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: 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

Journal:

Article Title: Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

doi:

Figure Lengend Snippet: The protocatechuate and catechol branches of the 3-oxoadipate pathway. Designations as gene products are given in parentheses. For the reactions common to both branches, some bacteria possess only a single set of genes, while others harbor separate pca and cat genes for one or more of the steps.

Article Snippet: Purified 3-oxoadipate enol-lactone hydrolase prior to sequencing was partially desalted by changing the buffer to 5 mM sodium phosphate buffer (pH 7) by ultrafiltration and repeated dilution, while selected peptide-containing fractions from reversed-phase HPLC as well as the blotted subunits of the protocatechuate 3,4-dioxygenase were sequenced directly in automated sequencers (Applied Biosystems model 473A or model 491, respectively).

Techniques:

Journal:

Article Title: Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

doi:

Figure Lengend Snippet: Sequences of N termini, tryptic peptides, and deduced primers

Article Snippet: Purified 3-oxoadipate enol-lactone hydrolase prior to sequencing was partially desalted by changing the buffer to 5 mM sodium phosphate buffer (pH 7) by ultrafiltration and repeated dilution, while selected peptide-containing fractions from reversed-phase HPLC as well as the blotted subunits of the protocatechuate 3,4-dioxygenase were sequenced directly in automated sequencers (Applied Biosystems model 473A or model 491, respectively).

Techniques: Sequencing

Journal:

Article Title: Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

doi:

Figure Lengend Snippet: Silver-stained SDS-polyacrylamide gel showing purified 4-carboxymuconolactone decarboxylase:3-oxoadipate enol-lactone hydrolase from R. opacus 1CP in lane 2. Lane 1, Pharmacia low-molecular-weight markers: bovine α-lactalbumin (14.4 kDa), soybean trypsin inhibitor (20.1 kDa), bovine carbonic anhydrase (30 kDa), chicken egg white ovalbumin (43 kDa), bovine serum albumin (67 kDa), and rabbit phosphorylase b (94 kDa).

Article Snippet: Purified 3-oxoadipate enol-lactone hydrolase prior to sequencing was partially desalted by changing the buffer to 5 mM sodium phosphate buffer (pH 7) by ultrafiltration and repeated dilution, while selected peptide-containing fractions from reversed-phase HPLC as well as the blotted subunits of the protocatechuate 3,4-dioxygenase were sequenced directly in automated sequencers (Applied Biosystems model 473A or model 491, respectively).

Techniques: Staining, Purification, Molecular Weight

Journal:

Article Title: Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

doi:

Figure Lengend Snippet: Sequence alignment of the 4-carboxymuconolactone decarboxylase:3-oxoadipate enol-lactone hydrolase PcaL of R. opacus 1CP to the 4-carboxymuconolactone decarboxylases PcaC of A. calcoaceticus (M33798 [24]) and B. japonicum (Y10223 [3]), to the N terminus (term.) of PcaC from P. putida (73), and to the 3-oxoadipate enol-lactone hydrolases PcaD of B. japonicum (Y10223 [3]) and CatD and PcaD of A. calcoaceticus (L05770 [25] and M76991 [63]). Positions identical in all compared sequences are highlighted. Possible catalytic triad residues (25, 46) of the hydrolases as well as glycine residues allowing formation of the “nucleophile elbow” (46) are indicated by arrows. Numbers above the sequences refer to positions in the alignment, not in a single sequence.

Article Snippet: Purified 3-oxoadipate enol-lactone hydrolase prior to sequencing was partially desalted by changing the buffer to 5 mM sodium phosphate buffer (pH 7) by ultrafiltration and repeated dilution, while selected peptide-containing fractions from reversed-phase HPLC as well as the blotted subunits of the protocatechuate 3,4-dioxygenase were sequenced directly in automated sequencers (Applied Biosystems model 473A or model 491, respectively).

Techniques: Sequencing

Journal:

Article Title: Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

doi:

Figure Lengend Snippet: Elution profiles of 3-oxoadipate enol-lactone-hydrolyzing and 4-carboxymuconolactone-decarboxylating activities during chromatography on Q-Sepharose High Performance (A) and Phenyl Superose (B). Experimental details are given in Table ​Table3.3. Closed and open circles, 3-oxoadipate enol-lactone hydrolase and 4-carboxymuconolactone decarboxylase activities, respectively; dotted lines, NaCl or (NH4)2SO4 concentration; solid lines, absorption at 280 nm.

Article Snippet: Purified 3-oxoadipate enol-lactone hydrolase prior to sequencing was partially desalted by changing the buffer to 5 mM sodium phosphate buffer (pH 7) by ultrafiltration and repeated dilution, while selected peptide-containing fractions from reversed-phase HPLC as well as the blotted subunits of the protocatechuate 3,4-dioxygenase were sequenced directly in automated sequencers (Applied Biosystems model 473A or model 491, respectively).

Techniques: Chromatography, Concentration Assay

Journal: PLoS Pathogens

Article Title: Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase

doi: 10.1371/journal.ppat.1005335

Figure Lengend Snippet: ( A ) Heterologous expression of r Sm Eno in E . coli BL21 Star (DE3). Coomassie-stained gel showing SDS-PAGE resolution of lysates of E . coli bacteria harboring pTrcHisB:: Sm Eno before (Gel, left lane) or 4 hours after (Gel, right lane) protein expression induction. The arrow indicates r Sm Eno in the induced lane. In western blot analysis of these lysates probed with monoclonal anti-His tag antibody, a prominent ~50 kDa protein ( Sm Eno) is detected (Western Blot, anti-His, arrow). Recombinant Sm Eno protein was purified from bacterial lysate by immobilized metal affinity chromatography. A single ~50 kDa pure protein ( Sm Eno) is resolved following Coomassie brilliant blue staining of an SDS-PAGE gel and this protein binds anti-ENO antibody, as determined by western blot analysis (right lane, arrow). Positions of migration of molecular mass markers are indicated on the left (kDa). Michaelis-Menten kinetic curve generated using PGA as substrate ( B , catalyzing the forward reaction) or using PEP as substrate ( C , catalyzing the reverse reaction). The apparent Km and Vmax values shown represent the mean +/- SD of three independent experiments. ( D ) Recombinant Sm Eno activity in a buffer system covering the pH range 5.5–9.5. Enzymatic activity is maximal at pH 7.5. ( E ) Impact of divalent ion (Mg 2+ or Ca 2+ , as indicated) concentration on mean r Sm Eno activity (± SD). The highest activity value (at 100 mM Mg 2+ ) was set at 100% and relative activities were calculated and are presented. Significant differences relative to equivalent measurements containing Ca 2+ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid. ( F ) r Sm Eno activity in the presence of increasing concentrations of NaF (white bars) compared to its activity in the absence of inhibitors (set at 100%, black bar). Significant differences relative to the untreated control are denoted by *** for p <0.001. ( G ) Influence of increasing concentrations of mefloquine (MFQ) on r Sm Eno activity (left bars) or on schistosomula lysate enolase activity (right bars). Activity measured in the absence of MFQ was set at 100% and relative activities were calculated. Significant differences relative to the untreated control are denoted by * for p<0.05 and ** for p<0.01. In E-F, bars represent mean relative activity ± SD, n = 3.

Article Snippet: Four hours later, bacteria were harvested by centrifugation and the cell pellet was resuspended in Bug Buster Lysis buffer (Life Technologies) and recombinant Sm Eno protein (r Sm Eno) was purified by affinity chromatography on a Ni-NTA Sepharose column following the manufacturer’s instructions (Life Technologies).

Techniques: Expressing, Staining, SDS Page, Bacteria, Western Blot, Recombinant, Purification, Affinity Chromatography, Migration, Generated, Activity Assay, Concentration Assay, Control

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: Proteins recognized by at least 3 CLL sera

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques: Sequencing

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: Immunoreactivity of patients with CLL

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques:

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: ENO1 expression was evaluated by immunohistochemistry and immunofluorescence confocal microscopy on CLL and R LN sections. Anti-ENO1 immunostaining of a representative CLL LN and R LN section was performed using an anti-mouse HRP-conjugated secondary Ab and 3,3′-diaminobenzidine (brown signal). Black arrows indicate paraimmunoblasts (A) and centroblasts (B) . Original magnification x4 (left panels) and x40 (right panels). Images were obtained using a Olympus BX41 microscope, equipped with Nikon Coolpix camera. Triple staining of representative CLL and R LN sections with anti-ENO1 (red), anti-CD23 (green) and anti-CD2 (white). Original magnification x63; zoom factor of 2.5 for images on the right. Scale bars represent 50 μm and 25 μm, respectively (C, D) . Cumulative data of ENO1 mean pixel intensity (arbitrary units) confirmed a significantly higher intensity of ENO1 expression in close proximity of B cells in CLL LN and T cells in R LN (always p<0.0001) (E) . Triple staining of representative CLL and R LN sections with anti-ENO1 (red), anti-CD23 (green) and anti-Ki67 (blue). Original magnification x63; zoom factor of 2.5 for images on the right. Scale bars represent 50 μm and 25 μm, respectively (F, G) . Quantitative measurements of ENO1 mean pixel intensity (arbitrary units) confirmed that CD23+/Ki67+ proliferating cells of the CLL LN expressed significantly higher level of ENO1 compared to the CD23+/Ki67- resting fraction (p<0.0001) and to CD23+/Ki67+ proliferating B cells of the R LN (p<0.0001) (H) . Triple staining of representative CLL LN and R LN sections with anti-ENO1 (red), anti-CD3 (green) and anti-Ki67 (blue). Original magnification x63; zoom factor of 2.5 for images on the right. Scale bars represent 50 μm and 25 μm, respectively (I, J) . Cumulative analysis of ENO1 mean pixel intensity (arbitrary units) confirmed that the CD3+/Ki67+ proliferating fraction of the T-cell compartment expressed significantly higher levels of ENO1 than CD3+/Ki67- resting fraction, in both CLL and R LN (p<0.001 and 0.03 respectively) (K) . For cumulative data, at least 3 randomly chosen fields from 3 different samples were analyzed. Samples were analyzed using a TCS SP5 laser scanning confocal microscope (Leica Microsystems) with an oil immersion 63x/1.5 objective lens, images were acquired with LAS AF Version Lite 2.4 software and processed with Photoshop (Adobe Systems). ENO1 mean pixel intensity was calculated with ImageJ software ( http://rsbweb.nih.gov/ij/ ). Box and whiskers plots represent median values, first and third quartiles, and minimum and maximum values for each dataset. Statistical analysis was performed using Mann-Whitney U test.

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Confocal Microscopy, Immunostaining, Microscopy, Staining, Software, MANN-WHITNEY

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: ENO1 expression was evaluated in PBMC isolated from patients with CLL, at baseline conditions and after 4-day in vitro culture by flow cytometry. A . ENO1 cytoplasmic expression. High percentages of ENO1 were expressed in the cytoplasm of CD19+/CD5+ CLL cells, CD14+ monocytes and CD3+ T cells. B . ENO1 surface expression. ENO1 was not expressed on the surface of CD19+/CD5+ CLL cells, whereas it was widely expressed by CD14+ monocytes and in a proportion of CD3+ lymphocytes. C, D . Surface ENO1 fold increase expression in CD19+ and CD19- cell fractions. Fold increase was calculated for each patient as a ratio between the percentage of CD19+/ENO1+ detected after 4-day in vitro culture and the percentage of CD19+/ENO1+ cells at baseline. After 4 days of culture, the fold increase in ENO1 expression was significantly higher in the apoptotic (AnnV+) than in the viable (AnnV-) fraction of CD19+ cells (p=0.02) (C). By contrast, there was no difference in the fold increase of ENO1 expression between the apoptotic (AnnV+) and the viable (AnnV-) fraction of CD19- cells (D). Box and whiskers plots represent median values, first and third quartiles, and minimum and maximum values for each dataset.

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques: Expressing, Isolation, In Vitro, Flow Cytometry

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: The presence of circulating anti-ENO1 Ab in sera of patients with CLL was correlated with parameters of disease progression and TTFT. Anti-ENO1 Ab were more frequently detected in sera from patients with progressive CLL than in sera from patients with stable CLL (p<0.0001) (A) . Kaplan-Meier survival curves for TTFT in our cohort of patients stratified on the basis of anti-ENO1 reactivity are shown. The presence of anti-ENO1 Ab correlated with shorter TTFT (ENO1 Ab+, n=19; ENO1 Ab-, n=16) (p=0.01) (B) .

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: A-D . Viability of CLL cells incubated for 1 hour with different concentrations of CLL serum, in the presence or in the absence of alemtuzumab. Cell viability was determined by Ann-V/PI staining and cytofluorimetric analysis on CLL cells purified from patients’ samples. A, B . Representative analyses of AnnV/PI expression on CLL cells left untreated or incubated with patient's serum at 25% (A) and 100% (B) concentration, in the presence or in the absence of alemtuzumab. Indicated is the percentage of AnnV/PI double negative (AnnV-/PI-) cells. C, D . Percentage of AnnV-/PI- viable CLL cells. Line plots show that cell viability was significantly reduced when CLL cells were exposed to alemtuzumab ( p <0.01). By contrast, there was no significant decrease in cell viability when CLL cells were exposed to sera alone, both at the 25% (C) and 100% (D) concentrations. E-G . CDC assay on the U937 cell line. E . ENO1 surface expression. Representative analyses of ENO1 expression on U937 cells. F . Representative analyses of AnnV/PI expression on U937 cells left untreated or incubated with ENO1-Ab+ patients’ sera at 100% concentration. Indicated is the percentage of AnnV-/PI- cells. G . Percentage of viable AnnV-/PI- U937 cells. Line plot shows no significant decrease in cell viability after exposure of U937 cells to 6 ENO1-Ab+ sera from CLL patients.

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques: Incubation, Staining, Purification, Expressing, Concentration Assay, CDC Assay

Journal: Oncotarget

Article Title: Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia

doi: 10.18632/oncotarget.13712

Figure Lengend Snippet: A-C . % of ADCC of CFSE labelled CLL cells co-cultured for 18 hours with HD PBMC, in presence or absence of 10% diluted anti-ENO1 Ab+ serum. Cell lysis was determined by PI staining and flow cytometry. % of ADCC was calculated by the formula % PI (T + E + serum) - % PI (T)/% PI (triton X treated T) – % PI (T). A, B . Representative analysis of CFSE/PI expression on CLL cells (gated on CFSE+ cells) purified from one patient's sample co-cultured with HD PBMC, used as effector cells, in the absence (A) or presence (B) of anti-ENO1 Ab+ serum at 10% concentration. Indicated is the % of CFSE/PI double positive cells. C . Bar graph show no significant differences in the % of ADCC of primary CLL cells cultured with PBMC and 10% diluted anti-ENO1 Ab+ serum (n=6) compared to target cells cultured with PBMC alone, in the absence of serum. D-F . ADCC assay on U937 cell line. D, E . Representative analysis of CFSE/PI expression on U937 cells (gated on CFSE+ cells) co-cultured with HD PBMC, used as effector cells, in the absence (D) or presence (E) of anti-ENO1 Ab+ serum at 10% concentration. Indicated is the % of CFSE/PI double positive cells. F . Bar graph show no significant differences in the % of ADCC of U937 cells cultured with PBMC and 10% diluted anti-ENO1 Ab+ serum (n=6) compared to target cells cultured with PBMC alone, in the absence of serum. Mean results from three different experiments.

Article Snippet: The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).

Techniques: Cell Culture, Lysis, Staining, Flow Cytometry, Expressing, Purification, Concentration Assay, ADCC Assay

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 1 | The expression of ENO1 on the cell-surface of a subpopulation of CSCs. (A) Representative FACS plots showing patterns of CD44, CD133, and surface ENO1 (sENO1) staining of primary prostate adenocarcinoma (PAC)-derived 22Rv-1 cells with the frequency of the boxed CD44+CD133+ cell population (representing CSCs in PAC; left) or sENO1+ cells in CD44+CD133+ CSCs (middle) or cells in the other subpopulations (representing non-CSCs; right) shown. (B) The percentages of sENO1+ cell subpopulation in CD44+CD133+ 22Rv-1 cells or cells in the other subpopulations (others). (C) The percentages of sENO1+ cell subpopulation in CD44+CD133+ PC-3 cells or cells in the other subpopulations. (D) The percentages of sENO1+ cell subpopulation in CD90+ gastric adenocarcinoma (GAC) AGS or NCI-N87 cells (representing CSCs in GAC) or CD90- cells (representing non-CSCs). Error bars represent mean ± SEM from three independent experiment (n = 3). Unpaired t-test was performed throughout where **p < 0.01; ***p < 0.001 in (B–D).

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Expressing, Staining, Derivative Assay

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 2 | sENO1 marks a subpopulation of mesenchymal-like and highly invasive CSCs. (A) The relative transcript levels of the mesenchymal- (left) or pluripotency- (right) associated genes in sENO1+ CSCs (represented by CD44+CD133+ 22Rv-1 cells), sENO1- CSCs, and non-CSCs (represented by cells in the other subpopulations) using qRT-PCR analysis. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05 versus non-CSCs; †p < 0.05 versus sENO1- CSCs. (B) Immunoblotting analysis of the indicated markers selected from (A) in non- CSCs, sENO+, and sENO- CSCs. Protein levels were quantified by densitometric analysis of the bands, normalized to b-tubulin (loading control). (C) Limiting dilution assay (LDA) demonstrating the tumorsphere-forming efficacy of each subset of tumor cells. Three independent experiments were performed (n = 6). Shown are maximum likelihood estimates with a 95% confidence interval, where **p < 0.01. (D) The invasive capacities of freshly sorted sENO1+ CSCs (represented by CD44+CD133+ 22Rv-1 cells), sENO1- CSCs, and non-CSCs in 22Rv-1 cells in a dual-chamber invasion assay. Shown are representative immunofluorescence images of the invaded cells, with cell nuclei stained with SYTOX-green (green). Scale bars = 500 µm. Right, the number of invaded cells. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where **p < 0.01.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Quantitative RT-PCR, Western Blot, Control, Limiting Dilution Assay, Invasion Assay, Staining

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 3 | sENO1+ CSCs are highly pro-metastatic. (A) Representative BLI of NOD/SCID mice receiving an intra-splenic injection of sENO1+ CSCs (represented by CD90+ NCI-N87 cells), sENO1- CSCs (CD90- NCI-N87 cells), and non-CSCs (represented by CD90- cells). at the indicated time following cell inoculation. (B) Tumor bulk quantified as BLI normalized photon counts as a function of time. Error bars represent mean ± SEM from one experiment (n = 8 mice per group). Unpaired t-test was performed throughout where **p < 0.01 versus non-CSCs. (C) Representative BLI of NOD/SCID mice receiving intra-splenic injection of sENO1+

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Injection

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 4 | ENO1 is expressed on the invadopodial surface of CSCs. (A) Confocal views of PAC CSCs (represented by CD44+CD133+ PC-3 cells) showing the cross-section of invadopodia structures (represented by cortactin+F-acin+ puncta) with the colocalized surface ENO1 (sENO1; green), cortactin (red), and F-actin (magenta) that penetrate into the underlying gelatin matrix. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale, 10 µm. (B) Top, a pie chart showing the percentage of sENO1+ invadopodia per PC-3 CSC. Bottom, a pie chart showing the percentage of sENO1+ invadopodia per GAC AGS CSC (represented by CD90+ AGS cells). (C) Left, representative three-dimensional (3D) reconstructed confocal image of CD44+CD133+ PC-3 CSCs showing the co- localization of sENO1 (green) and cortactin (red) at the ventral side of cell. Scale, 8 µm. Right upper, digital zoom-in image from serial Z sections (yellow rectangle) showing the spatial colocalization of sENO1 (green) and cortactin (red) at invadopodia. Scale, 5 µm. Right lower, the orthogonal view of the magnified areas (yellow squares at top) shown the distribution and localization of sENO1 and cortactin at the base of invadopodia. 3D rendered images of the invadopodia (arrows) were processed by using Imaris software. Scale, 1 µm.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Software

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 5 | sENO1+ CSCs generate more invadopodia than their sENO1- counterparts. (A) Confocal views of sENO1+ PC-3 CSCs (represented by CD44+CD133+

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques:

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 6 | sENO1 contributes to the invadopodial formation and the matrix-degradative function of CSCs. (A) Immunoblotting analysis showing the effect of lentivirus shRNA-mediated knockdown (KD) of ENO1 expression in PC-3 cells. Protein levels were quantified by densitometric analysis of the bands, normalized to b- tubulin (loading control). (B) Bar graph showing the percentage of sENO1+ PC-3 cells with KD of ENO1 expression or control KD. (C) Bar graph showing the density of invadopodia (represented by cortactin+F-actin+ puncta) per cells in PC-3 CSCs (represented by CD44+CD133+ cells) or non-CSCs (represented by cells in other subpopulations) with ENO1 KD or control KD. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed where **p < 0.01, ***p < 0.001 in (B, C). (D) PC-3 cells with KD of ENO1 expression or the control KD cells were seeded on top of a fluorescein-conjugated gelatin matrix and immunostained with cortactin (green) or phalloidin (F-actin; red). Nuclei were counterstained with DAPI (blue). Right, the fluorescence intensity of fluorescein- conjugated gelatin within the boundary (determined by F-actin staining) of PC-3 cells with ENO1 KD or control KD (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001. (E) Bar graph showing the invasive capacity of PC-3 CSCs with ENO1 KD or control KD in a dual-chamber invasion assay. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed where **p < 0.01 versus non-CSCs. (F) Representative immunofluorescence images of CD44+CD133+ PC-3 cells (representing CSCs) that had invaded the type I collagen matrix in the presence of an increasing concentration (0.1-1.0 µg/ml) of the anti-ENO1 polyclonal antibody (pAb; a-ENO1) in a dual-chamber invasion assay. The nuclei of the invaded cells were stained with SYTOX-green. Scale bars, 500 µm. Right, the number of invaded cells. Cells in other subpopulations (representing non-CSCs) were included as a control. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05, **p < 0.01 versus non-CSCs. (G) The invadopodia density per cell in PC-3 CSCs or non-CSCs exposed to an increasing concentration of a-ENO1. Error bars represent mean ± SEM from three independent experiments (n = 50 cells counted per sample). Unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001 versus non-CSCs. (H) PC-3 CSCs were seeded on top of a gelatin matrix in the presence or absence of a-ENO1 (20 µg/ml). Shown are the extent of matrix degradation as reflected by immunostaining with anti-Col1-3/4C (red). Right, the total cell fluorescence intensity of Col1-3/4C in PC-3 CSCs treated with a-ENO1 or a control IgG (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Control, Staining, Invasion Assay, Concentration Assay, Immunostaining

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 7 | CAV1 is indispensable for the surface localization of sENO1 on CSCs and its pro-invadopodia and pro-invasive functions. (A) Immunoblotting analysis showing the effect of lentivirus shRNA-mediated knockdown (KD) of CAV1 (top) or HSP70 (bottom) expression in PC-3 cells. Protein levels were quantified by densitometric analysis of the bands, normalized to b-tubulin (loading control). (B) Bar graph showing the percentage of sENO1+ cells in PC-3 CSCs (represented by CD44+CD33+ cells) with KD of CAV1 or HSP70 expression or control-KD. Unpaired t-test was performed throughout where ***p < 0.001 versus control KD. (C) Bar graph showing the density of invadopodia (represented by coractin+F-actin+ puncta) per cell in PC-3 CSCs or non-CSCs (represented by cells in other subpopulations) with CAV1 KD or control KD. Unpaired t-test was performed throughout where ***p < 0.001. (D) Bar graph showing the invasive capacity of PC-3 CSCs or non-CSCs with CAV1 KD or control KD in a dual-chamber invasion assay. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05 versus non-CSCs; †p < 0.05 versus control KD.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Control, Invasion Assay