Anti ctgf

PANC-1 or AsPC-1 cells were cultured in six-well plates and total protein was extracted by RIPA lysis buffer (Beyotime, China). After the cell lysates were sonicated, and treated with the BCA Protein Analysis kit (Beyotime, China) to detect the concentration of protein by using an enzyme-labeled instrument (Molecular Devices, USA). Proteins were used to SDS-page and transferred to nitrocellulose filter membrane (Pall Corporation, USA), blocked with rapid blocking buffer (GenScript, USA) for 17 min and incubated with primary antibody overnight at 4 °C. The next day it was washed with TBST and incubated with secondary antibodies for 40 min, the membranes were scanned and analyzed with Odyssey infrared imaging instrument (LI-COR, USA). Antibodies used in this study include: anti-β-actin (1:20,000, ABclonal, China), anti-YAP1 (1:1500, Cell Signaling Technology, USA), anti-CTGF (1:1000, Novus Biologicals, USA), and secondary antibody (800R rabbit antibody, 1:1000, LI-COR, USA).

Assessment of 3D cell invasion was pursued as describes earlier with minor changes⁶⁹ . Briefly 1 × 10³ breast cancer cells were seeded in 100 µL in a well of an ultra-low-adherence 96-well plate (ULA; Nexcelom, Cenibra GmbH, Bramsche, Germany). After 48 h spheroid formation was visually confirmed and 50 µL of media was removed. Thereafter, 50 µL Matrigel were added to wells with spheroids. Central position of spheroids was checked visually and Matrigel was allowed to solidify for 1 h at 37 °C and 5% CO². Afterwards 50 µL media were added to each well and a picture was taken marking time point 0 (t0h). When indicated rhCTGF (recombinant human connective tissue growth factor; 1 µg/ml; R&D systems), pdhFN1 (plasma-derived human fibronectin 1; 1 µg/ml; R&D systems), rhMMP2 (recombinant human matrix metalloproteinase 2; 0.01 µg/ml; R&D systems, Minneapolis, MN, USA), Batimastat (BB-94, 4 nM; Selleckchem, Munich, Germany) polyclonal rabbit IgG control (15 µg/ml; R&D systems) or anti-CTGF (15 µg/ml; Novus Biologicals). Spheroid growth area was analyzed using ImageJ polygonal selection and measurement. Mean values were calculated and compared to respective control.

Cell-ECM adherence was examined by coating 96-well plates with bovine collagen I (30 µL; 0.04 mg/ml; BD Bioscience) for 12 h at 4 °C. Solution was aspirated and plate was left to dry under bench. Cells were washed 3 times with FBS-free DMEM and cultured for 8 h in DMEM-FBS prior to adhesion assessment. Cells were detached using 10 mM EDTA-PBS solution. Cells were pelleted (1300 rpm for 5 min) and washed twice with DMEM supplemented with 0.1% BSA. Cells were seeded (2 × 10⁴) in DMEM supplemented with 0.1% BSA, when indicated treated with rhCTGF (1 µg/ml; R&D systems), Triptorelin (10^–7 M), polyclonal rabbit IgG control (15 µg/ml; R&D systems) or anti-CTGF (15 µg/ml; Novus Biologicals), and incubated at 37 °C with 5% CO₂ for 20 min. Non-adherent cells were washed of by adding 100 µL of DMEM four times. Adherent cells were counter-stained with crystal violet solution (0.5%) for 20 min at RT shaking. Wells were washed four times with ddH₂O and dried for at least 2 h. Pictures were taken and afterwards 200 µL Methanol were added to each well, incubated for 20 min shaking and absorbance was assessed at 570 nm using Synergy (BioTek Instruments, Bad Friedrichshall, Germany). Each experiment was performed in six replicates. Mean values were compared to respective control.