Pereira TA et al.1
Supplemental Material and Methods
Human samples
An experienced pathologist staged the biopsies based on MassonTrichrome staining. Further analysis of these anonymized tissue sections was
performed at Duke University. Isolation of liver cells from other de-identified
residual liver tissue from healthy liver allograft donors was also done at Duke
University.
Control human livers (n=2) were obtained from excess tissue that was
“cut-down” from donor livers that were used as allografts in pediatric recipients.
The healthy liver tissue fragments were processed in a similar fashion to other
biopsy tissues (formalin-fixation, paraffin-embedding, and immunostaining).
Histology and Immunohistochemistry (IHC)
IHC was performed to localize expression of Hh signaling components
(Shh, Ihh, Ptc, Gli2), the mesenchymal markers alpha smooth muscle actin
(SMA) and S100A4, a marker of activated endothelial cells (CD31), and several
epithelial progenitor markers (Keratin (Krt) 7, CD133, EpCam, and Survivin).
Antibody sources, dilutions and antigen retrieval treatments used for IHC are
listed in Supplemental Table 1. Antigens were demonstrated by
diaminobenzidine (DAB) or Ferangi BlueTM (FB8125, Biocare Medical).
Ptc, αSMA and Sirius red staining were assessed by morphometry
(Universal Imaging Corp, Downington, PA). To quantify Ptc, αSMA and Sirius red
staining, three 200 fields per section containing similar size portal tract were
Pereira TA et al.2
evaluated (n=3 per stage of fibrosis). The number of Shh(+) cells were counted in
10 representative 40x fields/section from each patient (n=3 per stage of fibrosis);
data are expressed as %Shh(+) cells/fibrosis stage
RNA and Protein Analysis
Briefly, RNA was extracted and analyzed by quantitative reverse
transcriptase Real time PCR (qRT-PCR). Total RNA was collected using TRIzol®
(Invitrogen). Following DNase I treatment, 5μg of RNA was reverse transcribed to
cDNA using random hexamers and SuperScript II Reverse Transcriptase
(Invitrogen). Following reverse transcription, cDNA was resuspended in pure
water at the ratio of 1μg of starting RNA to 100μl of water. Amplification was
performed using a SYBR Green PCR master mix (Applied Biosystems, Foster
City, CA). Each sample was analyzed in triplicate. Target gene levels in treated
cells or tissues are shown as a ratio to levels detected in corresponding control
tissue, according to the ΔΔCt method. β-actin and S9 were used as control genes
for human and rat samples, respectively. The primers are detailed in
Supplemental Table 3.
For protein analysis, liver tissue was lysed in 500µl of RIPA buffer
containing protease and phosphatase inhibitors. Proteins (50μg) were separated
by polyacrylamide gel electrophoresis and transferred to nylon membranes.
Primary antibodies employed were against αSMA (1:2000, Sigma-Aldrich), Gli2
(1:2000, Santa Cruz Biotechnologies, Santa Cruz, CA), and β-actin (1:2000,
Pereira TA et al.3
Sigma-Aldrich). Appropriate secondary antibodies were used with antigens
demonstrated
by
enhanced
chemiluminescence
(Pierce
Biotechnology,
Rockford, IL). β-actin was used to demonstrate equal protein loading.
RNA isolation from FFPE tissue and qRT-PCR
Briefly, approximately tissue was digested with lysis buffer containing
proteinase K for 1h, followed by removal of genomic DNA with gDNA Eliminator
spin columns. Next, RNA was eluted using RNeasy MinElute spin columns and
directly transcribed to cDNA using SuperScript II Reverse Transcriptase. cDNA
was diluted to 100µl with pure water. Standard qRT-PCR amplification was
performed using a SYBR Green PCR master mix (Applied Biosystems). β-actin
was used as internal control gene. Target gene levels in tissues were presented
as a ratio to housekeeping gene expression (β-actin ) levels according to the Δ
Ct method. Each sample was analyzed in triplicate.