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HEP_24091_sm_suppfigure1.tif10510KProliferative and oncogenic potential of HBx in a selectively repopulating liver. (A) Transgene vectors used for hydrodynamic delivery. (B) Luciferase imaging of mice injected with either HBx (top) or Gfp (bottom) at 48-days post-hydrodynamic injection (PHI). (C) Livers injected with HBx at 74-days (left) and 139-days (right) PHI were rough in texture with hyperplastic nodules. Scale bars, 0.5 cm. (D) Representative liver of control mice injected with Gfp at 113-days PHI (left). Fluorescent imaging of a representative lobe displayed uniform Gfp signal throughout the lobe (right). Scale bar, 0.5 cm.
HEP_24091_sm_suppfigure2.tif7427KAugmented oncogenic potential of HBx with shp53 in a selectively repopulating liver. (A) Control animals coinjected with empty vector and vector containing a short hairpin RNA directed against Trp53 (shp53) (empty/shp53) were normal at 63-days PHI (left). Animals coinjected with HBx and shp53 (HBx/ shp53) had rough surface texture with nodular appearance (middle). Hyperplastic nodules were detected at 71-days PHI for HBx/ shp53 animals (right). Scale bars, 0.5 cm. (B) Fluorescent imaging of representative lobes taken from livers described in (A) for Gfp signal. Empty/shp53 liver lobe had uniform Gfp signal (left). However, liver lobes taken from HBx/ shp53 animals at 63- and 71-days PHI had nodular Gfp signals (middle and right, respectively). (C) RT-PCR analyses of various genes-of-interest in macroscopically normal liver and hyperplastic nodules taken from 72-days PHI HBx/ shp53 experimental mice (n = 3). Dashed lines separate different mouse sample groups. (D) Semi-quantitative analysis of RT-PCR from (D) demonstrated significantly higher Afp expression levels in (i) normal HBx/ shp53 (8 livers from 3 animals) compared with normal empty/shp53 (15 livers from 5 animals) livers (P = 0.0035) and (ii) hyperplastic HBx/ shp53 nodules (7 nodules from 2 animals) compared with normal empty/shp53 livers (P < 0.0001). No statistical difference in Afp expression relative to Actb levels was seen between normal liver and hyperplastic nodules isolated from HBx/ shp53 animals (P = 0.3818). RT-PCR for Afp expression levels in normal empty/ shp53 livers data not shown. P, unpaired t test.
HEP_24091_sm_suppfigure3.tif7193KNRAS does not cooperate with HBx in liver tumorigenesis during selective repopulation. (A) NRAS induced hyperplasia at 82-days PHI (left) and its oncogenic potential was augmented by the detection of hyperplastic nodules in all experimental animals at 71-days PHI when coinjected with shp53 (NRAS/shp53) (middle). In contrast, only 1 hyperplastic nodule was detected in animals coinjected with HBx and NRAS (HBx/ NRAS) at 72-days PHI (right). Scale bars, 0.5 cm. (B) When HBx, NRAS and shp53 were coinjected (HBx/ NRAS/ shp53), mice sacrificed at 61- and 71-days PHI had multiple hyperplastic nodules detected (left and right, respectively). Scale bars, 0.5 cm. (C) Fluorescent imaging of representative lobes taken from livers described in (A) and (B) for Gfp signal. Lobes taken from HBx/ shp53 animals or HBx/ NRAS/ shp53 had nodular Gfp signals (left and right, respectively). (D) RT-PCR analyses of various genes-of-interest in normal livers and hyperplastic nodules taken from HBx/ NRAS/ shp53 and NRAS/ shp53 mice sacrificed at 61-days PHI.
HEP_24091_sm_suppfigure4.tif11108KDetection of CD45+ lymphocyte infiltration into livers of various transgene injected mice by immunohistochemical analysis. HBx, 78-day PHI; HBx/ shp53, 72-day PHI; HBx/ NRAS, 72-day PHI; NRAS/ shp53, 61-day PHI; NRAS, 82-day PHI; Gfp, 113-day PHI. Left column, sections were incubated with anti-CD45. Right column, no primary antibody was used. Scale bars, 100 μm.
HEP_24091_sm_suppinfo.doc43KSupplementary information

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