SUPPLEMENTARY MATERIAL
a
a
b
a,*
c,*,** a
School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701,
Korea b Department of Environmental Engineering, Kumoh National Institute of Technology,
Daehak-ro 61, Gumi, Gyeongbuk 730-701, Korea c
School of Applied Sciences, Kyungpook National University, Daegu 702-701, Korea
*Authors contributed equally to this paper
**Correspondence to: Sung-Eun Lee, School of Applied Biosciences; Kyungpook National
University, Daegu 702-701, Korea.

SUPPLEMENTARY MATERIAL
AMP-activated protein kinase (AMPK) is a key regulator of fatty acid biosynthesis and fatty acid oxidation throughout the body. Piperlongumine (PL) isolated from Piper longum (L.) was shown to potently upregulate activation of AMPK via phosphorylation and inactivation of acetyl-CoA carboxylases in cultured HepG2 cells, presumably enhancing the transfer of fatty acids into mitochondrial cells by inhibiting malonyl-CoA production. PL showed cytotoxicity on HepG2 cell growth at the concentration of 5

M of PL, while more than 80% of HepG2 cells were survived at the concentration of 2

M of PL. Overall, the results of this study indicate that PL activates AMPK phosphorylation and possess cytotoxicity in HepG2 cells.
Keywords: Piperlongumine, AMP-activated protein kinase, phosphorylation, HepG2 cell
1. Experimental
1.1. Chemicals and reagents
HPLC solvents were obtained from Burdick & Jackson, USA. The active forms of recombinant AMPKa1, b1, c1 protein and antibodies against phospho-AMPKa1/2 (Thr172), total AMPK, phospho-ACC (Ser79), and total ACC antibodies were purchased from Cell
Signaling Technology (New England BioLabs, Inc., Ipswich, MA). G6PDH and

-actin antibodies were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX). Reagents for polyacrylamide gel electrophoresis were obtained from BioRad (Hercules, CA). All other reagents were of the highest analytical grade. HepG2 cells were obtained from the American

Type Culture Collection.
1.2. Plant material, extraction, and isolation
Extraction and isolation of PL was conducted as previously described (Lee et al. 2002, Park et al. 2007). Fruits of Piper longum L. (Piperaceae) were purchased from a traditional herbal market in Daegu, Korea, in October 2012. The plant material was identified by Prof. Tae-Wan
Kim at Department of Plant & Environmental Science, Hankyung National University
(Ansung, Korea). Dried fruits of P. longum were grinded and macerated with MeOH at the room temperature for 3 days. The MeOH extract was resuspended with four volumes of water and then submitted to liquid-liquid fractionation with hexane, dichloromethane, and ethyl acetate. The dichloromethane fraction was then subjected to a Flash chromatography (Buchi,
Flawil, Switzerland) equipped with a Sepacore column packed with silica gel (Buchi, Flawil,
Switzerland) using a gradient elution system consisting of MeOH in dichloromethane. The fractions were further separated by Water Delta preparative HPLC (Waters, Milford, MA) using a

Bondapak C18 column (19 mm
×
300 mm, Waters, Milford, MA) using methanolwater (3:7). The pure compound was obtained and its structure was identified by spectroscopic methods with a comparison to an authentic standard compound
(piperlongumine) purchased from Sigma Co. (St. Louis, MO).
1.3. Cell culture and Western blot analysis
HepG2 cells were obtained from the American Type Culture Collection. HepG2 cells were cultured in Dulbecco’s Modified Eagle Media containing 10% FBS (v/v), 100 U/ml of penicillin and 100 μg/ml of streptomycin in 5% CO
2
at 37 °C.
Cell growth rates were determined by MTT assay in 96-well plates. Cultured HepG2 cells were lysed with cell lysis

buffer A (50 mM Tris–HCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF and 1 mM protease inhibitor cocktail, pH 8.0). Isolated mouse soleus muscle tissues were homogenized with a glass homogenizer in HEPES buffer (20 mM HEPES, 250 mM sucrose, 1 mM EDTA, pH 7.4) containing protease inhibitor cocktail. Total membrane fractions from HepG2 cells were prepared as previously described (Gauhar et al. 2012). For Western blot analysis, total cells lysate were prepared as previously described (Hwang et al. 2008). Protein samples were subjected to Western blot analysis according to the standard procedure using respective antibodies.
References
Gauhar R, Hwang SR, Jeong SS, Kim JE, Song H, Park DC, Song KS. Kim TY, Oh WK,
Huh TL. 2012. Heat-processed Gynostemma pentaphyllum extract improves obesity in ob / ob mice by activating AMP-activated protein kinase. Biotechnol Lett. 34:1607-1616.
Hwang SL, Kim HN, Jung HH, Kim JE, Choi DK, Hur JM, Lee JY, Song H, Song KS, Huh
TL. 2008. Beneficial effects of

-sitosterol on glucose and lipid metabolism in L6 myotube cells are mediated by AMP-activated protein kinase. Biochem Biophys Res
Commun. 377:1253-1258.
Lee SE, Mahoney NE, Campbell BC. 2002. Inhibition of aflatoxin B
1
biosynthesis by piperlongumine isolated from Piper longum L. J. Microbiol Biotechnol. 12:679-682.
Park BS, Son DJ, Park YH, Kim TW, Lee SE. 2007. Antiplatelet effects of acidamides isolated from the fruits of Piper longum L. Phytomedicine . 14:853-855.

Figure S1. Effects of piperlongumine (PL) on cell proliferation. HepG2 cells were exposed to
PL (2

M and 5

M) for 4 days. During cell culture, viable cell numbers were counted by
MTT assay at each day. Cell growth rate versus control were expressed as cell proliferation
(%). Data is a mean of at least three separate experiments. Closed rectangle and triangle represents 2

M and 5

M PL treatment, respectively. Closed diamond indicates no addition of PL in cell medium as a control.

PL (

M) p-ACC t-ACC p-AMPK
HepG2
0 0.5
1 2 t-AMPK
β-Actin
GAPDH
Figure S2. Effects of piperlongumine (PL) on phosphorylation of AMPK and expression of key factors in lipid metabolism in HepG2 cells. HepG2 cells were treated with PL for 2 h.
Cell lysates were subjected to Western blotting using

-actin and GAPDH as loading controls.
AMPK, AMP-activated protein kinase; p-AMPK, phosphorylated AMPK

subunit; t-AMPK, total AMPK

subunit; p-ACC, phosphorylated ACC; t-ACC, total ACC; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

PL (2

M)
CC (10

M) p-ACC t-ACC
－ －
－ +
+ +
－ + p-AMPK t-AMPK
β-Actin
GAPDH
Figure S3. AMPK activation by piperlongumine (PL) in HepG2 was abrogated by pretreatment (10 min) with compound C, an AMPK phosphorylation inhibitor. Cell lysates were subjected to Western blotting using

-actin and GAPDH as a loading control. CC, compound C; AMPK, AMP-activated protein kinase; p-AMPK, phosphorylated AMPK
 subunit; t-AMPK, total AMPK

subunit; p-ACC, phosphorylated ACC; t-ACC, total ACC;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase.