27.16 1.84 × 10−06 3.23 × 10−04 Gm42603 −0.64 2.79 26.95 1.99 × 10−06 3.31 × 10−04 Orm1 −0.64 2.89 26.91 2.15 × 10−06 3.51 × 10−04 Gm6093 −0.98 0.97 26.61 2.26 × 10−06 3.60 × 10−04 B230322F03Rik −0.85 1.87 26.99 2.42 × 10−06 3.76 × 10−04 Sorbs3 −0.60 2.72 25.98 2.86 × 10−06 4.30 × 10−04 Apof −1.20 1.94 28.08 3.79 × 10−06 5.13 × 10−04 Dbn1 −0.80 1.48 24.21 5.62 × 10−06 6.93 × 10−04 Ltbp1 −0.63 2.51 23.44 7.56 × 10−06 8.05 × 10−04 Ltbp4 −0.69 3.09 28.57 1.05 × 10−05 9.60 × 10−04 Gm27184 −0.81 1.38 22.11 1.27 × 10−05 1.12 × 10−03 Gm13257 −0.64 2.44 20.64 2.46 × 10−05 1.76 × 10−03 Gm9905 −0.71 2.01 20.50 2.58 × 10−05 1.82 × 10−03 Plxnb3 −0.64 2.27 20.20 2.72 × 10−05 1.88 × 10−03 BC064078 −0.68 2.11 20.15 2.78 × 10−05 1.90 × 10−03 Muc5b −0.65 2.99 24.69 2.80 × 10−05 1.91 × 10−03 Fbp2 −0.59 2.34 19.76 3.26 × 10−05 2.11 × 10−03 Hrct1 −0.62 2.22 19.57 3.51 × 10−05 2.22 × 10−03 Pparg −0.68 1.76 19.52 3.59 × 10−05 2.26 × 10−03 Cym −0.84 1.13 19.44 3.72 × 10−05 2.31 × 10−03 Apoc2 −0.59 5.09 30.85 4.47 × 10−05 2.64 × 10−03 X2310040G24Rik −0.62 2.59 20.17 4.85 × 10−05 2.81 × 10−03 Gm8130 −0.69 2.30 20.20 5.59 × 10−05 3.06 × 10−03 A730090N16Rik −0.65 1.83 18.43 5.61 × 10−05 3.06 × 10−03 Gm47577 −0.75 1.38 18.38 5.73 × 10−05 3.10 × 10−03 Atp6v0a4 −0.65 1.71 18.13 6.36 × 10−05 3.33 × 10−03 Orm3 −0.73 1.71 18.03 6.64 × 10−05 3.42 × 10−03 Cbln3 −0.60 4.83 29.63 6.92 × 10−05 3.45 × 10−03 Tmprss6 −0.66 4.52 31.54 7.04 × 10−05 3.47 × 10−03 Cyp2c65 −0.75 1.27 17.59 7.99 × 10−05 3.77 × 10−03 X9130230N09Rik −0.71 1.56 17.34 8.84 × 10−05 3.97 × 10−03 Lmcd1 −0.65 1.88 16.64 1.19 × 10−04 4.74 × 10−03 Crb2 −0.73 1.28 16.47 1.28 × 10−04 4.95 × 10−03 Ifi204 −0.61 8.23 25.04 1.33 × 10−04 5.09 × 10−03 B430218F22Rik −0.84 1.78 19.46 1.42 × 10−04 5.33 × 10−03 Gm47528 −0.61 1.92 15.78 1.72 × 10−04 6.09 × 10−03 Viruses 2020, 12, x 5 of 180 X4930471E19Rik −0.67 1.49 15.53 1.92 × 10−04 6.53 × 10−03 Tmem236 −0.77 1.39 15.89 2.00 × 10−04 6.75 × 10−03 Gm28536 −0.64 2.05 15.98 2.06 × 10−04 6.88 × 10−03 Gm36033 −0.74 1.10 15.32 2.09 × 10−04 6.95 × 10−03 Gjb4 −0.73 1.81 16.86 2.10 × 10−04 6.95 × 10−03 Acnat2 −0.68 1.49 15.06 2.35 × 10−04 7.49 × 10−03 Pnmt −0.62 1.61 15.00 2.41 × 10−04 7.61 × 10−03 Dctd −0.68 1.32 14.80 2.63 × 10−04 8.14 × 10−03 Gm35853 −0.64 1.43 14.45 3.07 × 10−04 9.12 × 10−03 X1200007C13Rik −0.90 1.17 16.14 3.14 × 10−04 9.28 × 10−03 Hpx −0.90 7.37 33.48 3.21 × 10−04 9.39 × 10−03 Gm19935 −0.64 1.37 14.31 3.26 × 10−04 9.44 × 10−03 Hist1h1d 1.54 2.64 131.63 1.13 × 10−17 1.53 × 10−13 Mphosph9 0.59 4.27 48.07 2.94 × 10−09 2.48 × 10−06 Ckap2 1.10 1.93 45.74 5.84 × 10−09 4.16 × 10−06 Casc4 0.66 3.09 36.11 7.78 × 10−08 3.19 × 10−05 Cenpe 0.85 2.47 35.07 2.42 × 10−07 6.85 × 10−05 Pfn2 0.72 2.39 30.20 6.07 × 10−07 1.42 × 10−04 Gm15834 0.62 2.91 27.86 1.42 × 10−06 2.78 × 10−04 Gen1 0.93 1.17 26.68 2.20 × 10−06 3.54 × 10−04 Nanos1 0.75 2.04 26.35 2.49 × 10−06 3.83 × 10−04 Kif15 0.64 2.08 20.03 2.92 × 10−05 1.95 × 10−03 Gm47572 0.64 1.71 17.26 9.17 × 10−05 4.03 × 10−03 Mybl1 0.63 2.13 17.10 9.80 × 10−05 4.14 × 10−03 Gm16042 0.78 1.02 15.76 1.74 × 10−04 6.10 × 10−03 Egr1 1.69 6.12 36.88 2.74 × 10−04 8.35 × 10−03 Differentially expressed genes in DENV−infected cells compared to Untreated cells Gene logFC logCPM F PValue FDR Hsd17b4 −0.99 7.29 182.34 4.42 × 10−21 1.75 × 10−19 Gnmt −1.12 4.24 174.07 1.41 × 10−20 5.49 × 10−19 Pccb −0.96 5.56 171.55 2.04 × 10−20 7.80 × 10−19 Acot12 −1.42 3.35 180.86 2.30 × 10−20 8.75 × 10−19 Kifc2 −1.15 4.20 172.42 2.44 × 10−20 9.22 × 10−19 Acad11 −0.92 6.67 158.18 1.49 × 10−19 5.43 × 10−18 Uroc1 −1.45 2.97 155.33 2.31 × 10−19 8.28 × 10−18 Acaa1b −1.73 4.83 360.28 5.75 × 10−19 2.00 × 10−17 Mturn −1.30 6.76 251.67 1.07 × 10−18 3.69 × 10−17 Ackr4 −0.89 5.60 142.94 1.67 × 10−18 5.66 × 10−17 Aldh1a7 −1.08 8.45 179.39 2.56 × 10−18 8.59 × 10−17 Tpmt −1.17 4.45 184.21 1.17 × 10−17 3.82 × 10−16 mt.Tl1 −1.03 4.71 155.91 1.21 × 10−17 3.95 × 10−16 S100a13 −0.92 4.21 130.99 1.26 × 10−17 4.08 × 10−16 Epas1 −1.05 3.87 129.66 1.59 × 10−17 5.01 × 10−16 Hist2h2be −1.10 3.41 128.47 1.96 × 10−17 6.11 × 10−16 X4930486I03Rik −0.90 4.57 126.20 2.94 × 10−17 9.09 × 10−16 Cyp2c55 −1.40 3.80 194.10 3.33 × 10−17 1.03 × 10−15 Ces2a −0.96 4.22 126.24 3.70 × 10−17 1.13 × 10−15 Dbi −0.83 8.95 123.37 4.88 × 10−17 1.48 × 10−15 Mt1 −0.86 10.05 119.23 1.04 × 10−16 3.09 × 10−15 Uqcr11 −0.77 6.09 116.65 1.69 × 10−16 4.89 × 10−15 Eif4b −0.81 9.67 115.51 2.09 × 10−16 6.00 × 10−15 Ndufs2 −0.76 7.35 115.30 2.18 × 10−16 6.24 × 10−15 Mrpl34 −1.03 3.43 113.95 2.81 × 10−16 7.96 × 10−15 Tkfc −0.76 5.77 112.81 3.49 × 10−16 9.78 × 10−15 Klhl13 −0.86 4.35 112.39