PMC

Effect of recombinant ShhN treatment on Gli1 mRNA expression in pancreatic CAFs. (a) Gli1 mRNA levels were assessed 24 hours after treatment of CAF27 cells with indicated concentrations of recombinant ShhN. (b) Comparison of ShhN treatment in control pancreatic fibroblasts and pancreatic CAFs. Gli1 mRNA levels were assessed 24 hours after treatment of HPNE, SC2, or CAF cells with indicated concentrations of recombinant ShhN. Relative Gli1 mRNA levels after normalization to the corresponding 18S rRNA levels are shown. Data are means of four independent experiments; bars are SD values.

Effects of Smo siRNA and recombinant ShhN treatment on Gli1 mRNA expression in pancreatic CAFs. (a) Smo and (b) Gli1 levels were assessed by qRT-PCR in CAF26 cells transfected with 2μM non-targeting or Smo siRNA for 72 hours. (c) CAF27 cells were transfected with 2μM non-targeting or Smo siRNA and treated with the indicated concentrations of recombinant ShhN 48 hours later. Gli1 mRNA levels were assessed at 72 hours post-transfection. Relative Smo or Gli1 mRNA levels after normalization to the corresponding 18S rRNA levels are shown. Data represent the mean of three independent experiments; error bars are SD values.

Immunohistochemical staining of Smo protein. Cells were stained with an anti-SMO antibody and an HRP-labeled secondary antibody (brown) then counterstained with hematoxylin and eosin. Staining revealed (a) strong Smo expression in Purkinje neurons of cerebellar tissue; (b) lack of Smo expression in normal pancreatic stromal fibroblasts (arrows) (c) no Smo staining in a primary pancreatic cancer section incubated with secondary antibody alone; and (d) strong SMO expression in pancreatic cancer associated stromal fibroblasts (arrows). Smo expression is also detected in pancreatic cancer cells. Representative samples from each type of tissue are shown. Magnification x40.

Analysis of Smo mRNA expression in fibroblast cultures. (a) Affymetrix exon array analysis of Smo mRNA expression in fibroblast cultures established from chronic pancreatitis tissue (SC3), IPMN tissue (SC2), normal pancreas (HPNE), and primary pancreatic adenocarcinomas (CAFs). Relative Smo expression values were obtained after using the Robust Multichip Average (RMA) method to normalize the raw intensity measurements of all probe sets. (b) Quantitative RT-PCR analysis of Smo mRNA expression in fibroblast cultures established from normal pancreas (HPNE), IPMN tissue (SC2), chronic pancreatitis tissue (SC3), and primary pancreatic adenocarcinomas (CAFs). Relative Smo mRNA levels after normalization to the corresponding 18S rRNA levels are expressed. Each assay was performed in triplicate. Data are means of three independent experiments; bars are SD values.