Journal: PLoS ONE

Article Title: Jujuboside B Reduces Vascular Tension by Increasing Ca 2+ Influx and Activating Endothelial Nitric Oxide Synthase

doi: 10.1371/journal.pone.0149386

Figure Lengend Snippet: (A)Jujuboside B increased NO generation in HAECs concentration-dependently. * P < 0.05, ** P < 0.01 compared with Control, n = 3. (B) Jujuboside B increased NO generation in HAECs negative time-dependently. * P < 0.05, ** P < 0.01 compared with 15 min group, n = 3. (C) L-NAME significantly inhibited Jujuboside B-induced NO generation. * P < 0.05, ** P < 0.01, n = 3. (D) L-NAME significantly inhibited Jujuboside B-induced eNOS activation. ** P < 0.01, ## P < 0.01, n = 4. (E) Western blot analysis of eNOS and p-eNOS at Serine-1177 in HAECs showed that Jujuboside B significantly increased p-eNOS Serine-1177 expression. (F) Densitometric analysis showed that Jujuboside B significantly increased p-eNOS Serine-1177 expression. * P < 0.05 compared with Control, n = 3.

Article Snippet: The residual protein bands were blocked with 5% milk and the membrane was incubated with rabbit p-eNOS (Serine-1177) antibody and rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, USA) dilution overnight.

Techniques: Concentration Assay, Control, Activation Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Jujuboside B Reduces Vascular Tension by Increasing Ca 2+ Influx and Activating Endothelial Nitric Oxide Synthase

doi: 10.1371/journal.pone.0149386

Figure Lengend Snippet: (A)EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS activation. ** P < 0.01 compared with Control, # P < 0.05, ## P <0.01 compared with Jujuboside B, n = 4. (B) Western blot analysis showed that EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS phosphorylation at Serine-1177. (C) Densitometric analysis showed that EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS phosphorylation at Serine-1177. # P < 0.05 compared with Control, * P < 0.05 compared with Jujuboside B, n = 3.

Article Snippet: The residual protein bands were blocked with 5% milk and the membrane was incubated with rabbit p-eNOS (Serine-1177) antibody and rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, USA) dilution overnight.

Techniques: Activation Assay, Control, Western Blot, Phospho-proteomics

Journal: Hypertension

Article Title: Renal Collecting Duct NOS1 Maintains Fluid–Electrolyte Homeostasis and Blood Pressure

doi: 10.1161/hypertensionaha.113.01291

Figure Lengend Snippet: Figure 1. NOS1 splice variant expression of the inner medulla (IM) and cerebellum (CB) from wild-type mouse (WT) and NOS1αKO mouse as determined by Western blotting with anti- NOS1 (C terminus). A, NOS1α is expressed in the cerebellum of the WT mouse but not in the cerebellum of NOS1αKO mice. NOS1β is expressed in the IM of WT and NOS1αKO mice. The molecular weight marker (MW) was run on the same gel, but it was noncontiguous. B, NOS1β and NOS3 coexpressed in the IM, and NOS1α and NOS3 coexpressed in the cerebellum of the WT mouse. The molecular weight marker (MW) was run on the same gel, but it was noncontiguous. C, Collecting duct expression of NOS1β in the WT and NOS1αKO mice.

Article Snippet: IHC - immunohistochemistry Antibody Sequence Amino Acids Host Application Concentration Company Location NOS1 rat C-terminus rabbit polyclonal IB 20 μg/10ml Santa Cruz Santa Cruz, CA NOS1 human 1414-1434 rabbit polyclonal IB, IHC 1.4 μg Biomol Plymouth Meeting, PA NOS3 human 1025-1203 Mouse monoclonal IB 12.5 μg/ 10ml BD Biosciences San Jose, CA CD3-ε mouse C-terminus goat polyclonal IHC 0.2 μg Santa Cruz Santa Cruz, CA F4/80 mouse ? rat monoclonal IHC 0.1 μg AbD Serotech Raleigh, NC 3 NOS1!"

Techniques: Variant Assay, Expressing, Western Blot, Molecular Weight, Marker

Journal: Nutrients

Article Title: Analysis of the Combined Effects of a Novel Combination of Hypersmin, Pumpkin Seed and Amaranthus Extracts in an In Vitro Model of Chronic Venous Insufficiency.

doi: 10.3390/nu17111807

Figure Lengend Snippet: Figure 3. Vascular tone analysis on in vitro model of vein after intestinal passage. In (A) NO production; in (B) eNOS levels; in (C) endothelin-1 levels. From (A) to (C) all the data are obtained with specific ELISA kits; * p < 0.05 vs. Control; α p < 0.05 vs. single agents; β p < 0.05 vs. Commercial product; γ p < 0.05 vs. KCl.

Article Snippet: The eNOS concentration was measured following the manufacturer’s instructions for the DuoSet ELISA kit for Human eNOS (R&D Systems, Minneapolis, MN, USA) in HUVEC cells [39].

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Control

Journal: The FASEB Journal

Article Title: PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis

doi: 10.1096/fj.201601291R

Figure Lengend Snippet: PHD3−/− mice show increased incidence of disc degeneration and decreased expression of select HIF-1 target genes in NP tissue. A) Schematic drawing of spinal motion segment and coronal cross section showing vertebral bodies and the intervertebral disc with its central NP, circumferential AF, and superior and inferior cartilaginous endplates (CEP). B, C) Representative Safranin-O/Fast Green/hematoxylin–stained sections of 12.5-mo-old WT (B) and PHD3−/− (C) mice. The PHD3−/− mouse showed a decreased number of NP cells and some changes in cell morphology. D) PHD3−/− mice showed increased incidence of discs with a higher grade of degeneration, as assessed by the Thompson grading scale. Four discs/animal were scored from 3 pairs of littermate animals. E–L') Representative immunofluorescence images from 12.5-mo-old WT (PHD3+/+) and knockout (PHD3−/−) mice showing NP tissue areas with a comparable number of cells. There was decreased staining of HIF-1 targets VEGF-A (E–F'), LDHA (G–H'), and GLUT1 (I, J) in knockout mice compared to WT mice. In contrast, another HIF-1 target, ENO1, shows comparable expression in PHD3+/+ (K, K') and PHD3−/− (L, L') mice. Krt19 was used as a marker to label the NP tissue compartment with VEGF-A, LDHA, and ENO1 (independent channel not shown). Scale bars, 100 μm (B, C) and 50 μm (E–L'). Images are representative from 3 independent littermate groups.

Article Snippet: Sections were then sequentially incubated with antibodies [VEGF, ab46154; LDHA, ab52488; and GLUT1, ab40084 (all from Abcam), or ENO1, NB100-65252 (Novus Biologics, Littleton CO, USA), all at a concentration of 1:100) with an NP marker anti-keratin (Krt)19 antibody (1:3, TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) in 5% normal donkey serum in 1× PBS, 0.4% Triton-X at 4°C overnight ( 22 ).

Techniques: Expressing, Staining, Immunofluorescence, Knock-Out, Marker

Journal: The FASEB Journal

Article Title: PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis

doi: 10.1096/fj.201601291R

Figure Lengend Snippet: PHD3 controls expression of a select set of HIF-1 targets in NP cells through a p300-dependent mechanism. A, B) Bright-field (A) and fluorescent (B) images of rat NP cells after transduction with lentivirus coexpressing shPHD3 and enhanced green fluorescent protein. Scale bars, 10 μm. C) Measurement of PHD3 mRNA expression after transduction of NP cells with shRNA targeting PHD3 (n = 7). D) Measurement of mRNA expression of HIF-1 target genes in PHD3-silenced NP cells cultured in NX or HX for 72 h (n = 7). E) Hypoxic expression of Pgk1, Gapdh, and Eno1 in PHD3-silenced cells did not change (n = 3). F) Measurement of activity of Eno1 promoter containing 2 well-characterized HIF-1 binding sites after PHD3 knockdown (n = 7). G, H) Western blot (G) and corresponding densitometric analysis (H) of select HIF-1 targets in NP cells after stable knockdown of PHD3 (n = 4). I) Location of HRE sites within promoters of Vegfa, Slc2a1, and Ldha and location of ChIP primers used in J. J) HIF-1α enrichment at HRE sites within Vegfa, Slc2a1, and Ldha promoters decreases after knockdown of PHD3 during 72 h HX (n = 3). Luciferase assays were performed with 3 technical replicates per experiment; quantitative RT-PCR assays were performed with 2 technical replicates per experiment. Data are means ± sem. *P < 0.05.

Article Snippet: Sections were then sequentially incubated with antibodies [VEGF, ab46154; LDHA, ab52488; and GLUT1, ab40084 (all from Abcam), or ENO1, NB100-65252 (Novus Biologics, Littleton CO, USA), all at a concentration of 1:100) with an NP marker anti-keratin (Krt)19 antibody (1:3, TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) in 5% normal donkey serum in 1× PBS, 0.4% Triton-X at 4°C overnight ( 22 ).

Techniques: Expressing, Transduction, shRNA, Cell Culture, Activity Assay, Binding Assay, Knockdown, Western Blot, Luciferase, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Specific activation of glycolytic enzyme enolase 2 in BRAF V600E‐mutated colorectal cancer

doi: 10.1111/cas.14929

Figure Lengend Snippet: Immunohistochemical analysis of ENO1 and ENO2 in BRAF wild‐type and BRAF V600E‐mutated CRC. A, Representative images of positive and negative immunohistochemical staining with anti–ENO2 antibody in BRAF wild‐type CRC and BRAF V600E‐mutated CRC. Scale bar: 50 µm. B, The positive rate of ENO1 and ENO2 expression in BRAF wild‐type and BRAF V600E‐mutated CRC based on immunohistochemical staining. C, The positive rate of ENO2 expression in BRAF wild‐type+nonV600E and BRAF V600E‐mutated CRC based on immunohistochemical staining in 121 CRC patient

Article Snippet: After blocking with 3% skim milk at room temperature for 1 hour, the membranes were incubated with primary antibodies at the appropriate concentrations at 4°C overnight: ENO1 (1:1000; #3810, Cell Signaling), ENO2 (1:1000; #9536, Cell Signaling), BRAF (1:1000; #14814, Cell Signaling), FOSL1 (1:1000; #D80B4, Cell Signaling), pERK (1:2000; #4370, Cell Signaling), ERK (1:1000; #4695, Cell Signaling), pAKT (1:2000; #4060, Cell Signaling), and AKT (1:1000; #9272, Cell Signaling).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Canagliflozin mitigates carfilzomib-induced endothelial apoptosis via an AMPK-dependent pathway.

doi: 10.1016/j.biopha.2023.114907

Figure Lengend Snippet: Fig. 1. Carfilzomib decreases the viability of endothelial cells by inducing apoptosis. EA.hy926 human endothelial-derived cells and human umbilical vein endothelial cells (HUVECs) were treated with increasing concentrations of carfilzomib (0, 0.05, 0.1, 0.2, 0.5, and 1 μM) for 24 h. Cell viability measured by the MTT assay in (A) EA.hy926 and (B) HUVECs. Cell viability was calculated relative to control wells and expressed as a percentage. Expression levels of the apoptotic markers: cleaved caspase-3 in (C) EA.hy926 and (D) HUVECs and cleaved PARP in (E) EA.hy926 and (F) HUVECs measured by western blotting. Values are presented as means ± SEM (n = 4–6). Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparison test. Note: * p < 0.05, * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001.

Article Snippet: Primary mouse antibodies against AMPK alpha (catalog #2793) and primary rabbit antibodies against phospho-AMPK alpha (catalog #4188), cleaved caspase-3 (catalog #9664), caspase-3 (catalog #9662), PARP (catalog #9542), eNOS (catalog #32027), phospho-eNOS (catalog #9571), VEGFR-2 (catalog #9698), ICAM-1 (catalog #4915), VCAM-1 (catalog #13662), phospho-p70 (catalog #9205), p70 (catalog #2708), phospho-Akt (catalog #4060), Akt (catalog #4691), phospho-mTOR (catalog #5536), mTOR (catalog #2983), phospho-JNK (catalog #4668), JNK (catalog #9252), phospho-p38 (catalog #4511), p38 (catalog #8690), phospho-ERK (catalog #4370), ERK (catalog #4695), and alpha-tubulin (catalog #2144) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Derivative Assay, MTT Assay, Control, Expressing, Western Blot, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Canagliflozin mitigates carfilzomib-induced endothelial apoptosis via an AMPK-dependent pathway.

doi: 10.1016/j.biopha.2023.114907

Figure Lengend Snippet: Fig. 4. Canagliflozin protects endothelial cells from carfilzomib-induced apoptosis. EA.hy926 human endothelial-derived cells and human umbilical vein endothelial cells (HUVECs) were treated with 0.5 μM carfilzomib (CFZ) in the absence or presence of SGLT2 inhibitors (canagliflozin (CANA), empagliflozin (EMPA), and dapagliflozin (DAPA)) in a concentration range of 0–100 µM in EA.hy926 cells and 0–20 µM in HUVECs. Cell viability measured by the MTT assay in (A) EA. hy926 and (B) HUVECs. Cell viability was calculated relative to control wells and expressed as a percentage. Expression levels of the apoptotic markers: cleaved caspase-3 in (C) EA.hy926 and (D) HUVECs and cleaved PARP in (E) EA.hy926 and (F) HUVECs measured by western blotting. Values are presented as means ± SEM (n = 4). Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparison test. Note: * p < 0.05, * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001.

Article Snippet: Primary mouse antibodies against AMPK alpha (catalog #2793) and primary rabbit antibodies against phospho-AMPK alpha (catalog #4188), cleaved caspase-3 (catalog #9664), caspase-3 (catalog #9662), PARP (catalog #9542), eNOS (catalog #32027), phospho-eNOS (catalog #9571), VEGFR-2 (catalog #9698), ICAM-1 (catalog #4915), VCAM-1 (catalog #13662), phospho-p70 (catalog #9205), p70 (catalog #2708), phospho-Akt (catalog #4060), Akt (catalog #4691), phospho-mTOR (catalog #5536), mTOR (catalog #2983), phospho-JNK (catalog #4668), JNK (catalog #9252), phospho-p38 (catalog #4511), p38 (catalog #8690), phospho-ERK (catalog #4370), ERK (catalog #4695), and alpha-tubulin (catalog #2144) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Derivative Assay, Concentration Assay, MTT Assay, Control, Expressing, Western Blot, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Canagliflozin mitigates carfilzomib-induced endothelial apoptosis via an AMPK-dependent pathway.

doi: 10.1016/j.biopha.2023.114907

Figure Lengend Snippet: Fig. 7. Canagliflozin and AICAR protects endothelial cells from CFZ-induced apoptosis by restoring AMPK. Human umbilical vein endothelial cells (HUVECs) were treated with 0.5 μM carfilzomib (CFZ) with or without 1 mM AICAR (AMPK activator), or 20 μM canagliflozin (CANA) ± 5 μM compound C (Cpd C). Results of western blots of 0.5 µM CFZ ± 1 mM AICAR for (A) cleaved caspase-3 and (B) cleaved PARP; µM CFZ with or without CANA ± Cpd C for (C) cleaved caspase-3 and (D) cleaved PARP; 0.5 µM CFZ ± 1 mM AICAR for (E) ICAM-1, (F) VCAM-1, (G) VEGFR-2. Values are presented as means ± SEM (n = 4–6). Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparison test. Note: * ** p < 0.001, * ** * p < 0.0001. Abbreviations; ICAM-1: intercellular adhesion molecule-1, VCAM-1: vascular cell adhesion molecule-1, VEGFR-2: vascular endothelial growth factor receptor 2.

Article Snippet: Primary mouse antibodies against AMPK alpha (catalog #2793) and primary rabbit antibodies against phospho-AMPK alpha (catalog #4188), cleaved caspase-3 (catalog #9664), caspase-3 (catalog #9662), PARP (catalog #9542), eNOS (catalog #32027), phospho-eNOS (catalog #9571), VEGFR-2 (catalog #9698), ICAM-1 (catalog #4915), VCAM-1 (catalog #13662), phospho-p70 (catalog #9205), p70 (catalog #2708), phospho-Akt (catalog #4060), Akt (catalog #4691), phospho-mTOR (catalog #5536), mTOR (catalog #2983), phospho-JNK (catalog #4668), JNK (catalog #9252), phospho-p38 (catalog #4511), p38 (catalog #8690), phospho-ERK (catalog #4370), ERK (catalog #4695), and alpha-tubulin (catalog #2144) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Canagliflozin mitigates carfilzomib-induced endothelial apoptosis via an AMPK-dependent pathway.

doi: 10.1016/j.biopha.2023.114907

Figure Lengend Snippet: Fig. 8. Canagliflozin does not interfere with the anticancer effect of carfilzomib. U266B1 and RPMI8226 cells were treated with 0.5 µM carfilzomib (CFZ) with or without canagliflozin (CANA). Proteasome 20 S activity assay for (A) U266B1 and (B) RPMI8226 cells. Cell viability measured by the MTT assay for (C) U266B1 and (D) RPMI8226 cells. Both proteasome activity and cell viability were calculated relative to control wells and expressed as a percentage. Expression levels of the apoptotic markers: cleaved caspase-3 in (E) U266B1 and (F) RPMI8226 cells and cleaved PARP in (G) U266B1 and (H) RPMI8226 cells measured by western blotting. Values are presented as means ± SEM (n = 4). Data were analyzed by one-way ANOVA followed by Dun net’s multiple comparison test. Note: * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001.

Article Snippet: Primary mouse antibodies against AMPK alpha (catalog #2793) and primary rabbit antibodies against phospho-AMPK alpha (catalog #4188), cleaved caspase-3 (catalog #9664), caspase-3 (catalog #9662), PARP (catalog #9542), eNOS (catalog #32027), phospho-eNOS (catalog #9571), VEGFR-2 (catalog #9698), ICAM-1 (catalog #4915), VCAM-1 (catalog #13662), phospho-p70 (catalog #9205), p70 (catalog #2708), phospho-Akt (catalog #4060), Akt (catalog #4691), phospho-mTOR (catalog #5536), mTOR (catalog #2983), phospho-JNK (catalog #4668), JNK (catalog #9252), phospho-p38 (catalog #4511), p38 (catalog #8690), phospho-ERK (catalog #4370), ERK (catalog #4695), and alpha-tubulin (catalog #2144) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Activity Assay, MTT Assay, Control, Expressing, Western Blot, Comparison

Journal: Journal of Clinical Investigation

Article Title: P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction

doi: 10.1172/jci81067

Figure Lengend Snippet: Figure 1. Gq and G11 mediate endo- thelial response to fluid shear stress in vitro. The indicated cells were transfected with scrambled (control) siRNA, or an siRNA directed against Gαq and Gα11. (A) Fluo-4–loaded HUVECs (n = 21, control; n = 25, Gαq/11; 3 indepen- dent experiments) and BAECs (n = 23, control; n = 16, Gαq/11; 3 independent experiments) were exposed to the indicated shear forces, and [Ca2+]i was determined as fluorescence intensity (RFU, rel- ative fluorescence units). Graphs show the AUC. Data represent the mean ± SEM; ***P ≤ 0.001, by 2-tailed Student’s t test. (B–D) HUVECs and BAECs (n = 3) were exposed to fluid shear (12 and 20 dynes/cm2, respectively) for the indicated durations. AKT, eNOS, and SRC activation (B and D) was determined by Western blotting for phosphorylated AKT, eNOS, and SRC kinases and total AKT, eNOS, and SRC. PECAM-1 and VEGFR-2 activation (D) was deter- mined by IP and Western blotting for tyrosine phosphorylated PECAM-1 and VEGFR-2 . Knock- down of Gαq/Gα11 was verified by anti-Gαq/Gα11 immunoblotting. Graphs show the densitometric evaluation. C, Nitrate concentra- tion in the cell medium. Data rep- resent the mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by 2-way ANOVA, with Bonferroni’s post-hoc test. p, phosphorylated; S, serine.

Article Snippet: Antiphosphorylated eNOS (mouse S1176, human S1177, and bovine S1179; catalog 9571); antiphosphorylated ADT (S473; catalog 4060); anti-AKT (catalog 9272); anti-GAPDH (catalog 2118); and antiphosphorylated SRC kinase (Y416; catalog 2113) Abs were obtained from Cell Signaling Technology.

Techniques: Shear, In Vitro, Transfection, Control, Fluorescence, Activation Assay, Western Blot, Knockdown

Journal: Journal of Clinical Investigation

Article Title: P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction

doi: 10.1172/jci81067

Figure Lengend Snippet: Figure 3. Induction of endothelial Gq/G11 deficiency results in reduced eNOS activation and hypertension. (A) Telemetrically recorded blood pressure in WT (n = 9) and EC-q/11–KO (n = 8) mice before, during, and after administration of tamoxifen for 5 days. Average blood pressure 5 days before induction was set to 100%. Graphs show systolic and diastolic arterial blood pressure 4 days before tamoxifen treatment and in the second week after induction. (B) Plasma nitrate levels in WT (n = 12) and EC-q/11–KO mice (n = 12) before and 10 days after induction. (C) Phosphorylation of eNOS at serine 1176 in lysates from mesenteric arteries prepared before (–) or 3 days after (+) tamoxifen induction in WT and EC-q/11–KO. Graph shows the densitometric evalua- tion (n = 4). Data represent the mean ± SEM; **P ≤ 0.01 and ***P ≤ 0.001, by 2-tailed Student’s t test.

Article Snippet: Antiphosphorylated eNOS (mouse S1176, human S1177, and bovine S1179; catalog 9571); antiphosphorylated ADT (S473; catalog 4060); anti-AKT (catalog 9272); anti-GAPDH (catalog 2118); and antiphosphorylated SRC kinase (Y416; catalog 2113) Abs were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Clinical Proteomics, Phospho-proteomics

Journal: Journal of Clinical Investigation

Article Title: P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction

doi: 10.1172/jci81067

Figure Lengend Snippet: Figure 4. P2Y2 mediates endothelial fluid shear stress response in vitro. (A–D) BAECs were transfected with scrambled siRNA (control) or siRNA directed against P2Y2 (A, B, and D) or were preincubated without or with 30 μM of the P2Y2 antagonist AR-C118925 (C). In the Fluo-4–loaded cells (A; n = 28 [control], n = 18 [P2Y2]; 3 independent experiments), the effect of the indicated shear forces on [Ca2+]i was determined as AUC and RFU fluorescence intensity. In B and C, cells were exposed to fluid shear (20 dynes/cm2) for the indicated durations, and AKT, eNOS, SRC, PECAM-1, and VEGFR-2 activation was determined as described. Graphs show the densitometric evaluation (n = 3). (D) Nitrate concentration in the cell medium (n = 3). (E) Amount of ATP in the supernatant of BAECs kept under static conditions or under flow (20 dynes/m2) for the indicated durations (n = 4). (F–H) BAECs (n = 4) were exposed to fluid shear (F and H; 20 dynes/cm2) or to 1 μM insulin for 10 minutes (G) in the absence or presence of 10 U/ml apyrase. AKT and eNOS activation was determined by Western blotting for phosphorylated and total AKT and eNOS (F and G). Bar diagrams show the densitometric evaluation. (H) Nitrate con- centration in the cell medium. Data represent the mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by 2-tailed Student’s t test (A) and 2-way ANOVA, with Bonferroni’s post-hoc test (B–H).

Article Snippet: Antiphosphorylated eNOS (mouse S1176, human S1177, and bovine S1179; catalog 9571); antiphosphorylated ADT (S473; catalog 4060); anti-AKT (catalog 9272); anti-GAPDH (catalog 2118); and antiphosphorylated SRC kinase (Y416; catalog 2113) Abs were obtained from Cell Signaling Technology.

Techniques: Shear, In Vitro, Transfection, Control, Fluorescence, Activation Assay, Concentration Assay, Western Blot

Journal: Journal of Clinical Investigation

Article Title: P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction

doi: 10.1172/jci81067

Figure Lengend Snippet: Figure 5. Endothelial P2Y2 controls vascular tone and blood pressure. (A) Effect of increased perfusion flow (left) and increasing SNP (middle) and ACh concentrations (right) on the diameter of mesenteric arteries from WT (left: n = 9; middle: n = 3; right: n = 6) and EC-P2Y2–KO (left: n = 13; middle: n = 3; right: n = 6) mice. Vessels were precontracted with 100 nM U46619. (B) Blood pressure in WT (n = 9) and EC-P2Y2–KO mice (n = 8) before, during, and after induction. Average blood pressure 5 days before induction was set to 100%. Graphs show systolic and diastolic arterial blood pressure 4 days before tamoxifen treatment and in the second week after induction. (C) Phosphorylation of eNOS at serine 1176 in lysates from mesenteric arteries prepared 3 days after induction in WT and EC-P2Y2–KO mice. Graph shows a densitometric evaluation (n = 4). Data represent the mean ± SEM; *P ≤ 0.05. **P ≤ 0.01, and ***P ≤ 0.001, by 2-tailed Student’s t test (B and C) and 2-way ANOVA, with Bonferroni’s post-hoc test (A).

Article Snippet: Antiphosphorylated eNOS (mouse S1176, human S1177, and bovine S1179; catalog 9571); antiphosphorylated ADT (S473; catalog 4060); anti-AKT (catalog 9272); anti-GAPDH (catalog 2118); and antiphosphorylated SRC kinase (Y416; catalog 2113) Abs were obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated intracellular NO production in H441 cells grown at the air–liquid interface (ALI). ( A ) : Representative image of DAF-FM-loaded H441 ALIs stimulated for 10 min with 1 mM sodium benzoate (NaBenz.) or denatonium benzoate (denat benz.); fluorescence increased with denatonium benzoate but not sodium benzoate. ( B ) : Average trace and bar graph (mean ± SEM) of four experiments as in ( A ). Significance determined by Student’s t -test; ** p < 0.01. ( C ) : Average trace and bar graph (mean ± SEM of three experiments) showing response in cultures pre-loaded with BAPTA-AM and stimulated in the absence of extracellular Ca 2+ (0-Ca 2+ o ) vs. control cultures pre-incubated with 0.1% DMSO only and stimulated in the presence of extracellular Ca 2+ . ( D ) : Denatonium-induced DAF-FM fluorescence increases in H441 ALIs were inhibited by pretreatment with geldanamycin or L-NAME but not HSP70 inhibitor VER-15508. Average trace and bar graph of results from four independent experiments are shown. Significance determined by one-way ANOVA with Dunnett’s post-test comparing all values to control (denatonium only); ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Fluorescence, Incubation

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated NO diffusion into the airway surface liquid (ASL) in H441 cells grown at the air–liquid interface (ALI) ( A ): Representative images and bar graph of 4 independent experiments of fluorescence at the apical plane of ALI when 100 µL of solution containing cell impermeable DAF-2 was placed on top (1.1 cm 2 Transwell) either containing sodium benzoate (top) or denatonium benzoate (bottom). Cultures were either pretreated with 0.1% DMSO (vehicle control), 10 µM PLC inhibitor U73122, or 10 µM inactive analogue U73343 prior to the experiment. Significance determined by one-way ANOVA with Dunnett’s post-test comparing all values to HBSS only control. ( B ): Bar graph of experiments performed as in ( A ) but testing inhibition of denatonium-induced or quinine-induced ASL DAF-2 fluorescence ± NOS inhibitor L-NAME or inactive D-NAME (10 µM). Bar graph shows the mean ± SEM of 3–5 independent experiments imaged at identical conditions. Significance by one-way ANOVA with Bonferroni post-test comparing all values to respective HBSS control; ** p < 0.01. ( C ) : Denatonium-stimulated H441 DAF-2 ASL fluorescence increases were reduced in the presence of GPCR signaling inhibitor YM254890 or HSP90 inhibitors geldanamycin, 17-AAG, or BIIB 021. HSP70 inhibitor VER-15508 had no effect. Bar graph shows the mean ± SEM of four independent experiments. Significance by one-way ANOVA with Dunnett’s post-test comparing all values to control (0.1% DMSO only); * p < 0.05 and ** p < 0.01. ( D ): H441s were treated with siRNA as described in the methods. ASL DAF-2 responses during denatonium stimulation were reduced by eNOS siRNA but not with scramble, nNOS, or PAR-2 siRNA. Bar graph shows the mean ± SEM of four independent experiments (separate siRNA transfections). Significance by one-way ANOVA with Dunnett’s post-test comparing all values to control; ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Diffusion-based Assay, Fluorescence, Transfection

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated intracellular NO production in primary sinonasal epithelial cells grown at the air–liquid interface (ALI). ( A ): Intracellular DAF-FM increases were measured in response to T2R38-agonist PTC (1 mM) followed by NO donor SNAP (25 µM) as positive control. PTC stimulated NO production in ALIs from PAV/PAV (homozygous functional T2R38) but not AVI/AVI (homozygous non-functional T2R38) ALIs (nonfunctional T2R38) patients. Geldanamycin pretreatment inhibited the NO production in PAV/PAV ALIs. Trace and bar graph show the mean ± SEM of 8–10 experiments per condition using ALIs from 4–5 patients. Significance determined by one-way ANOVA with Tukey–Kramer post-test comparing all values; ** p < 0.01. ( B ): Traces of DAF-FM fluorescence in PAV/AVI (heterozygous T2R38) cultures stimulated with T2R14/39 agonist apigenin (100 µM) shown with 0.1% DMSO vehicle control. Pretreatment with T2R14/39 antagonist 4′-fluoro-6-methoxyflavanone (4′-F-6-MF) or HSP90 inhibitor geldanamycin but not 0.1% DMSO (inhibitor vehicle control) reduced apigenin-induced but not SNAP-induced DAF-FM fluorescence increases. ( C ): Traces of DAF-FM fluorescence in PAV/AVI (heterozygous T2R38) cultures stimulated with T2R14 agonist quercetin (50 µM). Pretreatment with T2R14/39 antagonist 4′-fluoro-6-methoxyflavanone (4′-F-6-MF) or HSP90 inhibitor geldanamycin but not 0.1% DMSO (inhibitor vehicle control) reduced quercetin-induced but not SNAP-induced DAF-FM fluorescence increases. ( D ): Bar graph of intracellular DAF-FM fluorescence increases after 2 min stimulation from experiments as in ( C , D ). Stimulation (DMSO vehicle control, apigenin, quercetin, or SNAP) listed on top and pretreatment (DMSO vehicle control, 4′-F-6-MF, or geldanamycin) listed on the bottom. Each data point is an independent experiment ( n = 4–8 per condition). Significance by Bonferroni post-test; * p < 0.05 vs. bracketed bars; # p < 0.05 vs. DMSO alone.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Positive Control, Functional Assay, Fluorescence

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated NO diffusion into the airway surface liquid (ASL) in primary sinonasal epithelial cells grown at the air–liquid interface (ALI) Experiments were performed as in to measure NO diffusion into the ASL but with primary nasal ALIs. ( A ): PTC (500 µM) or 3oxoC12HSL (100 µM) stimulated extracellular DAF-2 fluorescence in PAV/PAV and AVI/AVI cultures, as indicated. PAV/PAV cultures were also pretreated with HSP90 inhibitors geldanamycin, 17-AAG, or BIIB 021 or HSP70 inhibitor VER-155008. ( B ): shows experiments with apigenin ± 4′-F-6-MF, geldanamycin, 17-AAG, or PLC inhibitor U73122 and inactive analogue U73343. Control Transwells containing no cells were similarly incubated with vehicle only or apigenin to test for any cell-independent reaction of apigenin with DAF-2. Significance by one way ANOVA with Bonferroni post-test; * p < 0.05 vs. bracketed bars; ** p < 0.01 vs. bracketed bars; ## p < 0.05 for the same condition in PAV/PAV vs AVI/AVI cultures.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Diffusion-based Assay, Fluorescence, Incubation

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated ciliary beating in primary sinonasal epithelial cells. ( A ): Left shows representative normalized CBF responses (representative experiments shown) to T2R14/39 agonist apigenin in human primary sinonasal ALIs ± T2R14/39 inhibitor 4′-fluoro-6-methoxyflavanone. Right shows normalized CBF responses (representative experiments shown) to apigenin ± geldanamycin (10 µM; 5 min pretreatment). Mean baseline CBF was not with vehicle or 4′-fluoro-6-methoxyflavanone pretreatment (7.5 ± 1.1 Hz or 8.2 ± 0.9 Hz, respectively; not significant by Students’ t -test). Mean baseline CBF was also not different before or after vehicle or geldanamycin pretreatment (6.9 ± 1.7 Hz or 7.9 ± 1.2 Hz, respectively; not significant by Students’ t -test). ( B ): Bar graph of the mean ± SEM of CBF responses from five independent experiments as shown in ( A ) using ALIs from four different patients. Significance determined by one-way ANOVA with Bonferroni post-test; * p < 0.05. ( C ): Left shows representative normalized CBF responses (representative experiments shown) to T2R14/39 agonist quercetin in human ALIs ± T2R14/39 inhibitor 4′-fluoro-6-methoxyflavanone. Mean baseline CBF was not with vehicle or 4′-fluoro-6-methoxyflavanone pretreatment (7.3 ± 1.2 Hz or 7.9 ± 0.6 Hz, respectively; not significant by Students’ t -test). Right shows normalized CBF responses (representative experiments shown) to quercetin ± geldanamycin (10 µM; 5 min pretreatment). Mean baseline CBF was not different before or after vehicle or geldanamycin pretreatment (7.4 ± 1.3 Hz or 7.0 ± 0.9 Hz, respectively; not significant by Students’ t -test). ( D ): Bar graph of the mean ± SEM of CBF responses from five independent experiments as shown in C using ALIs from five different patients. Significance determined by one-way ANOVA with Bonferroni post-test; ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces epithelial ciliary response to P. aeruginosa conditioned medium. ( A ): Graph shows real-time measurement of CBF (mean ± SEM of six independent experiments using ALIs from three patients) during prolonged geldanamycin treatment, followed by stimulation with purinergic agonist ATP. ( B ): Primary nasal ALIs genotyped for functional T2R38 (TAS2R38 PAV/PAV) or non-functional T2R38 (TAS2R38 AVI/AVI) were stimulated with diluted HBSS in which P. aeruginosa PAO-1 had been incubated overnight (conditioned HBSS; cHBSS, diluted with unconditioned HBSS). Peak CBF responses to PAO-1 cHBSS were greater in PAV/PAV cells vs. AVI/AVI cells. Representative trace shown from five experiments using cultures from separate individual patients. ( C ): PAV/PAV cells were stimulated with cHBSS from PAO-1 or PAO-JP2, which lacks the ability to produce AHLs. PAO-1 cHBSS stimulated CBF increases that were greater than CBF increases observed with PAO-JP2 cHBSS. Representative trace shown from five experiments using cultures from separate individual patients. ( D ): PAV/PAV cells were stimulated with PAO-1 cHBSS ± geldanamycin pretreatment. Representative trace shown from five experiments using cultures from separate individual patients. ( E ): Bar graph showing peak CBF (mean ± SEM with individual data points showing individual experiments) observed from experiments as in F-H . Asterisks represent significance compared with PAV/PAV + PAO-1 cHBSS at each individual concentration, determined by Sidak’s multiple comparison test; * p < 0.05 and ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Functional Assay, Incubation, Concentration Assay, Comparison

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces nasal epithelial bacterial killing mediated by T2Rs and NO. P. aeruginosa PAO-1 bacteria were incubated with nasal ALI cultures as described in the methods. ( A ): Bar graph showing live (Syto9)/dead (propidium iodide [PI]) staining quantified by fluorescence plate reader. First two bars represent bacteria incubated in the absence of nasal cells treated with saline only or saline + colistin. This illustrates max (saline) and min (colistin) live/dead ratios. Significance by one way ANOVA with Bonferroni post-test; ** p < 0.01 between bracketed groups; # p < 0.05 and ## p < 0.01 vs. PAV/PAV cultures with no inhibitor. ( B ): Representative image (left) and bar graph (right) showing CFU counts from experiments as shown in ( A ). HSP90 inhibitor geldanamycin reduced bacterial killing (increased CFUs) while HSP70 inhibitor VER 155008 did not. Significance by one-way ANOVA with Dunnett’s post test comparing all values to PAV/PAV control (no inhibitor); ## p < 0.01 vs. PAV/PAV control.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Bacteria, Incubation, Staining, Fluorescence, Saline

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated NO production in primary human M0 MΦs. ( A ): DAF-FM-loaded MΦs exhibited increases in fluorescence in response to 1 mM denatonium benzoate that were strongly inhibited by geldanamycin. Left shows average traces and right shows bar graphs (mean ± SEM) from eight independent experiments using MΦs from two donors. DAF-FM fluorescence increase was also inhibited by BIIB 021. Control = denatonium benzoate after pretreatment with 0.1% DMSO. Significance by one way ANOVA with Bonferroni posttest; * p < 0.05. ( B ): Low-level Ca 2+ responses to denatonium benzoate were not affected by geldanamycin. Top shows representative traces in the absence or presence of 1 µM geldanamycin. Bottom shows bar graph of six independent experiments using MΦs from three different donors. Response to purinergic agonist ATP shown as control. ( C ): NO production in MΦs treated with HSP90 or control non-targeting siRNAs. Left shows representative traces and right shows bar graph of data from four independent experiments per condition. Significance by Student’s t -test; ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Fluorescence

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated FITC- E. coli phagocytosis in primary human M0 MΦs. ( A ): Representative image of MΦs with phagocytosed FITC-labeled E. coli . ( B ): Left shows time course of phagocytosis responses during 30 min incubation in HBSS as described in the methods after pretreatment with geldanamycin or HBSS for times indicated on the y-axis. Each data point is the mean ± SEM of three independent experiments using MΦs from three different donors. Right shows separate experiments of baseline phagocytosis over 30 min (HBSS only) of FITC- E. coli after 2 h pretreatment with HBSS only (containing 0.1% DMSO as vehicle control), 1 µM VER-15508, 1 µM geldanamycin, or geldanamycin plus VER-15508. Significance determined by one-way ANOVA with Dunnett’s post-test comparing values to HBSS pretreatment; * p < 0.05, ** p < 0.01. Bar graph shows the mean ± SEM of six experiments using MΦs from three donors. ( C ): Stimulated 30 min phagocytosis of FITC- E. coli (HBSS only control or 1 mM denat. benz. ± pertussis toxin [PTX]) was measured after pre-incubation with HBSS + 0.1% DMSO or 1 µM geldanamycin. PTX and geldanamycin both inhibited denatonium-induced phagocytosis. Significance determined by one-way ANOVA with Bonferroni post-test; ** p < 0.01 vs. HBSS control and ## p < 0.01 vs. bracketed groups. ( D ): Geldanamycin reduced phagocytosis increases observed with both denatonium and quinine. Bar graph shows the mean ± SEM of six independent experiments using cells from six different individual patients. Significance by one way ANOVA with Tukey–Kramer post-test comparing all bars; ** p < 0.01 vs. HBSS alone; ## p < 0.01 vs. bracketed bar. ( E ): Assays were carried out in MΦs previously treated with siRNAs directed against eNOS, iNOS, HSP90, or non-targeting control sequences. Bar graph shows increase in phagocytosis relative to HBSS in the same macrophage background over four independent experiments. Significance compared with no siRNA control using one-way ANOVA with Bonferroni post-test and pairwise comparisons; * p < 0.05 and ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Labeling, Incubation

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated pHrodo- S. aureus phagocytic responses in primary human M0 MΦs. ( A ): Representative images of pHrodo-labeled S. aureus phagocytosis in primary human MΦs ± denatonium benzoate (1 mM) stimulation after D-NAME or L-NAME pretreatment (10 µM; 45 min). ( B ) : Bar graph of pHrodo- S. aureus fluorescence after experiments as in A . Significance by Bonferroni post-test with paired comparisons; ** p < 0.01. ( C ): Representative images of pHrodo-labeled S. aureus phagocytosis in primary human MΦs ± denatonium benzoate (1 mM) or 3oxoC12HSL (100 µM) after no-pretreatment (0.1% DMSO only as vehicle control)) or pretreatment with HSP90 inhibitors geldanamycin or BIIB 021 (pretreatment as in ). ( D ): Bar graph of pHrodo- S. aureus phagocytosis during stimulation with HBSS only (unstimulated control), 1 mM denatonium benzoate, or 100 µM 3oxoC12HSL ± geldanamycin or BIIB 021 (pretreatment as in ). Significance by one-way ANOVA with Bonferroni post-test; * p < 0.05 or ** p < 0.01. ( E ): Bar graph of pHrodo- S. aureus phagocytosis during stimulation with HBSS only (unstimulated control) or 1 mM denatonium benzoate ± pertussis toxin (PTX), geldanamycin, BIIB 021, 17-AAG, or VER 15508. PTX (500 ng/mL) pretreatment was 18 h. MΦs were pretreated with other inhibitors as in . Significance by one-way ANOVA with Bonferroni post-test; ** p < 0.01.

Article Snippet: For transfection, cells were seeded onto 8-well chambered glass coverslips (CellVis, Mountain View, CA, USA; precoated with poly-D-lysine) and transfected with eNOS-RFP, Wt HSP90, and/or D88N HSP90, kindly provided by W. Sessa (plasmids # 22497 [RRID:Addgene_22497], #22487 [RRID:Addgene_22487], and/or #22480 [RRID:Addgene_22480], respectively; Addgene, Watertown, MA, USA).

Techniques: Inhibition, Labeling, Fluorescence