Fig. 1
PAR2 knockdown in LX-2 cells inhibits tumour growth in a SCID mouse model. a-c Expression and function of PAR2 in LX-2 cells. a RT-PCR of PAR2 expression. Extraction of total RNA from the LX-2-wt cells and synthesis of cDNA was performed as described in the Methods section. PCR reactions without cDNAs were run as a negative control (Primer). Integrity of the cDNA was independently confirmed by amplification of beta-actin (Actin). MW marker, molecular-weight marker. Representative results of three independent experiments are shown. b PAR2 immunofluorescence was detected using the confocal laser scanning microscope LSM-510 Meta (Carl Zeiss, Germany). Localization of immunofluorescence labelled PAR2 is shown in permeabilized LX-2-wt cells using SAM-11 (1:100) and a FITC-conjugated anti-mouse IgG (1:200) as secondary antibody. c LX-2-wt cells grown on Lab Tek chambered borosilicate cover glass were loaded with fluo-4-AM as described in Methods. For calcium measurements, an inverted confocal laser scanning microscope LSM 510 was used. Fluorescence was monitored at 488 nm. (a) PAR2-AP (10 µM) and (b) trypsin (10 nM) induce Ca2+ rise in LX-2 cells. (c) Fluorescence images, in pseudocolor, from single LX-2 cells. The sequence shows a fast and transient fluorescence increase from 15 s to 40 s after PAR2-AP addition (0 s). Data represent the mean ± SD from calcium measurements in 20 individual cells, respectively. (d-f) PAR2 knockdown in LX-2 cells inhibits tumour growth in a SCID mouse model. SCID mice were randomized into five groups, each consisting of 8 animals. Hep3B and LX-2 cells were subcutaneously (co)injected at the right flank of the mice [(1): 5 × 105 LX-2-wt; (2): 105 Hep3B cells; (3): 105 Hep3B cells plus 5 × 105 LX-2-wt, (4): 105 Hep3B cells plus 5 × 105 LX-2-shCo cells, (5): 105 Hep3B cells plus 5 × 105 LX-2-shPAR2 cells]. d Tumours prepared 16 days after xenotransplantation. Data for body weight and tumour volume are indicated as mean ± SD (n = 8/group); **p < 0.01). e Representative micro CT images from the tumours 16 days after xenotransplantation. For 3D reconstruction of the tumours the software “Imalytics” (Philips GmbH, Aachen, Germany) was used. f Paraffin sections obtained from tumours and stained for hematoxylin/eosin, cytokeratin, Ki67 or CD31. Images were taken at magnification = × 5 (left panel) and at magnification = × 200