Cleaved parp 1

PDAC cells were treated with nab-paclitaxel (10 μM) and BMS-754807 (10 μM), and lysed after 16 hours to prepare cell lysate for Western blotting. Tumor lysates were prepared by snap freezing tumor tissues in liquid nitrogen and stored at −80°C. These tumor tissues were suspended in lysis buffer and homogenized using the Bullet Blender Homogenizer (Next Generation, Averill Park, NY), and extracts were sonicated on ice. Proteins in supernatants were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were incubated overnight at 4°C with the following antibodies: total IGF-1R, phospho-IGF-1R (Tyr1135/1136)/IR (Tyr1150/1151; #3024), total AKT, phospho-AKT (Ser473), total ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), cleaved caspase-3, cleaved PARP-1 and GAPDH (Cell Signaling Technology, Beverly, MA). The membranes were then incubated with the corresponding HRP-conjugated secondary antibodies (Pierce Biotechnologies, Santa Cruz, CA) for 1 to 2 hour. Specific protein bands were visualized using the enhanced chemiluminescence reagent (SignalFire, Cell Signaling) with Image360 system and quantitated by densitometry.

Cells were lysed in RIPA buffer (Sigma‐Aldrich) supplemented with a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche). Protein concentration was measured using a BCA protein assay kit (Thermo Scientific, USA). Antibodies against PARP1 (#9542), cleaved PARP1 (#5625), caspase‐3 (#9665), cleaved caspase‐3 (#9664), cyclin D1 (#2978), p27 (#2552) and GAPDH (#2118) were purchased from Cell Signaling Technology (Cambridge, MA, USA). Isolated proteins were probed with the indicated primary antibodies followed by incubation with HRP‐linked secondary antibodies and detection using an ECL system (Thermo Fisher, USA). Protein expression levels were normalized to that of GAPDH (Cell Signaling Technology).

After harvesting the cells, the lysates were prepared by resuspending the cells in RIPA buffer (Sigma Aldrich, Oakville, ON, Canada, #R0278) supplemented with protease inhibitor (Sigma Aldrich, #4693124001). To rupture the cells, the resuspended cells passed through a 25 G needle 15 times. The protein content in the samples was measured using BCA Protein Assay Kit (Thermo Scientific, Saint-Laurent, QC, Canada, #23225). The generated lysates were used for immunoblotting with following antibodies: GAPDH 1:2000 (Abcam, Waltham, MA, USA, #ab9485), cleaved caspase-3 1:500 (Cell signaling, # 9664S), cleaved PARP-1 1:500 (Cell signaling, # 5625S), ARP2/3 1:1000 (Millipore, #MABT95), Vimentin 1:1000 (Abcam, Waltham, MA, USA, #ab16700), RUNX1 1:500 (LS Bio, Seattle WA, USA, #LS-C353932), TGFβ1 1:500 (Abcam, Waltham, MA, USA, #ab215715).

Western blot analysis was performed using standard procedures for whole-cell extracts from cell lines. Antibodies used include Chk2 (1:5000, Becton Dickinson and Millipore), SIRT1 (1:2000, Millipore), acetyl-lysine (1:1000), phospho-CHK2-T68 (1:1000), phospho-CHK2-Thr387 (1:1000), phospho-p53-Ser20 (1:1000), acetyl-p53-Thr382 (1:1000), phospho-histone H2A.X (Ser139) (1:1000), phospho-ATM-Ser1981 (1:1000), ATM (1:1000), phospho-histone H3-S10 (1:1000), p-CDC25C (ser216) (1:1000), CDC25C (1:1000), cleaved PARP-1 (1:1000) and cleaved caspase-3 (1:1000) (Cell Signaling Technology), phospho-CHK2-Thr432 (1:500, Invitrogen), P53 (Do-1, 1:1000, Santa Cruz Biotechnology), FLAG (clone M2) (1:2000), α-tubulin (1:5000, Sigma), and β-actin (1:5000, Sigma). For immunoprecipitation analysis, cell lysates (1–4 mg) after preclearing were mixed with antibodies (2 μg) at 4 °C overnight followed by the addition of 30 μl of protein-G (for mouse antibodies)- or protein-A (for rabbit antibodies)-coupled sepharose beads (GE) for 3 h at 4 °C. Immune complexes were washed three times with lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, protease, phosphatase inhibitor mixture (Sigma)]. After boiling in 2× loading buffer, samples were subjected to SDS-PAGE, and then scanned using ECL.