Immunohistochemical staining images of cells in duodenal and foci areas of 90%PX rats.

<p>5 rats were sacrificed for this study. (A) Representative immune staining images of amylase (aciner cells), insulin (islet) and sox9 (centro-acinar cells) in duodenal area. (B) Representative immuno-staining images of amylase, insulin and sox in foci area. Note, unlike those in duodenal area, relatively weak staining of ductule-like cells for amylase, very little staining of islet cells for insulin and clear staining of ductule-like for sox9 cells.</p

Effect of treatment with VA-liposome siRNA HSP47 on BrdU uptake, ECM deposition and HSP47 positive cell number in duodenal area.

<p>Specomen from 5 PX rats were analyzed. (A) Representative immune staining images of BrdU uptake by PACs in the remnant pancreas of non-treated rat (day3 NT; upper panel), and that of treated rat (day3 TR; lower panel). (B) Representative immuno-staining images of azan staining of the remnant pancreas. Upper panel; non-treated rat (day3 NT), lower panel; treated rat (day3 TR). (C) Representative immuno-staining images of HSP47 stained specimen of duodenal area of non-treated rat (day3 NT; upper panel), and that of treated rat (day3 TR; lower panel). (D) Histograms of average BrdU uptake rate of non-treated rat (NT) and treated rat (TR). (E) Histograms of average Azan positive areas of non-treated (NT) and treated rat (TR). (F) Histogram of average HSP47 positive areas of non-treated rat (NT) and treated rat (TR).</p

Schematic drawing of PX procedure, and gross appearance and histological feature of remnant pancreas.

<p>(A) Schematic drawing of remnant pancreas of 90% PX (rat). 5 rats were used for this experiment. Gross and histological appearances of these 5 rats were essentially the same. Thus representative gross appearance of remnant pancreas from 90% PX rat is shown in (B). (C)(D) Representative histological images of HE stained tissue specimen of duodenal area (C) and foci (D) of remnant pancreas from 90% PX rat. Note totally distinct histological feature of C from D.</p

Increased extracellular matrix (ECM) deposition, αSMA positivity and HSP47 positive cells in duodenal area of remnant pancreas of 90% PX rat.

<p>On this investigation, data from 5 rats were also studied. (A) Representative Azan mallory staining images of sham pancreas and remnant pancreas at day 3 after 90% PX, and immuno-staining images of αSMA and HSP47 in interstitial area, and that of HSP47 in islet. (B) Azan positive areas and HSP47 positive cells in tissue specimen of (A),were highlighted by binarization with BzⅡ analyzer software (see for details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165747#sec006" target="_blank">material & Method</a> section.) (C)(D) Histograms of Azan positive area and HSP47 positive area in interstitial area (C), and in islet (D) employing BzⅡ analyzer software on the figures shown in Fig 4(B). Monochrome dark area in a whole specimen was computed and average value on 5 randomly selected specimen from each 5 rat was calculated. The average values of PX rats were normalized to those of sham rats. Note that both Azan positive area and HSP47 positive cell number significantly increased after PX (day3) (**P<0.01).</p

BrdU staining of acinar cells, islet cells in duodenal area and ductule-like cells in foci area of sham rat and 90% PX rat.

<p>5 rats in each sham and 90% PX group were sacrificed in this experiment. (A) Representative immuno-staining images of acinar cells for BrdU in duodenal area of sham rat (upper panel), 90% PX rat at day3 (middle panel) and 90% PX rat at day 5 (lower panel). (B) Representative immuno-staining images of islet cells for BrdU in duodenal area of sham rat (upper panel), 90% PX rat at day3 (middle panel) and 90% PX rat at day 5 (lower panel). (C) Representative immuno-staining images of ductule-like cells for BrdU in foci area of 90% PX rat at day3 (middle panel) and at day5 (lower panel). Histogrames of average BrdU uptake rate of acinar cells (D), islet cells (E), ductule-like cells (F). BrdU uptake rates were by expressed number of positive staining per nuclei, counting 8000 nuclei per tissue specimen from each 5 rat for acinar cells, all nuclei per two specimen from each 5 rat for islet cells and 2000 nuclei per specimen from each 5 rat for ductule-like cells. Note that at day 3 and 5 after PX, BrdU uptake into acinar and islet cells significantly increased as compared to that of sham (**P<0.01) and that at day5 after PX, BrdU uptake by islet cells and ductule-like cells significantly decreased as compared that of day3 (**P<0.01).</p

Effect of siRNA HSP47 on stimulatory activity of aPSCs to BrdU uptake by PACs in co-culture system.

<p>(A)Ⅰ BrdU uptake by PACs was analyzed by flow cytometry for GFP fluorescence, and expression of BrdU and amylase using each corresponding antibody. (A)Ⅱ BrdU uptake by PACs co-cultured with PSCs isolated from GFP rat was analyzed by flow cytometry for GFP fluorescence, and expression of BrdU and amylase using each corresponding antibody. (A)Ⅲ BrdU uptake by PACs co-cultured with siRNAHSP47 transfected PSCs from GFP rat was analyzed by flow cytometry for GFP fluorescence, and expression of BrdU and amylase using each corresponding antibody. (B) Histogram of HSP47 mRNA expression levels of siRNA HSP47 transfected and non-treated PSCs from GFP rat. Experiment was carried out three times and average value of the HSP47 mRNA levels in PSCs as measured by qPCR was calculated. Values were normalized to that of non-transfected PSCs. Note that almost complete knock down of HSP47 mRNA by siRNA HSP47. (C) Histogram of Mean fluorescence intensity (MFI) of BrdU stained PACs alone (Pa), PACs co-cultured with non-transfected PSCs (Co) and PACs co-cultured with transfected PSCs (t-Co). Experiment was carried out three times and average value of the HSP47 mRNA levels in PSCs as measured by qPCR was calculated. Note that BrdU-MFI of PACs which was significantly augmented by co-culture with PSCs was suppressed to the level of PACs alone.</p

Effect of treatment with VA-lip siRNA HSP47 on BrdU uptake and HSP47 expression in islet of duodenal area.

<p>Quantification of BrdU uptake rate was carried out as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165747#pone.0165747.g003" target="_blank">Fig 3</a>. Quantification of Azan positive area and HSP47 positive area was conducted as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165747#pone.0165747.g004" target="_blank">Fig 4</a>. Specimens from 5 PX rats were investigated. (A) Representative immuno-staining images of BrdU positive cells in the islet of non-treated rat (day3 NT; upper panel) and that of treated rat (day3 TR; lower panel). (B) Representative immuno-staining images of HSP47 stained islet of non-treated rat (day 3 NT; upper panel) and of treated rat (day3 TR; lower Panel). (C) Representative immuno-staining images of insulin positive islet in the specimen of non-treated rat (day3 NT; upper panel) and of treated rat (day3 TR; lower panel). (D) Histogram of average BrdU uptake rate of islet cells in duodenal area of non-treated rat (NT) and treated rat (TR). BrdU uptake rate was quantified as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165747#pone.0165747.g003" target="_blank">Fig 3</a>. (E) Histogram of average HSP47 positive area in islet. Quantitative analysis of HSP47 positive area was carried out according to the method described in material and method. Note that after treatment, both BrdU and HSP47 positive cells significantly decreased.</p

Effect of VA-lip siRNA HSP47 treatment on BrdU uptake by ductule-like cells, Azan mallory staining intensity and HSP47 expression in the foci area.

<p>(A) Representative immuno-staining images of BrdU uptake by ductule-like cells in the foci area of non-treated rat (day3 NT; upper panel), treated rat (day3 TR; lower panel). (B) Representative immuno-staining images of Azan staining in the foci area. Upper panel; non-treated rat (day3 NT), lower panel; treated rat (day3 TR). (C) Representative immuno-staining images of HSP47 stained specimen of the foci area of non-treated rat (day3 NT; upper panel), treated rat (day3 TR; lower panel). (D) Histogram of average BrdU uptake rate of non-treated rat (NT) and treated rat (TR). (E) Histogram of average Azan positive area of non-treated (NT) and treated rat (TR). (F) Histogram of average HSP47 positive area of non-treated rat (NT) and treated rat (TR). Quantification of BrdU uptake rate, Azan positive area and HSP47 positive area was performed as described above.</p