Anti mouse and anti rabbit igg

DMEM and fetal bovine serum (FBS) were obtained from GIBCO/BRL Life Technologies (Grand Island, NY). Antibodies to caspase-3, PARP-1, α-tubulin, and anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to acetyl-α-tubulin^Lys40, p-Akt^Ser473 and p-Akt^Thr308 were from Cell Signaling Technologies (Boston, MA). Ouabain, propidium iodide (PI), sulforhodamine B, RNase, trichloroacetic acid (TCA), Triton X-100, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, sodium fluoride, sodium orthovanadate and proteinase K were obtained from Sigma-Aldrich (St. Louis, MO). Fluo-3/AM, JC-1 and carboxyfluorescein succinimidyl ester (CFSE) were from Molecular Probes Inc. (Eugene, OR, USA). Epi-reevesioside F was isolated from the root of Reevesia formosana. Purification and identification of Epi-reevesioside F were published elsewhere [20 (link)].

RPMI 1640 medium and fetal bovine serum (FBS) were obtained from GIBCO/BRL Life Technologies (Grand Island, NY). Antibodies to cyclin D1, cyclin E, cyclin A, cyclin B1, Cdk4, Cdk2, Cdk1, p-Rb^Ser807/811, Cdc25A, PARP-1, AMPK siRNA and anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to p-Chk1^Ser345, p-Chk2^Thr68, p-AMPKα^Thr172, p-mTOR^Ser2448, p-p70S6K^Thr389 (p70 ribosomal S6 kinase), p62, p-H2AX^Ser139 and GAPDH were from Cell Signaling Technologies (Boston, MA). Antibody against LC3 was purchased from Novus Biologicals (Littleton, CO). The siRNA specifically targeting Chk2 mRNA was purchased from Dharmacon (Lafayette, CO). SRB, PI, CFSE, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), nitroxoline, paclitaxel, etoposide, Chk2 inhibitor and all other chemical compounds were obtained from Sigma-Aldrich (St. Louis, MO).

RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin and 2′,7′ indicating the DNA damage response.-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from GIBCO/BRL Life Technologies (Grand Island, NY). Antibodies of PARP-1, Bax, Bcl-2, Bcl-xL, Bak, Mcl-1, Rad51, α-tubulin, DNA-PKcs, anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies of γ-H2A.X, Bid, caspase-8, cleaved caspase-9, and Ku80 were from Cell Signaling Technologies (Boston, MA). Caspase-3 was from Imgenex, Corp. (San Diego, CA). Antibodies of p-Chk2^Thr68 and p-DNA-PKcs^Thr2609 were from Abcam PLC, Inc. (Massachusetts, US). Antibody of RPA32 was from GeneTex Inc. Antibody of PDE5 was from OriGene Technologies, Inc. (Rockville, MD, USA). PDE5 small interfering RNA (siRNA) was from GE Healthcare Dharmacon Inc. (Chicago, USA). All chemical compounds and anticancer drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Heteronemin was extracted from marine sponge Hyrtios erecta and purified in Professor Ping-Jyun Sung's Lab. Minimum Essential Medium (MEM), RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco BRL Life Technologies (Grand Island, NY). EGTA, EDTA, leupeptin, dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), propidium iodide (PI), dimethyl sulfoxide (DMSO), MTT (3-[4,5]-2,5-diphenyltetrazolium bromide), 4′-6-diamidino-2-phenylindole (DAPI), SB203580, SP600125, and chloroquine were obtained from Sigma (St. Louis, MO). Antibodies to various proteins were obtained from the following sources: anti-mouse and anti-rabbit IgGs, poly-ADP-ribose polymerase (PARP), Bcl-2, Bcl-xL, Bax, and p62 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); p-AKT (Ser 473), AKT, p-ERK (Thr 202/Tyr 204), ERK, p-p70S6K (Thr 421/Ser 424), p70S6K, p-4EBP1 (Thr 37/46), 4EBP1, p-JNK (Thr 183/Tyr 185), JNK, p-p38 (Thr 180/Tyr 182), p38, p-HSP27 (Ser 78), Atg5, cleaved caspase-3, caspase-9, and caspase-8 were purchased from Cell Signaling Technology (Boston, MA); cytochrome c was purchased from BD Biosciences (San Diego, CA); caspase-3 was purchased from Imgenex (San Diego, CA); LC3 was purchased from Novus (Littleton, CO); actin and GAPDH were purchased from Millipore (Billerica, MA).

A549 cells (approximately ~5 × 10⁶ cells/mL) were grown in complete medium with 1 to 3 μM concentration of ECPU-0001 and control cells were treated with DMSO. Next day, cells were collected by centrifuge (Hanil Scientific, Inc., Gyeonggi-do, South Korea) at 1200 rpm for 3 min at 4 °C. Cells were rinsed with 1× cold PBS. The pellet was downed and harvested cells were lysed in cell lysis RIPA buffer containing DTT, NaVO3 and PI respectively. After quantification of protein concentration using the Bradford method, the lysate was mixed with 4× protein sample buffer then boiled in 95 °C boiling water bath for 10 min followed entire steps as mentioned elsewhere [61 (link)]. The membranes were immunoblotted with the following antibodies: mouse monoclonal anti-BAX, anti-VDAC1, anti-c-MYC, anti-BID, anti-BCL-2, anti-Mcl-1, anti-Bcl-XL, anti-APAF-1, anti-NF-κB, anti-Iκκβ, anti-caspase-3, and anti-Caspase-9, rabbit polyclonal anti-CytoC, anti-XIAP, anti-IL-4, anti-PARP-1 and anti-BAK. The information of the supplier and dilution for each antibody have provided in the table (Table S1). The PVDF membranes were put at 4 °C in rotating shaker for counter immunoblotted with the anti-mouse and anti-rabbit IgG (1:2500 dilution, Santa Cruz Biotechnology Inc., Delaware Ave, Santa Cruz, CA, USA). Each protein bands were detected by chemiluminescence enhancing solution.

TAA and silymarin were obtained from Sigma Aldrich (St. Louis, Missouri, USA). The ELISA kits used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and r-glutamyl transferase (r-GPT) were acquired from Abcam (Cambridge, UK). Malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) assay kits were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Antibodies against collagen-1, TGF-β1, α-SMA, vimentin, Smad2/3, p-Smad2/3, Smad4, Smad7, p-PTEN, PTEN, p-Akt, Akt, p-PI3K, PI3K, and β-actin were purchased from Abcam (Cambridge, Massachusetts, USA) and Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-mouse and anti-rabbit IgG and HRP-linked-conjugated secondary antibodies were procured from Santa Cruz Biotech. (Santa Cruz, California, USA).