Comparison of in vitro transfection efficiency of HBV plasmids between genotypes HBV expressing plasmids (5
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1 Supplemental Materials Figures legend Supplemental Table 1, 2 and 3 Supplemental Figure 1, Figure 2 and Figure 3 Supplemental Figure 1 Comparison of in vitro transfection efficiency of HBV plasmids between genotypes HBV expressing plasmids (5 ug, genotype Aa, Ae, Bj35, Bj56, C22, and CAT or empty vector) and pmaxgfp (0.5 ug, Lonza) were electroporated in 2-4 millions of HepG2 or Huh7 cells using 4D-Nucleofector X Kit (Lonza). After 24 hours of transfection, live cells were examined with a fluorescent microscope (Bz-9000 Keyence) and pictures of the phase-contrast images and fluorescent images were obtained. Numbers of GFP positive cells and total viable cells were counted in 5 different areas and the transfection efficiency was calculated as the percentage of GFP positive cells among viable cells. The bars express mean percentage ± SE of GFP positive cells in 5 different areas in each genotype. ns, not significant with evaluated ANOVA followed by Fisher s PLSD (A). Representative pictures of green fluorescent images and phase-contrast images of HepG2 cells transfected with HBV plasmids and pmaxgfp (Lonza) (B). HBV expressing-plasmids (5 ug) and pmaxgfp (0.5 ug) were electroporated in 2-4 millions of HepG2 or Huh7 cells using a 4D-Nucleofector X Kit (Lonza). After 24 hours
2 of transfection, cells were replated in a 96-well cell culture plate. After 6 hours of incubation the fluorescence intensity of GFP was measured using a fluorometer (Fluoroskan Ascent FL, Thermo). After measuring the GFP intensity, cell viability of each well was measured using an MTS assay (CellTiter 96 AQueous One, Promega). Relative transfection efficiency was calculated as GFP intensity/absorbance in each well. The bars represent mean ± SE of transfection intensity relative to empty vector control. ns, not significant evaluated with ANOVA followed by Fisher s PLSD (C). Supplemental Figure 2 Real-time PCR array targeting chemokine-related mrna was carried out. The 3D-bar graphs indicate the relative expression of chemokine-related genes in HBV-replicating Huh-7 cells in comparison to those with control plasmids. A list of good expression genes and intermediate genes significantly induced in HBV-replicating Huh-7 cells in comparison to those with control plasmids is shown. Supplemental Figure 3 The expression of MICA/MICB on the HCC cell lines with HBV replication MICA/MICB were stained with MICA/MICB antibody and analyzed by FACS Canto-II. The MFIs of MICA/MICB are showed in bar graphs. Five independent experiments were carried out. Error bar indicated
3 standard deviation. Independent student t test were carried out.
4 S.Table1 Real time PCR-array column and gene name Gene Name /Row Gene Name /Row Gene Name /Row Gene Name /Row AGTRL1 A01 CCR4 C01 CXCL6 E01 SDF2 G01 BDNF A02 CCR5 C02 CXCL9 E02 SLIT2 G02 CXCR5 A03 CCR6 C03 CXCR3 E03 TCP10 G03 C5 A04 CCR7 C04 CXCR4 E04 TLR2 G04 C5AR1 A05 CCR8 C05 CXCR6 E05 TLR4 G05 CCBP2 A06 CCRL1 C06 CYFIP2 E06 TNF G06 CCL1 A07 CCRL2 C07 TYMP E07 TNFRSF1A G07 CCL11 A08 CKLF C08 GDF5 E08 TNFSF14 G08 CCL13 A09 CMTM1 C09 GPR31 E09 TREM1 G09 CCL15 A10 CMTM2 C10 GPR81 E10 VHL G10 CCL16 A11 CMTM3 C11 HIF1A E11 XCL1 G11 CCL17 A12 CMTM4 C12 IL13 E12 XCR1 G12 CCL18 B01 CMKLR1 D01 IL16 F01 B2M H01 CCL19 B02 CSF3 D02 IL18 F02 HPRT1 H02 CCL2 B03 CX3CL1 D03 IL1A F03 RPL13A H03 CCL3 B04 CX3CR1 D04 IL4 F04 GAPDH H04 CCL4 B05 CXCL1 D05 IL8 F05 ACTB H05 CCL5 B06 CXCL10 D06 IL8RA F06 HGDC H06 CCL7 B07 CXCL11 D07 LTB4R F07 CCL8 B08 CXCL12 D08 MMP2 F08 CCR1 B09 CXCL13 D09 MMP7 F09 CCR10 B10 CXCL2 D10 MYD88 F10 CCR2 B11 CXCL3 D11 NFKB1 F11 CCR3 B12 CXCL5 D12 SCYE1 F12
5 S. Table2 The characteristic of patients for the serum CX3CL1 quantification Healthy CH-C CH-B CH-B CH-B (n=15) (n=15) (Genotyep A) (Genotype B) (Genotype C) (n=5) (n=15) (n=15) Age (Mean/SD) 38.8 (3.1) 49.9 (10.6) 45 (4.8) 48.2 (9.1) 48.7 (8.7) Gender(M/F) (8/7) (9/6) (3/2) (9/6) (8/7) HBV-DNA (Mean/SD) NA NA 5.9 (0.63) 6.22 (0.65) 6.11 (0.62) HBeAg (+/-) NA NA (3/2) (9/6) (9/6) HBsAg titer (Mean/SD) NA NA 7172 (3784) 7794 (4080) 7672 (3815) ALT (Mean/SD) NA 65.2 (10.5) 60.4 (9.2) 65.5 (9.6) 62.1 (8.9) PLT (Mean/SD) NA (23835) (21916) (12984) (20488) Presence of Liver Cirrhosis Presence of HCC
6 Supplemental Table 3 Characteristics of patients included in immunohistochemical analysis HBV HCV NBNC number age 57.8± ± ±1.1 p= Sex(male/female) 11/2 13/6 6/2 p= Tumor size(mm) 56.3± ± ±15.4 p= ALT(IU/L) 45.8± ±8.2 18±2.4 p= γgtp (IU/L) 63.8± ± ±27.2 p= Alb (g/dl) 4.18± ± ±0.08 p= Plt ( 10 3 /µl) 158.5± ± ±33.4 p= PT (%) 91.5± ± ±3.4 p= AFP (ng/ml) 1515± ±719 81±45 p= PIVKA-II (AU/L) 56650± ± ±3309 p= Stage (I/II/IIIA) 5/4/4 6/7/6 6/0/2 p= HBV genotype(a/b/c/na) 0/0/4/9 HBV-DNA(LC/ml) 3.87±0.55 mean±se
7 Supplemental Figure 1 A C HepG2& Huh7& GFP&posi;ve&cells GFP&posi;ve&cells (%) (%) HBV&expressing&plasmids&5&ug& pmaxgfp&0.5&ug Transfec;on Repla;ng&cells&in&a&96&well&plate& &at&10,000&cells/well Measurement&of&GFP&intensity& Measurement&of&viability& MTS&assay& HepG2&or&Huh7,&2R4&millions Calcula;on&of&efficiency& as&gfp&intensity/absorbance ns ns empty&vector HepG2& Huh7& Aa Bj35 C22 GFP phase&&contrast ns.0 Supplemental figure. In vitro transfection efficiency of HBV plasmids between genotypes. HBV expressing plasmids (5 ug, genotype Aa, Ae, Bj35, Bj56, C22, and CAT or empty vector) and pmaxgfp (0.5 ug, Lonza) were electroporated in 2-4 millions of HepG2 or Huh7 cells using 4D-Nucleofector X Kit (Lonza). After 24 hours of transfection, live cells were examined with a fluorescent microscope (Bz-9000 Keyence) and pictures of phase-contrast images and fluorescent images were obtained. Number of GFP positive cells and total viable cells were counted in 5 different areas and the transfection efficiency was calculated as a percentage of GFP positive cells in viable cells. The bars express mean percentage ± SE of GFP positive cells in 5 different areas in each genotype. ns, not significant with evaluated ANOVA followed by Fisher s PSLD (A). Representative pictures of green fluorescent images and phase-contrast images of HepG2 cells transfected with HBV plasmids and pmaxgfp (Lonza) (B). HBV expressing plasmids (5 ug) and pmaxgfp (0.5 ug) were electroporated in 2-4 millions of HepG2 or Huh7cells using a 4D-Nucleofector X Kit (Lonza). After 24 hours of transfection, cells were replated in a 96-well cell culture plate. After 6 hours of incubation the fluorescence intensity of GFP was measured using a fluorometer (Fluoroskan Ascent FL, Thermo). After measuring the GFP intensity, cell viability of each well was measured using an MTS assay (CellTiter 96 AQueous One, Promega). Relative transfection efficiency was calculated as GFP intensity/absorbance in each well. The bars represent mean ± SE of transfection intensity relative to empty vector control. ns, not significant evaluated with ANOVA followed by Fisher s PSLD (C). B GFP&intensity/cell&viability&& rel.&to&empty&vector GFP&intensity/cell&viability&& rel.&to&empty&vector ns.0
8 Supplemental Figure2 Genotype(A(HBV(Replica2ng(Huh7(cell( Ae Fold Difference (Test/ Control) A B C D E F G H Row Genotype(B(HBV(Replica2ng(Huh7(cell( Bj Fold Difference (Test/ Control) A B C D E F G H Row Genotype(C(HBV(Replica2ng(Huh7(cell( C;AT Fold Difference (Test/ Control) A B C D E F G H Row Threshold(cycle(value(range<28 28<Threshold(cycle(value(range<33
9 Supplemental*Figure*3 (MFI) 4600 HepG * * * * * * Vector Bj-stop Ae Aa Bj35 Bj56 C-AT C-22 * p<0.05 (MFI) Huh7 * * * * * * Vector Bj-stop Ae Aa Bj35 Bj56 C-AT C-22 * p<0.05
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
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