γh2ax

MEFs were treated with either KU-55933 (10 μM, Selleckchem) or vehicle for 72 hours prior to harvesting for analysis of protein expression. Cell lysates were prepared with RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 μM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 μM sodium pyrophosphate, 1 μM β-glycerophosphate, 1 μM Na₃VO₄ and 1 μg/ml leupeptin), supplemented with 1X of protease inhibitor cocktails (Sigma) and Halt phosphatase inhibitor cocktail (Thermo #78420). Snap-frozen liver and kidney samples were homogenized using FastPrep-24 instrument in RIPA buffer. Equal amounts of proteins (40 μg) were resolved on 4-15% Mini-PROTEAN TGX precast protein gels (Bio-Rad). Primary antibodies used are as follows: p-ATM Ser 1981 (cell lysates: Rockland cat. no. 200-301-400; tissue lysates: Santa Cruz sc-47739), ATM (CST #2873), p-KAP1 Ser 824(Abcam ab70369), KAP1 (Abcam ab22553), γ-H2AX (Novus NB100-384), PARP1 (CST #9532), p-p65 Ser 536 (CST #3033), p65 (CST #8242), p-IκBα Ser 32/36 (CST #9246), IκBα (Santa Cruz sc-371), p16^INK4a (Santa Cruz sc-1207), p21^Cip1 (Santa Cruz sc-6246), LaminA/C (Santa Cruz sc-20681), anti-tubulin (CST #2146), and GAPDH (CST #5174). Blots were exposed to X-ray film in a dark room or developed on iBright™ FL1000 Imaging System (Figure 1E and 1F). Protein levels were quantified by Image J software.

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 15 min each at RT. After blocking samples with 2% BSA for 30 min at RT, cells were subjected to immunostaining with antibodies against phospho H3 at Ser 10 (Cell Signaling, Leiden, The Netherlands, cat no. 3377) and γH2AX (Novus Biologicals, Centennial, CO, USA, cat no. NB100-384). The next day, cells were incubated with Flamma^®552-conjugated goat anti-rabbit IgG (bioacts, Incheon, Korea) for 30 min at RT. The fluorescence signals were visualized with EVOS FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
For bromodeoxyuridine (BrdU) staining, cells were labeled with BrdU by incubation with 10 μM of BrdU for 30 min. After fixation and permeabilization as described above, cells were treated with 2M HCl for 20 min and neutralized with 0.1M sodium borate buffer for 30 min. After blocking, cells were subjected to immunofluorescence staining with anti-BrdU (Thermo Fisher Scientific, Waltham, MA, USA, cat no. MA3-071) primary antibody. The next day, cells were incubated with Flamma^®488-conjugated goat anti-mouse IgG (bioacts, Incheon, Korea) for 30 min at RT. Signal visualization was performed as described above.

Paraffin (3 μm) sections of pancreas samples were stained with Hematoxylin/Eosin, Masson trichrome, or various primary and secondary antibodies. Paraformaldehyde (4%) fixed and paraffin embedded tissues were stained on an Autostainer Link 48 (Dako, Glostrup, Denmark). Primary antibodies used in this study were: Human/Mouse Gkn1 antibody from bio-techne R&D Systems (AF7287), anti-Gastrokine 2 from Abcam (ab188866), GAPDH from Santa Cruz (sc-25778), α-Tubulin from Cell Signaling (11H10), α-SMA from Cell Signaling (2125), Cleaved Caspase-3 from Cell Signaling (9661 S), Cleaved Caspase-8 from Cell Signaling (8592), total Caspase-8 from Cell Signaling (4927), FAS from Santa Cruz (sc-1024), Ki-67 from Abcam (16667), Bcl-xl from Cell Signaling (2762), Bcl-2 from Cell Signaling (2876), Mcl-1 from Cell Signaling (5453) and γH2AX from Novus Biologicals (NB100-384). Secondary antibodies used in this study were: anti-Rabbit from Dako EnVision+ System-HRP (K4011), rabbit-anti-Sheep Immunoglobulins/HRP from Dako (P0163).

MIA PaCa-2 and PANC-1 cells were seeded and treated with 0.4 µM CM03, SAHA (1 µM for MIA PaCa-2 and 4 µM for PANC-1) or in combination of both compounds, at these concentrations. The treatment continued for 24 h in MIA PaCa-2 cells and for 48 h in PANC-1 cells because 24 h treatment was insufficient for observing its effect on PANC-1 cells. Untreated or vehicle treated (DMSO) cells were used as a control. Cells were collected and lysed with RIPA lysis buffer (ThermoFisher, Cat. No. 89900) supplemented with protease and phosphatase inhibitors (ThermoFisher, Cat. No. 78442). The lysate concentration was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher, Cat. No. 23227). Capillary-based automated Western blotting was performed on a Wes machine (https://www.proteinsimple.com/wes.html) to run the cell lysates and detect proteins of interest according to the manufacturer’s instructions. Antibodies used were for cleaved PARP (Cell Signaling, Cat. No. 5625), γ-H2AX (Novus Biologicals, Cat. No. NB100-384), and GAPDH (Novus Biologicals, Cat. No. NB300-325).

The cell and tissue sections were washed with cold PBS and immediately fixed with cold methanol (−20°C) for 10 min, followed by incubation in 3% bovine serum albumin in PBS for 1 hr to block the nonspecific binding sites. A primary antibody to 8-OHdG (Santa Cruz) or γH2AX (Novus Biologicals) was added overnight at 4°C. After washing 3 times with 1X PBST, secondary antibodies and Alexa Fluor® 488-conjugated goat anti-mouse (1:1000, Life Technologies) were added for 1 hr at room temperature (Additional file 2: Table S3). Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for analysis. For the DHE staining, the slides were incubated with the 5 μM DHE for 10 to 15 minutes in a dark chamber, on an orbital shaker at room temperature. After washing 3 times for 5 minutes/wash with 1X PBS and slight fixation for 4 to 8 minutes in 7% formaldehyde in 1X PBS, the slides were counterstained with DAPI, and images were captured using a fluorescence microscope.

After cells were fixed with 4% paraformaldehyde for 15 min, they were washed with permeabilization buffer (0.05% Triton-X in PBS) for 5 min three times at room temperature. The fixed cells were incubated with blocking buffer (BiogeneX, Fremont, CA, USA, cat# HK085) for 45 min, and then incubated with primary antibodies against Gata4 (Thermo Scientific, Waltham, MA, USA, cat# PA1-102, 1:400 dilution), Ki67 (Abcam, cat# ab92742, 1:1000 dilution), αSMA (Sigma, cat# A5228, 1:1000 dilution), or γH2AX (Novus Biologicals, Centennial, CO, USA, cat#NB100-384, 1:200) overnight at 4 °C. After being washed with permeabilization buffer three times for 5 min, cells were incubated with Alexa fluorogenic secondary antibodies (Thermofisher) at 1:400 dilution at room temperature for 1 h. For each animal, five slides ranging from the top to the bottom of the heart were selected for staining. Cells were rewashed with permeabilization buffer three times and then incubated with DAPI solution at a final concentration of 2 µM in permeabilization buffer for 15 min. After cells were washed three times with permeabilization buffer, cell images were captured with ImageXpress Micro XL Automated Cell Imaging system (Molecular Device), n = 3.