Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Phosphorylation of STAT3 (A) and ERK (B) in 3D culture.
  (A) Addition of OSM induced phosphorylation of STAT3. Cells were incubated with or without 10 ng/ml OSM in the presence of EGF and HGF for 2 days. Anti-STAT3 and anti-phospho-STAT3 (Cell signaling, Beverly, MA) were used to detect total and phosphorylated STAT3, respectively.
  (B) Addition of U0126, a MEK inhibitor, reduced phosphorylation of ERK. Cells were incubated with or without 10 μM U0126 in the presence of EGF and HGF for 2 days. Anti-ERK and anti-phospho-ERK were used to detect total and phosphorylated ERK, respectively.
• Supplemental Figure 2 - Effect of inhibition of p38 MAPK and JNK on cyst formation. HPPL formed cysts with a central lumen in the presence of 10 μM SB202190, a p38 MAPK inhibitor (A), whereas HPPL formed aggregates but not cysts in the presence of 10 μM SP600125, a JNK inhibitor (B). HPPL were cultured with either SB202190 or SP600125 in the presence of EGF and HGF for 7 days. After fixation, they were stained with AlexaFluor 546 conjugated phalloidin.
• Supplemental Figure 3 - Increase of proliferation does not affect the size of the central lumen of HPPL cysts.
  (A) Ki67⁺ cells were most frequently detected in the presence of both EGF and HGF. HPPL were cultured without growth factors and with EGF, HGF, or EGF plus HGF for 2days. Samples were stained with anti-Ki67 antibody. Five areas were randomly selected in each sample and the number of Ki67⁺ was counted under a fluorescence microscope. Experiments were repeated 3 times and average values are shown in the graph. Error bars refer standard deviations. Asterisks (*), and double asterisks (**) represent p<0.05 and p <0.01, respectively.
  (B) Size of the central lumen of cysts is similar among culture with EGF, HGF, and EGF plus HGF. HPPL were grown in 40% Matrigel for 7 days and then stained with AlexaFluor 546 conjugated phalloidin. Pictures of 10~20 cysts that were selected from three samples cultured in the same condition were taken by a confocal microscope and diameters of the central lumen were measured on a Zeiss LSM software. Average values are shown in the graph. Error bars refer standard deviations.