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

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

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

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 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

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 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

Suppressive effects of kaempferol on induction and activation of SMAD4, IRE1α and GRP78 (A), and XBP-1 protein induction (B) and XBP-1 mRNA transcription and splicing (C and D) in TGF-β-stimulated BEAS-2B cells.

<p>Cells were treated with 1–20 μM kaempferol and simulated with 10 ng/ml TGF-β. Cell lysates were prepared for Western blot analysis with a primary antibody against SMAD4, phospho-IRE1α, GRP78 and XBP-1. Representative blot data were obtained from 3 experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) represent quantitative results of blots. Values in bar graphs not sharing a letter indicate significant different at P<0.05. RT-PCR was conducted to determine XBP-1 mRNA splicing (C and D). GAPDH was used as a housekeeping gene for the co-amplification with XBP-1.</p

Inhibitory effects of kaempferol on tunicamycin-induced expression and activation of ATF6 and IRE1α (A), and GRP78 and HSP70 (B) in BEAS-2B cells.

<p>Cells were treated with 1–20 μM kaempferol and simulated with 1 μM tunicamycin. Cell lysates were prepared for Western blot analysis with a primary antibody against ATF6, phospho-IRE1α, GRP78 and HSP70. Representative blot data were obtained from 3 experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of upper blots. Values in bar graphs not sharing a letter indicate significant different at P<0.05.</p

Immunofluorescent data showing inhibition of XBP-1 induction in OVA-challenged mouse lung tissues by kaempferol.

<p>Epithelial XBP-1 protein was identified as reddish and/or pinkish staining. XBP-1 was visualized with a Cy3-conjugated secondary antibody and nuclear staining was done with DAPI. Each photograph is representative of four mice. Magnification: 200-fold. The bar graphs (mean ± SEM) represent quantitative results of Cy3 staining. Values in bar graphs not sharing a letter indicate significant different at P<0.05.</p