Time course response of XBP1 induction (A) and its inhibition by 1–10 µg/ml PPE (B) and nuclear translocation (C) in 1 µM tunicamycin-exposed macrophages.

<p>For the measurement of XBP1 induction, total cell lysates were subject to Western blot analysis with a primary antibody against XBP1. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05. Nuclear translocation of XBP1 was detected by immunofluorocytochemical staining with Cy3-conjugated XBP1 antibody and nuclear counter-staining was carried out with DAPI (C). Microscopic observation was done by fluorescent microscopy. Magnification: 200-fold.</p

Time course responses of ATF6, BiP/GRP78 and XBP1 (A) and their inhibition by 1–10 µg/ml PPE (B) in 1 µM tunicamycin-treated macrophages.

<p>For the measurement of induction of ATF6, BiP/GRP78 and XBP1 (B), total cell lysates were subject to Western blot analysis with a primary antibody against ATF6, BiP/GRP78 or XBP1. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05.</p

PPE restoration of induction of ABCA1(A), SR-B1 (B) and ICAM-1 (C) demoted in tunicamycin-treated J774A1 murine macrophages.

<p>Cells were stimulated with 1 µM T091317 for 12 h, 50 µg/ml Cu²⁺-oxidized LDL for 6 h, and 10 ng/ml TNF-α for 6 h in the absence and presence of 1 µM tunicamycin and 10 µg/ml PPE. For the measurement of expression of ABCA1, SR-B1 and ICAM-1, total cell lysates were subject to Western blot analysis with a primary antibody against ABCA1, SR-B1 and ICAM-1. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05. Values (mean ± SEM, n = 3) not sharing a common letter are different at P<0.05.</p

Time course responses of ATF6 and BiP/GRP78 induction (A) and their inhibition by 1–10 µg/ml PPE (B) in 1 µM tunicamycin-treated macrophages.

<p>For the measurement of induction of ATF6 and BiP/GRP78 (B), total cell lysates were subject to Western blot analysis with a primary antibody against ATF6 or BiP/GRP78. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05.</p

Inhibition of nuclear translocation (A and B), and temporal response of XBP1 transcription and its inhibition by PPE (C and D).

<p>J774A.1 macrophages were treated with 50 µg/ml oxidized LDL for 18 h in the absence and presence of 1–10 µg/ml PPE. Nuclear extracts were isolated by nuclear fraction assay and Western blot analysis was conducted by using primary anti-XBP1 (A). Lamin B was used as a nuclear control. Values (mean ± SEM, n = 3) not sharing a common letter are different at P<0.05. Nuclear translocation of XBP1 was also detected by immunofluorocytochemical staining with Cy3-conjugated XBP1 antibody and nuclear counter-staining was carried out with DAPI (B). Microscopic observation was done by fluorescent microscopy. Magnification: 200-fold. XBP1 mRNA levels were measured by quantitative RT-PCR assay (C and D). GAPDH was used for the internal control (n = 3).</p

Time course response of cytotoxicity by tunicamycin (A), elevation of cell viability by PPE (B), and DNA laddering (C) and inhibition of nuclear condensation and DNA fragmentation (D) and by PPE in tunicamycin-treated macrophages.

<p>MTT assay was performed for the measurement of cell survival of 1 µM tunicamycin-exposed J774A1 murine macrophages by 1–10 µg/ml PPE (A and B). The bar graph data represent mean ± SEM from 4 independent experiments with multiple estimations. Values are expressed as percent cell survival relative to untreated control cells (cell viability = 100%). For the measurement of cellular apoptosis, DNA laddering, Hoechst33258 staining and TUNEL assay (C and D) were conducted with light microscopy and fluorescent microscopy, respectively. Histograms showing the relative fluorescent staining intensity of TUNEL-positive cells (D). Magnification: 200-fold. Means without a common letter differ, P<0.05.</p

Temporal induction of XBP1 transcription (A) and inhibition of XBP1 transcription (B) by PPE.

<p>J774A.1 macrophages were treated with 1 µM tunicamycin for 6 h in the absence and presence of 1–10 µg/ml PPE. XBP1 mRNA levels were measured by quantitative RT-PCR assay (A and B). GAPDH was used for the internal control (n = 3). The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05.</p

Inhibition of induction of VEGFR2, phospho-VEGFR2, Angpt1, Angpt2, Tie-2 and phospho-Tie-2 in diabetic kidneys by PCE.

<p>The db/db mice were orally supplemented with 10 mg/kg PCE daily for 8 weeks. The db/m mice were employed as diabetic controls. For the measurements of the tissue levels of VEGFR2 (A), histological sections of mouse kidneys were immunohistochemically stained by using anti-mouse VEGFR2 and color-fixed with substrate chromogens of 3,3′-diaminobenzidine. The sections were counter-stained with hematoxylin. The VEGFR2 level was identified as brown staining. Each photograph is representative of four animals. Magnification: x400. The bar graphs (mean ± SEM, n = 3) in the right panel represent quantitative staining intensity. For the Western blot analysis (B and C), tissue extracts were subjected to Western blot analysis with a primary antibody of phospho-VEGFR2, Angpt1, Angpt2, Tie-2 and phospho-Tie-2. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the bottom panels represent quantitative results obtained from a densitometer. Respective values not sharing a common letter differ, <i>P</i><0.05.</p

Western blot analysis showing inhibition of diabetic induction of Angpt1, Angpt2 and Tie-2 by PCE.

<p>HRMC were incubated in 5.5(A). HUVEC were incubated in HRMC conditioned media [with (w/) 27.5 mM mannitol and w/33 mM glucose] for 6 h in the absence and presence of 1–20 µg/ml PCE (B). Cell lysates were subjected to Western blot analysis with a primary antibody of Angpt1, Angpt2 and Tie-2. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the bottom panels represent quantitative results obtained from a densitometer. Respective values not sharing a common letter differ, <i>P</i><0.05.</p

Attenuation of diabetic induction of PECAM-1, integrin β3, VE-cadherin and TSP-1 by PCE.

<p>HRMC were incubated in 5.5[with (w/) 27.5 mM mannitol and w/33 mM glucose] for 6 h in the absence and presence of 1–20 µg/ml PCE (A). Cell lysates were subjected to Western blot analysis with a primary antibody of PECAM-1 and integrin β3 (A). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 5) in the bottom panels represent quantitative results obtained from a densitometer. The db/db mice were orally supplemented with 10 mg/kg PCE daily for 8 weeks (B and C). The db/m mice were employed as diabetic controls. The VE-cadherin induction in histological sections of mouse kidneys was immunohistochemically determined by using anti-mouse VE-cadherin and FITC-conjugated IgG (B). Each photograph is representative of four animals. Magnification: x400. The bar graphs (mean ± SEM, n = 4) in the right panel represent quantitative staining intensity. Plasma level of TSP-1 in mice was measured with an ELISA kit (C). Means not sharing a common letter differ, <i>P</i><0.05.</p