DT treatment efficiently depletes macrophages.

<p>For microscopic images of macrophages, macrophages were collected from peritoneal lavage of LysM^Cre/iDTR mice (A) 2 days after DT treatment (10 ng/g body weight) and (H) 7 days after stopping DT injection. Macrophages were collected from (B) peritoneal lavage or (C) bone-marrow of LysM^Cre/iDTR mice, then treated with saline or DT (0.25 ng/ml) for 8 hours. mRNA levels of macrophage marker F4/80 and CD68 were measured in (D) WAT, (E) liver, and (F) skeletal muscle of mice treated with saline or DT for 2 days. mRNA levels of M2 macrophage marker ARG1 were measured in (G) WAT of mice treated with saline or DT for 2 days and 7 days after stopping DT injection. Data are represented as mean ± SEM. Asterisks denote significant differences *<i>P</i><0.05, **<i>P</i><0.01 vs. saline-treated mice.</p

Macrophage depletion increases tissue NE infiltration.

<p>Male LysM^Cre/iDTR mice were fed a chow diet and were i.p. injected with DT (10 ng/g) or saline. (A) Blood NE concentration was assessed after 1 and 6 days of DT injection by a hematology test. (B–D) mRNA levels of NE markers LY-6G, LCN, and NE-chemoattractant chemokine MIP2 in WAT, liver, and skeletal muscle were measured by Q-PCR (n = 5). LY-6G, lymphocyte antigen 6 complex, locus G; MIP2, macrophage inflammatory protein 2; LCN2, lipocalin 2 (E) Infiltrated NEs in WAT were identified by immunohistochemisty using NE marker LY-6G antibody. Arrows indicate positive cells. (F) Protein levels of NE marker LY-6G in WAT, liver, and skeletal muscle were measured by Western blotting (n = 4 each). Representative images were shown in the figure. (G–I) Quantification of LY-6G/GAPDH protein levels in tissues. (J) The comparison of inflammatory cytokine expression with NE marker expression in WAT by Q-PCR during the different time points of DT treatment. (K and L) G-CSF mRNA levels were measured by Q-PCR using RNA samples from OP9 cells treated with 10% peritoneal (K) or bone-marrow derived macrophage (L) conditioned media (n = 4 each). Data are represented as mean ± SEM. Asterisks denote significant differences *<i>P</i><0.05, **<i>P</i><0.01 vs. saline-treated mice.</p

Macrophage depletion reduces body weight and fat mass.

<p>Male LysM^Cre/iDTR mice were fed (A, C, and E) a chow diet (n = 7) or (B, D, and F) a HF diet (n = 5) for 6 wks and i.p. injected with DT (10 ng/g) or saline every other day. For chow-fed mice, DT injection was stopped at day 25 which is indicated by arrows. (A and B) Body weight was measured every other day and calculated as a percent change compared to day 0 of saline or DT injection (C and D) Fat mass and (E and F) lean body mass were measured using an EchoMRI scanning device when mice were at fed state and these masses were calculated as a percent change compared to day 0 of saline or DT injection. Data are presented as mean ± SEM. *<i>P</i><0.05 vs. saline-treated mice.</p

DT depletes microglia in

<p>Microglia were isolated from day 1 newborn LysM^Cre/iDTR mice (n = 3). Isolated microglia were cultured in cell-culture plates. Saline or DT (0.25 ng/ml) was treated for 8 hrs. (A) Representative macroscopic images of isolated microglia treated with saline or DT for 8 hrs. (B) Total protein levels of microglia marker Iba1 in the hypothalamus of mice treated with saline or DT for 6 days. (C) Microglia in the brain were identified by immunohistochemisty using microglia marker Iba1 antibody. Arrows indicate positive cells (n = 3). 3 V represents the direction of third ventricle.</p

Macrophage depletion suppresses energy expenditure and food intake.

<p>The energy expenditure of male LysM^Cre/iDTR mice was assessed using metabolic cages 6 days after saline or DT injection. (A) O₂ consumption during the light and dark cycle (n = 4/each group) (B) Area under the curve of O2 consumption during the light and dark cycle (C) Food intake was measured every other day at 10 am during saline or DT injection. Injection was stopped at day 25, which is indicated by arrows. Cumulative food intake of (D) chow-fed (n = 5/group) or (E) HF-fed mice (n = 3/group) treated with saline or DT (F) Serum concentration of leptin (n = 5/group) was measured using ELISA kit after overnight fasting. (G) Phosphorylation of STAT3 protein was measured by Western blotting. (H) Quantified values of p-STAT3/STAT3. Data are represented as mean ± SEM. Asterisks denote significant differences *<i>P</i><0.05 vs. saline-treated mice.</p

Tentative compound identification by reverse-phase HPLC-ESI/MS in both negative and positive mode.

a<p>NP, fraction numbers.</p>b<p>ID, peak numbers.</p>c<p>t_R, retention time.</p>d<p>N_A, number of A-type linkage.</p>e<p>MW, molecular weight.</p>f<p>(E)GC, (E)GCG, (E)CG are abbreviations for (epi)gallocatechin, (epi)gallocatechin-3-O-gallate, (epi)catechin-3-O-gallate.</p

Calculated and Observed Mass Values for Na Adduct Ions of PDs Dimers to Tetradecamers in PCBLs by MALDI-TOF/MS.

a<p>N_A, number of A-type linkage.</p

Fractionation information by normal-phase preparative HPLC and tentative compound identification by normal-phase HPLC-ESI/MS in negative mode.

a<p>NP, fraction numbers.</p>b<p>CT, collecting time period.</p>c<p>Yield, yield of one injection of preparative HPLC, that is, milligram per 200 milligrams bayberry leaf proanthocyanidin extract (BLPEs).</p>d<p>ID, peak numbers.</p>e<p>t_R, retention time.</p>f<p>N_A, number of A-type linkage.</p>g<p>MW, molecular weight.</p>h<p>(E)GC, (E)GCG, (E)CG are abbreviations for (epi)gallocatechin, (epi)gallocatechin-3-O-gallate, (epi)catechin-3-O-gallate.</p

Reversed-phase HPLC chromatograms of fractions collected from preparative normal-phase HPLC.

<p>N1 to N6 referred to fractions collected from preparative normal-phase HPLC according to the collecting time in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096162#pone-0096162-t003" target="_blank">Table 3</a>.</p

¹³C (600 MHz) NMR Data of GC, ECG, GCG, EGCG in Methanol-d4.

<p>¹³C (600 MHz) NMR Data of GC, ECG, GCG, EGCG in Methanol-d4.</p