Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Enhanced large conductance K + channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

doi: 10.1152/ajpheart.00636.2013

Figure Lengend Snippet: Comparison of BK-α- and BK-β-subunit expression in cerebral vessel homogenates isolated from FHH and FHH.1BN rats. BK-α- and BK-β-subunits were probed on different blots. The same blots were then reprobed for β-actin. A: representative bands corresponding to the molecular weight of the BK-α-subunit (∼100 kDa), BK-β-subunit (∼25 kDa), and β-actin (∼42 kDa) in cerebral vessels obtained from FHH and FHH.1BN rats (n = 8 animals each; 3 sets of experiments). B: a comparison of the expression of α- and β-subunits. Numbers in parentheses indicate the number of samples studied.

Article Snippet: After transfer, the membrane was blocked with TBS-T buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween, and a 5% blocking powder (Bio-Rad) at 4°C for 1 h. The blot was probed with primary antibody against BK α- and β-subunit [1:500 and 1:200, respectively; polyclonal rabbit Anti-K Ca 1.1, to amino acids 1184–1200 and Anti-sloβ1 (KCNMB1); Alomone Labs, Jerusalem, Israel] overnight at 4°C.

Techniques: Expressing, Isolation, Molecular Weight

Journal: Diabetes

Article Title: Gene Silencing of Phogrin Unveils Its Essential Role in Glucose-Responsive Pancreatic β-Cell Growth

doi: 10.2337/db08-0970

Figure Lengend Snippet: Effects of shRNA-expressing adenoviruses on GSIS and the proliferation of MIN6 cells. A : MIN6 cells were infected with adenoviruses integrating shVector, shPh1, shPh2, shPh3, or shIA2 for 72 h. The expression levels of endogenous phogrin and IA-2 proteins were determined by immunoblotting with specific antibodies for phogrin and IA-2. α-Tubulin is a nontargeting control. B : MIN6 cells infected with Ad-shVec, shPh1, shPh2, or shPh3 were immunostained with anti-phogrin antibody. C : MIN6 cells expressing shPh3 were fixed at time points 24, 48, and 72 h. Cells were then immunostained with anti-phogrin and anti-insulin antibodies. D : The infected cells were incubated for 2 h in modified KRB (2 mmol/l glucose) before high-glucose stimulation. After stimulation for 30 min with 2 or 25 mmol/l glucose, culture media ( left ; secretion) and cells ( right ; content) were collected and subjected to RIA for insulin. Data are given as means ± SE of five independent experiments. E : Time course of MIN6 cell proliferation 24–96 h after infection with Ad-shVec, shPh1, shPh2, shPh3, or shIA2. Cell number was measured as indicated in research design and methods . Phogrin-KD or IA2-KD cells grew more slowly than the shVec-infected cells (* P < 0.001, ** P < 0.005). F : Cells infected by Ad-shVec or shPh3 for 72 h were assayed for BrdU incorporation by immunofluorescence analysis. Experiments were performed four times ( n = 12). (Please see http://dx.doi.org/10.2337/db08-0970 for a high-quality digital representation of this figure.)

Article Snippet: Anti-adaptin, anti–carboxypeptidase E (anti-CPE), anti–calcium-dependent activator protein (anti-CAPS), anti-munc18, anti-dynamin, anti–clathrin heavy chain, anti–phosphatidylinositol 3-kinase (anti-PI3K), anti-Grb2, and anti–extracellular signal–related kinase (anti-ERK) mouse monoclonal antibodies and anti–insulin receptor β-subunit (anti-IRβ) rabbit polyclonal antibodies were from BD Biosciences.

Techniques: shRNA, Expressing, Infection, Western Blot, Incubation, Modification, BrdU Incorporation Assay, Immunofluorescence

Journal: Oncology Letters

Article Title: PDGF-BB regulates the transformation of fibroblasts into cancer-associated fibroblasts via the lncRNA LURAP1L-AS1/LURAP1L/IKK/IκB/NF-κB signaling pathway

doi: 10.3892/ol.2021.12798

Figure Lengend Snippet: PDGF-BB induced the formation of the CAF phenotype in hOMF. (A) The cells were treated with PDGF-BB in a dose-dependent manner (10, 20, 30 ng/ml) in 10% fetal bovine serum for 72 h for three passages. The cells were subjected to western blot analysis with antibodies against CAF markers α-SMA, FAP-α, PDGFR-β and p-PDGFR-β. β-tubulin served as a loading control and sample loading was 20 µg. (B) Fluorescence microscopy of hOMF stained with α-SMA, FAP-α and PDGFR-β (probed with a primary and a secondary antibody). Cells were counterstained with DAPI. Data were expressed as means ± SEM (n=3). *P<0.05 vs. hOMF, # P<0.05 vs. 10 ng/ml PDGF-BB. PDGF, platelet-derived growth factor; CAF, cancer-associated fibroblast; hOMF, human oral mucosa (p3) 500K fibroblast; α-SMA, α-smooth muscle actin; FAP-α, fibroblast activation protein-α; PDGFR-β, platelet-derived growth factor receptor-β; p, phosphorylated.

Article Snippet: The following antibodies were used in the present study: α-SMA (cat. no. ab5694; 1:1,000), FAP-α (cat. no. ab53066; 1:500), IKKα (cat. no. ab32041; 1:1,000), IκBα (cat. no. ab32518; 1:1,000), NF-κB p65 (cat. no. ab16502; 1:1,000), p-NF-κBp65 (p-S536; cat. no. ab86299; 1:1,000), GAPDH (cat. no. ab181602; 1:10,000), β-tubulin (cat. no. ab179511; 1:1,000) and β-actin (cat. no. ab8227; 1:1,000) were obtained from Abcam; LURAP1L (cat. no. PA5-55072; 1:1,000) was obtained from Thermo Fisher Scientific, Inc., and PDGFR-β (cat. no. bs-0232R; 1:1,000) and p-PDGFR-β (Tyr740; cat. no. bs-3323R; 1:1,000) were obtained from BIOSS.

Techniques: Western Blot, Fluorescence, Microscopy, Staining, Derivative Assay, Activation Assay

Journal: Oncology Letters

Article Title: PDGF-BB regulates the transformation of fibroblasts into cancer-associated fibroblasts via the lncRNA LURAP1L-AS1/LURAP1L/IKK/IκB/NF-κB signaling pathway

doi: 10.3892/ol.2021.12798

Figure Lengend Snippet: Effects of the PDGFR-β inhibitor, CP-673451 on LURAP1L-AS1-LURAP1L/IKK/IκB/NF-κB signaling and CAF programming. (A) Reverse transcription-quantitative PCR analysis of LURAP1L and LURAP1L-AS1 expression levels. (B) Western blot analysis of FAP-α, α-SMA, IKKα, IκBα, NF-κB p65 and p-p65 expression following overexpression of LURAP1L-AS1 and treatment with PDGF-BB. *P<0.05 vs. PDGF-BB. PDGF, platelet-derived growth factor; LURAP1L, leucine-rich adaptor protein 1-like; LURAP1L-AS1, LURAP1L antisense RNA 1; IKKα, IκB kinase α; IKK, I-κB kinase; IκBα, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α; NF-κB, nuclear factor-κB; CAF, cancer-associated fibroblasts; p, phosphorylated; α-SMA, α-smooth muscle actin; FAP-α, fibroblast activation protein-α.

Article Snippet: The following antibodies were used in the present study: α-SMA (cat. no. ab5694; 1:1,000), FAP-α (cat. no. ab53066; 1:500), IKKα (cat. no. ab32041; 1:1,000), IκBα (cat. no. ab32518; 1:1,000), NF-κB p65 (cat. no. ab16502; 1:1,000), p-NF-κBp65 (p-S536; cat. no. ab86299; 1:1,000), GAPDH (cat. no. ab181602; 1:10,000), β-tubulin (cat. no. ab179511; 1:1,000) and β-actin (cat. no. ab8227; 1:1,000) were obtained from Abcam; LURAP1L (cat. no. PA5-55072; 1:1,000) was obtained from Thermo Fisher Scientific, Inc., and PDGFR-β (cat. no. bs-0232R; 1:1,000) and p-PDGFR-β (Tyr740; cat. no. bs-3323R; 1:1,000) were obtained from BIOSS.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Derivative Assay, Activation Assay

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Acyl-CoA synthetase 1 exacerbates lipotoxic inflammasome activation in primary macrophages

doi: 10.1002/JLB.3HI0219-045RR

Figure Lengend Snippet: (A, B) Peritoneal macrophages (pMACs) isolated from WT or TRIF KO mice (TRIF) were stimulated as indicated (LPS 100 ng, palmitate – 250 μM) for 16 h. Cell lysates (A) or mRNA (B) were prepared and ACSL1 protein and mRNA expression was assessed by Western blotting (A) and qRT-PCR (B), respectively. The bands are quantified and shown beneath the blot. Actin is shown as a loading control. (C, D) WT pMACs were stimulated as indicated for 16 h in the presence of an IFNAR-antibody (filled bars) or isotype control antibody (open bars). The expression of ACSL1 (C) and ISG15 (D) was quantified qRT-PCR and normalized to 36B4. (E) Protein was isolated from pMACs isolated from littermate WT or LysM-Cre X ACSL1flx/flx (KO) mice stimulated with control of palm-LPS for 16 h. ACSL1 protein expression was assessed by Western blotting. (F) WT (open bars) or mACSKO (filled bars) pMACs were treated with control (BSA-LPS) or palm (250 μM)-LPS (100 ng) for 20 h, and the release of IL-1β and TNF-α was determined by ELISA. (G) WT or mACSKO pMACs were treated with palm-LPS or stearate (stear; 150 μM)-LPS for 20 h and IL-1β release was quantified by ELISA. (F) pMACs from WT or mACSKO mice were stimulated with palm-LPS or treated with LPS 500 ng for 2 h followed by nigericin (nig; 1 μM) for 60 min or alum (500 μg/ml) for 6 h. IL-β release was quantified by ELISA. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. *, P < 0.05 for WT versus TRIFKO or mACSKO or control ab versus IFNAR ab; ns; nonsignificant

Article Snippet: Duoset ELISA kits (IL-1 β and TNF- α ) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Expressing, Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Acyl-CoA synthetase 1 exacerbates lipotoxic inflammasome activation in primary macrophages

doi: 10.1002/JLB.3HI0219-045RR

Figure Lengend Snippet: (A) WT (top panels) or mACSKO (bottom panels) pMACs were treated with the indicated stimuli for 16 h after which they were subjected to ORO staining to visualize crystals. Images were taken using confocal microscopy at 40× magnification. Nuclei were counterstained with DAPI (blue). (B) Quantification of ORO under all conditions is shown in A. (C) Electron microscopy of macrophages from WT and mACSKO mice demonstrating the ultrastructural appearance of intracellular crystals. (D) pMACs were treated with palm-LPS in the presence of the ACS inhibitor TC at 1 or 5 μM for 16 h after which the cells were stained with ORO, and the signal was quantified after extraction. (E) WT or mACSKO pMACs were treated with palm-LPS, and the indicated concentrations of TC and IL-1β release was quantified by ELISA. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate.*, P < 0.05 for WT versus mACSKO or veh versus TC

Article Snippet: Duoset ELISA kits (IL-1 β and TNF- α ) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Confocal Microscopy, Electron Microscopy, Extraction, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Acyl-CoA synthetase 1 exacerbates lipotoxic inflammasome activation in primary macrophages

doi: 10.1002/JLB.3HI0219-045RR

Figure Lengend Snippet: (A) BODIPY-palmitate uptake was quantified by flow cytometry in WT and mACSKO pMACs at baseline and following LPS activation for 16 h. (B) WT pMACS were stimulated as indicated for 16 h after which cells were lysed and partitioned in to cytosolic (c) and mitochondrial (m) fractions. ACSL1 protein expression was assessed by Western blotting. Tubulin and VDAC were used as controls for fraction of cytosolic and mitochondrial compartments, respectively. Band quantification is shown beneath the blots. (C, D) pMACs from WT or mACSKO mice were stimulated as indicated for 16 h after which cells were lysed and C16 ceramide (C) or C16 acylcarnitine (D) were quantified by LC-MS/MS. (E) WT (black, gray lines) or mACSKO (red, blue lines) were treated with PBS (black, red lines) or LPS (gray, blue lines) for 16 h and cellular OCR was assessed using a Seahorse flux analyzer. The arrows indicate drug injections occurring during the run. (F) pMACs were treated with palm-LPS ± 50 μM etomoxir (eto) for 20 h, and IL-1β release was quantified by ELISA. (G) Macrophages from WT and mACSKO mice were stimulated with BSA-PBS (red line) or palm-LPS (black line) for 16 h and mitochondrial ROS was assessed by mitosox staining coupled with flow cytometry. As a control for mitochondrial ROS generation, WT cells were treated with rotenone for 6 h (rot). Representative histograms are displayed. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. *, P < 0.05 for WT versus mACSKO

Article Snippet: Duoset ELISA kits (IL-1 β and TNF- α ) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Activation Assay, Expressing, Western Blot, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Journal of Endocrinology

Article Title: The activation state of macrophages alters their ability to suppress preadipocyte apoptosis

doi: 10.1530/joe-12-0114

Figure Lengend Snippet: Figure 1 Pro-inflammatory macrophage activation, PDGF release, and survival activity of J774A.1-MacCM. (A) 3T3-L1 preadipocytes were incubated for 6 h in serum-free control medium, J774A. 1-MacCM, or LPS-J774A.1-MacCM generated by LPS pretreatment of macrophages for the times indicated. Viable adherent cells (trypan blue negative) were enumerated and expressed as mean cell number GS.E.M. nZ4, *P!0.05. (B) PDGF BB in J774A.1-MacCM or LPS-J774A.1-MacCM, treated with vehicle or LPS for the indicated period before the preparation of MacCM, was quantified by ELISA. Data are expressed in picogram per milliliter. nZ3, *P!0.05.

Article Snippet: Membranes were incubated for 1 h in 5% skim milk or 3% BSA to block nonspecific binding sites and then probed as indicated with the following primary antibodies directed against: PDGF receptor b (PDGFRb; rabbit polyclonal, 1 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphotyrosine (PY20, mouse monoclonal, 1:1000; BD Biosciences, Mississauga, ON, Canada), ERK1/2 (rabbit polyclonal, 0.25 mg/ml; Upstate Biotechnology, Charlottesville, VA, USA), phospho-ERK1/2 (pERK1/2, Thr202/Tyr204, rabbit polyclonal, 1:2000), Akt (rabbit polyclonal, 1:1000), or phospho-Akt (pAkt, Ser473, rabbit polyclonal, 1:1000; all from Cell Signaling Technology, Beverly, MA, USA).

Techniques: Activation Assay, Activity Assay, Incubation, Control, Generated, Enzyme-linked Immunosorbent Assay

Journal: Journal of Endocrinology

Article Title: The activation state of macrophages alters their ability to suppress preadipocyte apoptosis

doi: 10.1530/joe-12-0114

Figure Lengend Snippet: Figure 2 LPS-J774A.1-MacCM, PDGF signaling, and 3T3-L1 preadipocyte death. 3T3-L1 preadipocytes, pretreated or not with 10 mM imatinib, as indicated, were incubated for 15 min in serum- free control medium, J774A.1-MacCM, or LPS-J774A. 1-MacCM (generated by LPS pretreatment of macrophages for 6 h). Whole cell lysates were immunoblotted with the indicated antibodies. nZ3 (A), nZ4 (B), *P!0.05, **P!0.01, ***P!0.001 vs control medium, or indicated pairs.

Article Snippet: Membranes were incubated for 1 h in 5% skim milk or 3% BSA to block nonspecific binding sites and then probed as indicated with the following primary antibodies directed against: PDGF receptor b (PDGFRb; rabbit polyclonal, 1 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphotyrosine (PY20, mouse monoclonal, 1:1000; BD Biosciences, Mississauga, ON, Canada), ERK1/2 (rabbit polyclonal, 0.25 mg/ml; Upstate Biotechnology, Charlottesville, VA, USA), phospho-ERK1/2 (pERK1/2, Thr202/Tyr204, rabbit polyclonal, 1:2000), Akt (rabbit polyclonal, 1:1000), or phospho-Akt (pAkt, Ser473, rabbit polyclonal, 1:1000; all from Cell Signaling Technology, Beverly, MA, USA).

Techniques: Incubation, Control, Generated

Journal: Journal of Endocrinology

Article Title: The activation state of macrophages alters their ability to suppress preadipocyte apoptosis

doi: 10.1530/joe-12-0114

Figure Lengend Snippet: Figure 3 LPS-induced macrophage TNFa and survival activity of MacCM. (A) 3T3-L1 preadipocytes were incubated for 15 min in serum-free control medium, LPS control medium, J774A.1-MacCM, or LPS-J774A.1-MacCM, prepared following 24 h incubation with 10 ng/ml LPS. Whole cell lysates were immunoblotted with the indicated antibodies. nZ3, *P!0.05, ***P!0.001. (B) 3T3-L1 preadipocytes were incubated for 6 h in serum-free control medium, J774A.1-MacCM, or LPS-J774A.1-MacCM, following preincubation of the media with anti-TNFa neutralizing antibody or control nonspecific rat IgG. Viable adherent cells (trypan blue negative) were enumerated and data expressed as mean cell number GS.E.M. nZ3, *P!0.05, **P!0.01. (C) 3T3-L1 preadipo- cytes were incubated for 6 h with serum-free control medium, 20 ng/ml PDGF BB, or J774A.1-MacCM, each supplemented or not with 10 ng/ml TNFa. Viable adherent cells (trypan blue negative) were enumerated and data expressed as mean cell number GS.E.M. nZ3, *P!0.05.

Article Snippet: Membranes were incubated for 1 h in 5% skim milk or 3% BSA to block nonspecific binding sites and then probed as indicated with the following primary antibodies directed against: PDGF receptor b (PDGFRb; rabbit polyclonal, 1 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphotyrosine (PY20, mouse monoclonal, 1:1000; BD Biosciences, Mississauga, ON, Canada), ERK1/2 (rabbit polyclonal, 0.25 mg/ml; Upstate Biotechnology, Charlottesville, VA, USA), phospho-ERK1/2 (pERK1/2, Thr202/Tyr204, rabbit polyclonal, 1:2000), Akt (rabbit polyclonal, 1:1000), or phospho-Akt (pAkt, Ser473, rabbit polyclonal, 1:1000; all from Cell Signaling Technology, Beverly, MA, USA).

Techniques: Activity Assay, Incubation, Control