Anti ki67

Primary antibodies for FACS were anti-CD73-PE (550741, 1:100), anti-CD90-FITC (555595, 1:200) from Biosciences and anti-CD105-APC (17-1057, 1:100) from eBioscience. Primary antibodies for Western blot were anti-WRN (sc-5629, 1:500), anti-β-Actin (sc-130301, 1:3,000), anti-β-Tubulin (sc-5274, 1:3,000) from Santa Cruz Biotechnology, anti-P21 (2947, 1:2,000), anti-HP1γ (2619, 1:1,000) from Cell Signaling Technology, anti-LAP2β (611000, 1:2,000) and anti-P16 (4828, 1:200) from BD Bioscience. Antibodies for immunofluorescent staining were anti-hSMA (ZM-0003) from ZSGB-Bio, anti-Progerin (sc-81611, 1:50), anti-Lamin A/C (sc-7293, 1:200) from Santa Cruz Biotechnology, anti-HP1γ (2619, 1:500) from Cell Signaling Technology, anti-53BP1 (A300-273A, 1:500) from Bethyl Laboratories, anti-γ-H2AX (05-636, 1:500) from Millipore, anti-LAP2β (611000, 1:500), anti-hCD31 (555445, 1:200) from BD Bioscience, and anti-Ki67 (VP-RM04, 1:1,000) from Vector.

For histological evaluation of treatment effect, tumors from CM, RM experimental, and PBS control groups were processed for paraffin embedding and sectioned at 7 µm. Immunostaining was performed using paraffin sections essentially as described with n = 3 mice per group^{49 (link)}. Following antigen unmasking in citrate solution (113.93 mM Na₃C₆H₅O₇, pH 6), incubation with combined primary antibodies including polyclonal anti-Ki67 (at 1:200, Abcam, Cambridge, UK) and monoclonal anti-vimentin (at 1:400, Sigma-Aldrich, St Louis, Mo.) was performed overnight at 4 °C. This was followed by amplification of signals from anti-Ki67 with biotinylated anti-rabbit Ig (Vector Laboratories). Detection was performed with streptavidin-Alexa 488 and anti-mouse Ig-Alexa 594 (Thermo Fisher Scientific, Waltham, MA). Nuclei were stained with 0.001% 4′, 6 diamidino-2-phenylindole (DAPI). Images were captured on confocal microscope Zeiss LSM510 under 40X/1.3 oil DIC M27 objective and images processed with the Zen software (Zeiss, Welwyn Garden City, and UK) and ImageJ application was used to analyze and generate images.

TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Human has-miR-20a mimics and control miRNA mimics were from Thermo Scientific Dharmacon (Lafayette, CO). Antibodies were purchased as follows: anti-Smad2, anti-Smad3, anti-phospho-Smad2 and anti-phospho-Smad3 from Cell Signaling (Denver, MA); anti-p21^CIP1, anti-Smad4, anti-c-Myc and anti-TβRII from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Ki67 from VECTOR (Burlingame, CA); and anti-β-actin from Sigma Biochemicals (St Louis, MO). The TRKI (LY2109761) was kindly provided by Dr. Jonathan Yingling (Eli Lilly Pharmaceuticals, Indianapolis, IN).

Mice were killed by carbon dioxide asphyxiation. Staining was performed in formalin-fixed, paraffin-embedded tumour sections (4-μm thickness). Fully automated immunostaining was achieved using a BenchMark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA). The following antibodies were used: monoclonal mouse anti-human CEACAM6 (9A6, 1:4,000; Santa Cruz Biotechnology Inc.), anti-CC3 (1:100, BioCare Medical, Pacheco, CA, USA), and anti-Ki-67 (1:100, VP-K452; Vector Laboratory, Burlington, Canada). The number of positive cells per tumour area was quantified.