Fusion fx5

Differences between mock-transduced and ADAM17-silenced LEC sublines in the expression and release of relevant proteins were analyzed using Human Soluble Receptor Array (R&D Systems) designed for non-hematopoietic cells. The LEC sublines (1×10⁷ cells each) were plated on six dishes (10-cm-diameter, ~1.6×10⁶ cells per dish in 10 ml of basal medium) and incubated overnight. Then the medium was changed (8 ml per dish) and, after a 16-h incubation, collected and concentrated to a volume of 600 μl, using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3 membrane (Millipore). The cells were washed with PBS, collected with Accutase, centrifuged and solubilized for 3 h at 4°C in 600 μl of lysis buffer provided with the array and supplemented with Complete Protease Inhibitor Cocktail (Roche Applied Science). The assay was performed according to the manufacturer's instructions. The membranes were incubated with the buffer containing 300 μl of the concentrated cell media or with 300 μg of the cell lysate proteins. Chemiluminescence signals were captured with Fusion FX5 (Vilber Lourmat) and analyzed with BIO1D software (Scientific Software Group).

Genomic DNA was extracted from ASCs cultured in two different conditions using the PureLink Genomic DNA Mini Kit (Invitrogen). Telomere length analysis was carried out using TeloTAGGG Telomere Length Assay kit (Roche) in accordance with the manufacturer’s instructions. The chemiluminescence was detected by gel documentation imaging system (Fusion FX5, Vilber Lourmat, France). By comparing the signal relative to a molecular weight standard, the mean telomere restriction fragment (TRF) length was quantified.

Identical amounts of membrane protein or cell lysate (15-20 µg) were separated on 10% SDS-PAGE gels, followed by transfer to nitrocellulose membranes. Loading was systematically verified using Red Ponceau staining. Immunoblotting was performed as previously described using the different antibodies as indicated [8] (link), [10] (link). For cell lysates, β-tubulin was used as loading control. Protein bands were revealed by chemiluminescence (GE Healthcare, Orsay, France) using a peroxidase-conjugated secondary antibody and a chemiluminescence kit (GE Healthcare), followed by imaging on a Bio-Rad Fusion FX5 (Vilber Lourmat, France). Densitometric analysis was performed using ImageJ software.

Tissue or cell total protein was extracted using Radio Immunoprecipitation Assay lysis buffer (R0010, Solarbio, Beijing, China) at 4 °C for 15 min followed by centrifugation at 15,000 r/min for 15 min. The protein concentration was measured using a bicinchoninic acid kit (20201ES76, Yeasen, Shanghai, China). Then, the proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto the PVDF membrane, and blocked in 5% bovine serum albumin for 1 h. Next, the membrane was incubated at 4 °C overnight with diluted primary antibodies: Fbxw7 (ab109617, 1: 200), EZH2 (ab186006, 1: 500), ZBTB16 (ab39354, 1: 1000), FLAG (ab1162, 1: 200), HA (ab9110, 1:2000), Myc (ab9106, 1:500), GAPDH (ab8245, 1:1000), and Ub (ab7780, 1:500). All the rabbit polyclonal antibodies were purchased from Abcam (Cambridge, UK) unless otherwise noted. After incubation, the membrane was re-probed with horseradish peroxidase-labeled anti-Rabbit immunoglobulin G (IgG) H&L (ab205718, 1:10,000) for 1 h at room temperature. The blots were visualized with enhanced chemiluminescence regents. The images were captured using FUSION FX5 (Vilber Lourmar, France), and analyzed by ImageJ 1.48u (National Institutes of Health, USA). GAPDH (ab9485, 1:500) was served as the internal reference.

PANC-1 cells and MIA PACA-2 cells were lysed with lysis buffer (Beyotime Institute of Biotechnology, China), and protein concentration was subsequently estimated by the bicinchoninic acid protein assay. Then, equal amounts of protein samples were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore, USA). After being blocked with 5% nonfat milk, membranes were incubated overnight at 4 °C with rabbit primary antibodies against human ADAM10 (Boster, China) and human actin (Zhongshan Golden Bridge Biotechnology, China). After being washed with Tris-buffered saline/0.1% Tween three times, membranes were incubated with secondary antibodies for 1 hours at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL; Pierce,USA) kit. The specific protein bands were captured and visualized using FUSION FX5 (Vilber Lourmat).

Human growth factor antibody array membrane kit was used (Abcam, Cambridge, UK) to evaluate the changes in growth factor profiles of DPCs by PM extract treatment. Total of 41types of human growth factors could be analyzed at once. Briefly, DPCs (1x10⁵cells/well) were seeded in 24well plates and cultured overnight. Cells were treated with 20 μg/ml of PM extract for 24 h, and then culture supernatants were collected for growth factor analysis. Fresh medium and culture supernatant from non-treated cells were used as blank and control, respectively. Conventional immunoblot process was performed following the manufacturer’s guide. Biotin-conjugated anti-cytokines antibodies were used as primary antibody and HRP-conjugated streptavidin was used for chemiluminescence detection. The resulting blots were analyzed under identical condition using chemiluminescence detector Fusion FX5 (Vilber Lourmat, France).