Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene 4′- β -Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

doi: 10.1155/2018/2747018

Figure Lengend Snippet: 4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H&E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p < 0.01 and ∗∗∗ p < 0.001. Comparisons were made by one-way ANOVA with Turkey post hoc tests.

Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v / v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v / v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Techniques: Expressing, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene 4′- β -Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

doi: 10.1155/2018/2747018

Figure Lengend Snippet: 4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. the vehicle control group.

Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v / v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v / v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Techniques: Expressing, MTT Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene 4′- β -Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

doi: 10.1155/2018/2747018

Figure Lengend Snippet: The antioxidant effect of 4-PG is mediated by HO-1 activation. (a and b) To evaluate whether the antioxidant effect of 4-PG was mediated by the activation of HO-1, RAW 264.7 cells were pretreated with the HO-1 inhibitor, ZnPP (5 μ M), for 30 min followed by the incubation of 4-PG (10 μ M) for 6 h. Then, cells were challenged with LPS (100 ng/ml) for another 4 h. The production of intracellular ROS was detected by staining DCF-DA using confocal microscopy (a) and flow cytometry (b). Scale bar = 10 μ m. (c) Mitochondrial ROS was stained with MitoSOX and measured by flow cytometry. Here, the fold change of fluorescence intensity is presented as mean ± SD ( n = 5 determined in five independent experiments); ∗∗ p < 0.01 and ∗∗∗ p < 0.001. Comparisons were made by one-way ANOVA with Turkey post hoc tests.

Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v / v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v / v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Techniques: Activation Assay, Incubation, Staining, Confocal Microscopy, Flow Cytometry, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene 4′- β -Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

doi: 10.1155/2018/2747018

Figure Lengend Snippet: HO-1 is necessary for antioxidant and anti-inflammatory effects of 4-PG. RAW 264.7 cells were transfected with scramble RNA ( scRNA ) and siRNA against HO-1 ( siHmox-1 ) for 36 h and then pretreated with either 4-PG (10 μ M) or PTER (10 μ M) for 6 h followed by stimulation of LPS (100 ng/ml) for another 4 h. (a) The production of ROS was determined by confocal microscopy. Scale bar = 10 μ m. (b) The mRNA and protein levels of HO-1 were determined by RT-PCR and Western blot, respectively. The conditioned supernatants were harvested, and the secreted levels of TNF- α (c) and IL-6 (d) in Hmox-1 +/+ and Hmox-1 −/− BMDM were assessed by ELISA. (e) mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. (f–h) BMDMs, extracted from Hmox-1 +/+ and Hmox-1 −/− mice, were pretreated with 4-PG (10 μ M) for 6 h and MitoTEMPO (100 μ M) for 30 min, respectively, and then cells were stimulated with LPS (100 ng/ml) for an additional 4 h. (i–k) A549 cells were pretreated with 4-PG (10 μ M) for 4 h or with MitoTEMPO (100 μ M) for 30 min, respectively, and then stimulated with LPS (10 μ g/ml) for an additional 6 h. (f and i) Mitochondrial ROS were stained with MitoSOX and measured by flow cytometry. (g and j) The fold change of fluorescence intensity is presented as mean ± SD ( n = 5, determined in five independent experiments). (h and k) The mRNA level of TNF- α was determined by RT-PCR. (l and m) BMDMs isolated from Hmox-1 +/+ and Hmox-1 −/− mice were pretreated with 4-PG (10 μ M) for 6 h followed by the challenge of LPS (100 ng/ml) for 4 h, and for Hmox-1 −/− BMDMs, cells were copretreated with 4-PG (10 μ M) and HO-1 catabolites such as CO-releasing molecule- (CORM-) 2 (20 μ M), bilirubin (5 μ M), and ferrous sulfate (10 μ M), respectively, in the presence of LPS stimulation. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments); ∗∗∗ p < 0.001. NS: not significant. Comparisons between scRNA and siHO-1-transfected groups were made by two-way ANOVA with Bonferroni post-tests.

Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v / v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v / v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Techniques: Transfection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Fluorescence, Isolation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene 4′- β -Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

doi: 10.1155/2018/2747018

Figure Lengend Snippet: HO-1 deficiency abolished the anti-inflammatory effect of 4-PG in a mouse model of acute lung injury. 10-week-old Hmox-1 +/+ and Hmox-1 −/− mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (a) Lung sections were stained with hematoxylin and eosin (H&E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. The mRNA and protein levels of HO-1 from lung tissues were determined by RT-PCR (b) and Western blotting (c), respectively. BAL fluid was recovered after 24 h stimulation of LPS, and secreted levels of IL-6 (d) and TNF- α (e) in BAL fluid were measured by ELISA. (f) Schematic diagram of proposed pathways. 4-PG has the protective and therapeutic effects in LPS-induced ALI by increasing HO-1 expression and reducing oxidative stress. Data were expressed as mean ± SD ( n = 5 per group); ∗∗∗ p < 0.001. NS: not significant. Comparisons between genotypes at HO-1 were made by two-way ANOVA with Bonferroni post-tests.

Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v / v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v / v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Techniques: Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Heme Oxygenase‐1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm

doi: 10.1161/JAHA.121.022747

Figure Lengend Snippet: A , Representative Western blot for HO‐1 protein expression. The 2 marked HO‐1 bands represent native (≈32 kDa) and a truncated (≈28 kDa) isoform of HO‐1. B , Comparison of HMOX1 mRNA expression and ( C ) HO‐1 protein expression in AOD, eAAA, and rAAA. Data are normalized to an internal control (=1). For HMOX1 mRNA expression in eAAA, aortic tissues from n=19 patients were available. This group included 2 to 3 different specimen from the same AAA. Because of this, the number of samples was increased to n=24. In rAAA, n=11 aortic specimen were available. This group included 2 different AAA samples from 3 patients and the number of analyzed samples increased to n=13. For HO‐1 protein in eAAA, n=17 patients were included and 3 different AAA specimens from the same patient were analyzed. Because of this, the number of samples increased to n=20. In rAAA, n=9 patients were included and 2 different samples from the same AAA were analyzed in 2 patients. D and F , Separate quantification of the upper (≈32 kDa) and lower (≈28 kDa) HO‐1 band. F , Representative slides for HO‐1 immunohistochemistry in eAAA and rAAA specimen. Redstained areas represent positive HO‐1 immunoreactivity. Cell nuclei (blue) were counterstained using Mayer's hemalum. The lumen (Lu), adventitia (Ad), and attaching thrombus (Th) are marked in red. G , Comparison of aortic bilirubin content in AOD, eAAA, and rAAA and ( H and I ) Spearman's correlation ( r S ) with HO‐1 protein expression in eAAA and rAAA. In eAAA, pairs of bilirubin and HO‐1 protein were available in n=11 patients only. B–E and G , All data are shown as scatter dot plots. The horizontal line depicts the median with range (B‐E, G). The number of analyzed samples is given in the figures. Kruskal – Wallis and Dunn's multiple comparison test. * P <0.05 AOD vs eAAA. # P <0.05 AOD vs rAAA. Ad indicates adventitia; AOD, arterial occlusive disease; eAAA, electively treated AAA; HO‐1, heme oxygenase‐1; HMOX‐1 , heme oxygenase‐1 gene; Lu, lumen; rAAA, ruptured AAA; and Th, thrombus.

Article Snippet: Membranes were incubated with primary antibodies against HO‐1 (#610713, BD Biosciences).

Techniques: Western Blot, Expressing, Immunohistochemistry

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Heme Oxygenase‐1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm

doi: 10.1161/JAHA.121.022747

Figure Lengend Snippet: A , Comparison of HO‐1 protein expression in eAAA samples with a median diameter less or higher than 84.5 mm. The minimum value was 56.0 mm, the maximum 90.0 mm. Only patients with a diameter higher than 55.0 mm were included because this diameter is chosen in the clinics for surgical removal of the AAA. B , Comparison of HO‐1 protein expression in rAAA samples with a diameter less or higher than 94.0 mm. The minimum value was 52.2 mm, the maximum value 110.0 mm. D and E , Elastin fiber degradation was graded into a score of 1 to 4 and HO‐1 protein expression was grouped. G , Comparison of aortic collagen content in AOD, eAAA, and rAAA and ( H and I ) Spearman's correlation ( r S ) with HO‐1 protein expression in eAAA and rAAA. Representative slides for ( C ) Elastica‐van‐Gieson and ( F ) Picro‐sirius Red staining. The black rectangles represent enlargements of the lower magnifications. All data are presented as scatter dot plots. The horizontal line depicts the median with range (A, B, D, E, and G). The number of analyzed samples is given in the figures. A , B , D , and E , Mann–Whitney U test. G , Kruskal–Wallis and Dunn's post hoc test. * P <0.05 56.0 to 84.4 vs 84.5 to 90.0 mm. Ad indicates adventitia; AOD, arterial occlusive disease; eAAA, electively treated AAA; HO‐1, heme oxygenase‐1; Lu, lumen; rAAA, ruptured AAA; and Th, thrombus.

Article Snippet: Membranes were incubated with primary antibodies against HO‐1 (#610713, BD Biosciences).

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Heme Oxygenase‐1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm

doi: 10.1161/JAHA.121.022747

Figure Lengend Snippet: A , Representative zymograms for detection of pro‐MMP9 and MMP9 in AOD, eAAA, and rAAA. B , Comparison of aortic pro‐MMP9 expression in AOD, eAAA, and rAAA and ( C and D ) Spearman's correlation ( r S ) with HO‐1 expression in eAAA and rAAA. E , Comparison of aortic MMP9 activity in AOD, eAAA, and rAAA and ( F and G ) Spearman's correlation ( r S ) with HO‐1 protein in eAAA and rAAA. Grubb test was performed and 1 significant outlier was detected in the data set for MMP9 ( B , rAAA). Data are presented as scatter dot plots. The horizontal line depicts the median with range (B and E). The number of analyzed samples is given in the figures. Kruskal–Wallis and Dunn's multiple comparison test. * P <0.05 eAAA vs rAAA. AOD indicates arterial occlusive disease; eAAA, electively treated AAA; HO‐1, heme oxygenase‐1; MMP, matrix metalloprotease; and rAAA, ruptured AAA.

Article Snippet: Membranes were incubated with primary antibodies against HO‐1 (#610713, BD Biosciences).

Techniques: Expressing, Activity Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Heme Oxygenase‐1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm

doi: 10.1161/JAHA.121.022747

Figure Lengend Snippet: A , Comparison of aortic H 2 O 2 release in AOD, eAAA, and rAAA. Extracellular H 2 O 2 was measured by Amplex Red assay and fluorescence was normalized to the protein content. B and C , Pearson's correlation ( r P ) of H 2 O 2 with HO‐1 expression in eAAA and rAAA. D , Comparison of aortic thiobarbituric acid reactive substances (TBARS) in AOD, eAAA, and rAAA samples. Values were normalized to the protein content. E and F , Pearson's correlation ( r P ) of TBARS with HO‐1 expression in eAAA and rAAA. G , Comparison of carbonyl residues in oxidized proteins in AOD, eAAA, and rAAA. Carbonyl residues were derivatized and quantified by Western blot. H and I , Spearman's correlation ( r P ) of oxidized proteins with HO‐1 expression in eAAA and rAAA. J , Representative Western blot for detection of carbonyl groups in AOD, eAAA, and rAAA. A and D , Because of scatter, data were log transformed. Data are shown as scatter dot plots. The horizontal line depicts the median with range (A, D, and G). The number of analyzed samples is given in the figures. Data were compared by Kruskal–Wallis and Dunn's multiple comparison test. AOD indicates arterial occlusive disease; DNP‐BSA, 2,4‐dinitrophenyl conjugated to BSA; eAAA, electively treated AAA; H 2 O 2 , hydrogen peroxide; HO‐1, heme oxygenase‐1; MDA, malondialdehyde; rAAA, ruptured AAA; and RFU, relative fluorescence units.

Article Snippet: Membranes were incubated with primary antibodies against HO‐1 (#610713, BD Biosciences).

Techniques: Amplex Red Assay, Fluorescence, Expressing, Western Blot, Transformation Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Heme Oxygenase‐1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm

doi: 10.1161/JAHA.121.022747

Figure Lengend Snippet: A , Serum HO‐1 concentrations were quantified in patients with eAAA and in venous vessel varicose in the preoperative state. Surgery because of rAAA was always an emergency procedure and blood was collected intraoperatively. To exclude effects caused by differences in the study protocol, serum HO‐1 was not determined in rAAA samples. C , The AAA diameter was grouped as <55.0, 55.0 to 70.0, and >70.0 mm. C and D , Spearman's correlation coefficient ( r S ) between serum HO‐1 and the AAA diameter and CRP concentrations. The horizontal line depicts the median with range (A and C) . The number of analyzed samples is given in the figures. A , Mann–Whitney U test. B , Kruskal–Wallis and Dunn's post hoc test. * P <0.05 <55.0 vs 55.0 to 70.0 mm. **** P <0.0001 varicose vs eAAA. CRP indicates C‐reactive protein; eAAA, electively treated abdominal aortic aneurysm; HO‐1, heme oxygenase‐1; and rAAA, ruptured abdominal aortic aneurysm.

Article Snippet: Membranes were incubated with primary antibodies against HO‐1 (#610713, BD Biosciences).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Experimental exposure to Lutzomyia longiplapis sand flies induces HO-1 and Nrf2 protein expression at bite sites in healthy human volunteers. (A,B,D,E) Immunohistochemistry of paraffin-embedded sections of skin specimens obtained by biopsy of the bite sites 48 h after sand fly exposure. (A) HO-1 staining in sand fly-exposed skin, 400X. (B) Higher magnification from image (A) (1000X). (C) Nrf-2 staining in sand fly-exposed skin, 400X. (D) Higher magnification from image (C) (1000X). (E) HO-1 staining in unbitten skin (1000X). (F) Nrf-2 staining in unbitten skin (1000X). Bar, 20 μm. Red arrows point to cells positively stained with HO-1 or Nrf2. Digital images 400 × (lower magnification) or 1000X (higher magnification) were captured using a Nikon E600 microscope and an Olympus Q-Color 1 digital camera with the Image Pro Plus software. (G) Plasma HO-1 levels in individuals ( n = 7) before and after nine exposures to sand fly bites were measured by ELISA. Plasma samples were collected 48 h after each exposure to sand fly bites. Each line represents one individual volunteer. Data represent individual values and were compared using the Kruskal-Wallis test ( p = 0.08).

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Expressing, Immunohistochemistry, Staining, Microscopy, Software, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Bites of Lutzomyia longipalpis sand flies induce Nfr2-dependent HO-1 protein expression by resident macrophages in mice. Ears of C57BL/6 mice were exposed to bites of 20 uninfected L. longipalpis sand flies (UB, uinfected bite) and tissue was processed for total protein extraction before or after sorting by flow cytometry. (A,B) Western blots of total protein extracts from mice ears. Hsp90 was used as a loading control. (A) kinetics of HO-1 expression following sand fly bites. Total protein extract from a mouse spleen was used as a positive control. (B) HO-1 expression in wild-type, Nrf2 knockout (Nrf2 −/− ) and IL-10 knockout (IL-10 −/− ) mice 24 h after exposure to sand fly bites. (C,D) Ear cells recovered 24 h after sand fly bites were stained with antibodies for population sorting by flow cytometry prior to total protein extraction. (C) Gating strategy for sorting populations of non-myeloid cells (I), neutrophils (II), resident macrophages (III) and inflammatory monocytes (IV). (D) Western blot of total protein extract from populations sorted according to (C) . Naïve total ear cells were used as a negative control (CT). Histone H3 was used as a loading control. Blots are representative of two to three independent experiments.

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Expressing, Protein Extraction, Flow Cytometry, Western Blot, Positive Control, Knock-Out, Staining, Negative Control

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Lutzomyia longipalpis saliva activates ARE, nuclear translocation of Nrf2, and HO-1 expression in human macrophages. (A) THP-1 derived macrophages were plated on coverslips and stimulated with either the equivalent of one pair of salivary gland sonicate (SGS) per well or 30 μM CoPP (cobalt protoporphyrin IX) for 24 h, then stained for HO-1. Cells were counter-stained with 1 μg/mL of DAPI to visualize the nucleus. Orange and Blue colors indicate positive staining for HO-1 and nuclei, respectively. Bar, 20 μm. Digital images (400 × original magnification) were captured on a Nikon E600 microscope coupled to an Olympus Q-Color 1 digital camera, and visualized using Image Pro Plus software. (B,C) THP-1 macrophages were treated with one pair SGS per well, followed by extraction of total protein, and detection by Western blot. (B) HO-1. (C) Nrf-2. β-actin and Lamin B were used as loading controls. (D) RAW 264.7 cells were transiently transfected with p3xARE- or pNrf2-promoterluciferase reporter plasmids constructs and treated with 30 μm CoPP or 10 mM DL-sulforaphane (SFN) or one pair SGS per well for 24 h post-transfection. Whole-cell lysates were analyzed for luciferase activity of p3xARE and Nrf2. Data are representative of three independent experiments.

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Translocation Assay, Expressing, Derivative Assay, Staining, Microscopy, Software, Western Blot, Transfection, Construct, Luciferase, Activity Assay

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Lutzomyia longipalpis saliva induces HO-1 protein expression at bite sites in mice. C57BL/6 mice were injected intradermally in the ear with the equivalent of 1 pair of L. longipalpis salivary gland sonicate (SGS). (A) Western blot of whole-tissue extract 24 h after sand fly exposure. β-actin was used as a loading control. (B) Optical density of the bands in (A) was quantified using ImageJ software. Data are presented as the ratio of HO-1 to β-actin. Data are representative of two independent experiments.

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Expressing, Injection, Western Blot, Software