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Materials and reagents
Culture media, Dulbecco/Vogt modified Eagle's minimal essential medium
(DMEM), HAM’s F12, fetal bovine serum, MEM non essential amino acids solution,
MEM vitamin solutions, glyceryl monostearate, chemically defined lipid concentrate,
soybean trypsin inhibitor, penicillin/streptomycin, gentamycin, glutamine and VEGF kit
(Biosource) were purchased from Invitrogen (Carlsbad, CA).
The substrate for γ-
glutamyltranspeptidase (γ-GT), N (γ-L-glutamyl)-4-methoxy-2-naphthylamide was
purchased from Polysciences (Warrington, PA).
Immunohistochemistry
After deparaffination, sections were hydrated in alcohol and endogenous peroxidase
activity was blocked by a 30-minute incubation in methanolic hydrogen peroxide (10%).
According to the instructions supplied by the vendor a proper retrieval was used to
unmask the antigen recognized by the primary antibody, then the samples were rinsed in
0.05% Tween 20 in phosphate-buffered saline (PBS, pH 7.4) and blocked with Ultra V
Block (LabVision), before applying the primary antibody. Sections were incubated
overnight at 4°C with the following primary antibodies: a pancytokeratin antibody
(56kDa and 64kDa keratins, DAKO; Carpinteria, CA) was used to identify the biliary
cysts.Primary antibodies against IGF1, IGF1-R, and proliferating cellular nuclear antigen
(PCNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, Inc., CA), pmTOR (Ser 2448) and p-ERK 1/2 Ttyr 202/Tyr204) were purchased from Cell Signaling
Technology (Danvers, MA) and the antibody for the cleaved, activated form of Caspase3 from Novus Biologicals (Littleton, CO). Following incubation with the selected
antibody, liver sections were rinsed with 0.05% Tween 20 in PBS or TBS for 5 minutes,
incubated for 30 minutes at room temperature with secondary biotinylated antibodies and
finally developed with 3-3_ diaminobenzidine. To avoid variability in the development,
the stainings were performed in parallel and on the same day. Negative controls, in which
the primary antibody was omitted, showed the absence of specific staining. Specimens
were read by two blinded investigators (CS and SO).
Morphometric quantitation of microvascular density.
To study the changes in microvascular density in Pkd2-KO vs Pkd1KO in the presence
and absence of SU5416, liver sections were stained with rat anti-CD341 (clone MEC14.7,
GeneTex, 1:500) and rabbit anti-Cow Cytokeratin Wide Spectrum Screening (WSS)
(DAKO, 1:300). Five non-overlapping random fields per specimen were taken at 200x
using a cooled digital camera (Nikon DS-U1, Nikon). To calculate the vascular and
biliary areas, two different thresholds were set out for CD34 (red fluorescence) and wide
spectrum cytokeratin (green fluorescence) positive structures respectively, and then
expressed as percentage of pixel above the threshold per field using the LuciaG 5.0
software (Nikon). Data were expressed as ratio between CD34 and panCK positive areas.
Western blot
Total cell lysates were extracted by using a lysate buffer (50 mM Tris-HCl, 1%
NP40, 0.1% SDS, 0.1% Deoxycholic acid, 0.1 mM EDTA, 0.1 mM EGTA) containing
fresh protease and phosphatase inhibitor cocktails (Sigma, St Louis, CA). Protein
concentration was measured using the Comassie protein assay reagent (Pierce, Rockford,
IL). Equal amounts of total lysate were applied to a 4-12% NuPAGE Novex Bis-Tris
gel (Invitrogen, Carlsbad, CA) and electrophoresed. Proteins were transferred to
nitrocellulose membrane (Invitrogen, Carlsbad, CA). Membranes were blocked with 5%
non-fat dry milk (Bio-Rad Laboratories) in phosphate-buffered saline containing 0.1 %
Tween-20 (PBST) for 1h and then incubated with specific primary antibodies.
Nitrocellulose membranes were washed three times with PBST and then incubated with
horseradish peroxidase-conjugated secondary antibodies for 1h. Proteins were visualized
by enhanced chemiluminescence (ECL Plus kit; AmershamBiosciences, Piscataway, NJ,
USA). The intensity of the bands was determined by scanning video densitometry using
the Total lab Tl120DM software (Non linear USA Inc, Durham NC, USA)
Cell isolation and characterization.
Cystic and WT cholangiocytes were isolated as described. Briefly, mice were
anaesthetized by isofluorane, the portal vein was cannulated and the liver perfused in
situ with 250 ml of cold ringer-HCO3 buffer (KRB) for 5 minutes2-4. After liver
perfusion, 2-3 ml of liquid trypan blue agar was injected into the portal vein. Under a
dissecting microscope, bile ducts or cysts were microdissected by using the blue agarfilled portal vein and hepatic artery for reference. The isolated cysts or bile ducts were
again microdissected, and then plated into collagen gel. In 1-2 weeks biliary cells
usually started to outgrow. When sub-confluent, cells were passaged and plated again on
the top of rat-tail collagen. Cells were positive for CK-19 (Troma III, Hybridoma Bank
University of Iowa) and acetylated tubulin (Sigma, St Louis, MI, USA). Establishment
of a confluent monolayer with competent tight junction was assessed by measuring
transepithelial resistance and membrane potential difference (Millicell ERS system). One
week after confluence, transepithelial resistance was above 1000
· cm2. (Values
obtained with collagen-coated inserts alone were subtracted from values recorded with
monolayers).4 Finally cells were characterized by electron microscopy as described ().
Legends for supporting figures:
Supporting Fig.1 Purity of nuclear and cytosolic fractions. Nuclear and cytosolic
fractions were obtained as described in the methods section. The purity of the two
fractions was confirmed by immunoblotting for Na+/K+-ATPase (cytosolic marker) and
Hystone 3 (nuclear marker) expression.
Supporting Fig 2 Rapamycin treatment affects liver weight but not the body weight.
Rapamycin treatment significantly reduced the liver weight in Pkd2KO mice while there
was a not significant effect on body weight. (*=p<0.05 vs controls mice; ^=p,0.05 versus
vehicle treated mice).
Supporting Fig. 3 In females mice cystic area is higher than in males. Cystic area,
assessed as reported in methods section, was significantly higher in females Pkd2KO
mice with respect to males. Rapamycin treatment was effective either in females and
males. (°=p<0.05 vs vehicle treated mice)
Supporting
Fig. 4 Rapamycin treatment significantly reduces P70S6K
phosphorylation and VEGF expression. (A) Micrographs are representative of Vehicle
and rapamycin (1.5 mg/Kg/day) treated mice. As shown, rapamycin clearly reduced the
phosphorylation of P70S6K and the expression of VEGF in liver section of Pkd2KO
mice. These data were confirmed by Western blot on total liver lysate (B). Bar graphs in
(C) show the data from 8 vehicle and 8 rapamycin treated mice. (*p<0.05 vs vehicle
treated mice; **p<0.001 vs vehicle treated mice).
Supporting Fig.5 The VEGFR2 inhibitor SU5416 does not inhibit IGFR-1. To
ascertain that SU5416 did not inhibit, the IGF1-R (a tyrosine-kinase receptor), we
analyzed the phosphorylation status, of IGF1-R by western blot (antibodies were from
Cell Signaling Technology, Danvers CA) after administration of IGF1, in the presence of
SU5416 or of the specific IGF1-R inhibitor AG1024, as a positive control. After IGF1
administration, the phospho IGF1-R/IGF1-R ratio as assessed by densitometry, was 1,11
± 0,34, while it was 1,07 ± 0,32 after IGF1 and SU5416 treatment and 0,58 ± 0,22 after
IGF1+AG1024 treatment (*p<0.05 as respect to IGF1 and to IGF-1+SU5416; n=5).
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