P h2ax

Western blotting was performed as described previously [34 (link), 35 (link)]. Antibodies used were as follows: p-Erk1/2, p-p38, Mnk1, eIF4E, p-eIF4E, Bcl-2, Mcl-1, PARP, cleaved PARP, p-H2AX, β-actin (all aforementioned antibodies from Cell Signalling Technology, Danvers, MA, USA), Mnk2 (Abcam, MA, USA), and Bcl-2 (Dako, Glostrap, Denmark). Both anti-mouse and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase-conjugated antibodies (Dako, Glostrap, Denmark) were used as secondary antibodies. Enhanced Chemiluminescence (ECL) reagents (GE Life Sciences, Australia) were used for detection.

For histopathological observation, tumor, lung, liver, spleen, heart, and kidney samples were promptly excised and fixed in 4% paraformaldehyde in PBS. Paraffin sections (5 mm) were prepared and stained with hematoxylin and eosin. For immunohistochemistry (IHC), paraffin sections were stained with antibodies against p-H2AX (#9718; Cell Signaling Technology), c-caspase-3 (#9664; Cell Signaling Technology), ki67 (ab16667; Abcam), PCNA (sc-56; Santa Cruz Biotechnology), snail and slug (ab180714; Abcam), and vimentin (#5741; Cell Signaling Technology). Mayer’s hematoxylin stain was used for nuclei counterstaining (Muto Pure Chemicals Co., Tokyo, Japan).

Whole cell lysates of adherent cells, spheres, or tumor tissues were prepared for western blot analysis as previously described [39 (link)]. The following primary antibodies were used: nanog (ab109250; Abcam), oct4 (ab109183; Abcam), sox2 (ab92494; Abcam), CD44 (#37259; Cell Signaling Technology), ALDH1A1 (#54135; Cell Signaling Technology), E-cadherin (#3195; Cell Signaling Technology), N-cadherin (#13116; Cell Signaling Technology), p-STAT3 (#4113; Cell Signaling Technology), STAT3 (#8768; Cell Signaling Technology), p-AKT (#4060; Cell Signaling Technology), AKT (#2920; Cell Signaling Technology), p-SRC (#2101; Cell Signaling Technology), SRC (#2110; Cell Signaling Technology), p-ERK (#4370; Cell Signaling Technology), ERK (#9107; Cell Signaling Technology), p-H2AX (#9718; Cell Signaling Technology), c-caspase-3 (#9664; Cell Signaling Technology), p-survivin (NB500–236; Novus Biologicals), snail and slug (ab180714; Abcam), vimentin (#5741; Cell Signaling Technology), and β-actin (#4970; Cell Signaling Technology).

Human epithelial cells were seeded (50,000 cells/well) in chamber slides (Nalge Nunc International 154526, Rochester, NY, USA), and treated with e-cig aerosolized media for 30 min. After e-cig treatment, cells were washed with PBS, fixed with methanol: acetone (1:1) for 10 min at room temperature. After fixation, cells were permeabilized with 0.5% Triton X-100 for 20 min and blocked with 1% BSA for 1 h. Slides were incubated with primary antibody overnight at 4 °C (pERK1/2 1:250; NF-KB 1:400; p-H2A.X 1:400, all antibody from Cell Signaling, Danvers, MA, USA), washed with PBS, and incubated with corresponding secondary antibody (Alexa Fluor 488 goat anti-mouse, Invitrogen by ThermoFisher A11001, Eugene, OR, USA; Goat anti-rabbit IgG DyLight 488 Conjugated, Invitrogen 35552, Rockford, IL, USA) overnight at 4 °C. Slides were mounted with DAPI (Southern Biotech 0100-20, Roskilde, Denmark).

Cells were seeded in 6-well plates and allowed to attach for 24 h. Cells were then incubated for 24 hours with indicated concentrations of AZ31 and/or SN38. Cells were then washed with PBS and lysed with RIPA buffer (Cell Signaling, Danvers, MA). After sonication and centrifugation, a total of 30 μg of protein lysate was loaded onto a NuPage gel (Life Technologies, Carlsbad, CA), electrophoresed, and transferred to a nitrocellulose membrane using the Pierce G2 FastBlotter (Thermo Fisher, Rockford, IL). The membrane was blocked and probed overnight with primary antibodies (Cell Signaling Technologies (Danvers, MA) at a concentration 1:1000: p-ATM (catalog# 13050), ATM (catalog# 2873), p-p53 (catalog# 9284), p53 (catalog# 2527), p-CHK2 (catalog# 2665), p-RAD50 (catalog# 14223), p-H2AX (catalog# 9718) and actin (catalog# 4970). The next day the membranes were washed for 10 minutes 3X with TBS/Tween 20, and then probed with DyLight secondary antibodies 1:15,000 (Cell signaling, Danvers, MA), and imaged using the Licor Odyssey (Licor, Lincoln, NE).

Mouse spinal cord tissues was homogenized with lysis buffer (20 mM Tris, pH 8.0; 150 mM NaCl; 0.5% Nonidet P-40; 0.5% sodium deoxycholate) and the proteins were extracted after removing cell debris. The total protein was quantified with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA), and then, 30 μg of protein was subjected to SDS-PAGE. After electrophoresis, the separated protein was transferred onto nitrocellulose membranes. After the membrane was blocked for 1 h in Tris-buffered saline (TBS) containing 0.1% Tween and 10% nonfat dried skim milk, it was incubated with each primary antibody for 2 h at room temperature. The primary antibodies used in western blotting analysis p53 (1:500, Abcam); pH2A.X (1:500, Cell signaling); and PUMA (1:200, Abcam). After washing with TBS-T three times for 15 min, the membrane was incubated for 1 h with the appropriate amount of horseradish peroxidase-conjugated secondary antibody. Anti-βII-tubulin antibody (1:4000) (Santa Cruz, Santa Cruz, CA, USA) was used to detect tubulin protein as the protein loading control, and the chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used to obtain protein bands.

The following antibodies were used for IF and diluted in TBS supplemented with 0.1% Tween20 and 2% BSA: CyclinA2 (1:400; #sc-751; Santa Cruz), CyclinA2 (1:400; #4656; Cell Signaling), PLK1 (1:400; ab14210; Abcam), pTCTP (1:400; #5251; Cell Signaling), pH2AX (1:800; #2577; Cell Signaling), pCyclinB1 (1:400; #ab55184; Abcam), pLaminA/C (1:400; #2026; Cell Signaling), Alexa Fluor 488-Goat anti-Rabbit (1:2000; #A11008 Life Technologies) and Alexa Fluor 555-Goat anti-Mouse (1: 2000; #A21422 Life Technologies). The following antibodies were used for WB and diluted in TBS supplemented with 0.1% Tween20 and 5% milk powder: CDC6 (1:400; #sc-9964 Santa Cruz), CDT1 (1:400; #sc-28262 Santa Cruz), GAPDH (1:20000; #G8795; Sigma), anti-Rabbit HRP (1:2000; #ab6721; Abcam) and anti-Mouse HRP (1:2000; #ab97023; Abcam).