The Lick AGN Monitoring Project 2016 : dynamical modeling of velocity-resolved Hβ lags in luminous Seyfert galaxies

K.H. acknowledges support from STFC grant ST/R000824/1.We have modeled the velocity-resolved reverberation response of the Hβ broad emission line in nine Seyfert 1 galaxies from the Lick Active Galactic Nucleus (AGN) Monitoring Project 2016 sample, drawing inferences on the geometry and structure of the low-ionization broad-line region (BLR) and the mass of the central supermassive black hole. Overall, we find that the Hβ BLR is generally a thick disk viewed at low to moderate inclination angles. We combine our sample with prior studies and investigate line-profile shape dependence, such as log10(FWHM/σ), on BLR structure and kinematics and search for any BLR luminosity-dependent trends. We find marginal evidence for an anticorrelation between the profile shape of the broad Hβ emission line and the Eddington ratio, when using the rms spectrum. However, we do not find any luminosity-dependent trends, and conclude that AGNs have diverse BLR structure and kinematics, consistent with the hypothesis of transient AGN/BLR conditions rather than systematic trends.Publisher PDFPeer reviewe

The Lick AGN Monitoring Project 2016: Dynamical Modeling of Velocity-Resolved H\b{eta} Lags in Luminous Seyfert Galaxies

We have modeled the velocity-resolved reverberation response of the H\b{eta} broad emission line in nine Seyfert 1 galaxies from the Lick Active Galactic Nucleus (AGN) Monitioring Project 2016 sample, drawing inferences on the geometry and structure of the low-ionization broad-line region (BLR) and the mass of the central supermassive black hole. Overall, we find that the H\b{eta} BLR is generally a thick disk viewed at low to moderate inclination angles. We combine our sample with prior studies and investigate line-profile shape dependence, such as log10(FWHM/{\sigma}), on BLR structure and kinematics and search for any BLR luminosity-dependent trends. We find marginal evidence for an anticorrelation between the profile shape of the broad H\b{eta} emission line and the Eddington ratio, when using the root-mean-square spectrum. However, we do not find any luminosity-dependent trends, and conclude that AGNs have diverse BLR structure and kinematics, consistent with the hypothesis of transient AGN/BLR conditions rather than systematic trends

The Lick AGN Monitoring Project 2016 : velocity-resolved Hβ lags in luminous Seyfert galaxies

Funding: K.H. acknowledges support from STFC grant ST/R000824/1.We carried out spectroscopic monitoring of 21 low-redshift Seyfert 1 galaxies using the Kast double spectrograph on the 3 m Shane telescope at Lick Observatory from April 2016 to May 2017. Targetingactive galactic nuclei (AGN) with luminosities of λLλ(5100 Å) ≈ 1044 erg s−1 and predicted Hβ lags of∼ 20–30 days or black hole masses of 107–108.5 M⊙, our campaign probes luminosity-dependent trends in broad-line region (BLR) structure and dynamics as well as to improve calibrations for single-epoch estimates of quasar black hole masses. Here we present the first results from the campaign, including Hβ emission-line light curves, integrated Hβ lag times (8–30 days) measured against V -band continuum light curves, velocity-resolved reverberation lags, line widths of the broad Hβ components, and virial black hole mass estimates (107.1–108.1 M⊙). Our results add significantly to the number of existing velocity-resolved lag measurements and reveal a diversity of BLR gas kinematics at moderately high AGN luminosities. AGN continuum luminosity appears not to be correlated with the type of kinematics that its BLR gas may exhibit. Follow-up direct modeling of this dataset will elucidate the detailed kinematics and provide robust dynamical black hole masses for several objects in this sample.Publisher PDFPeer reviewe

Morphologies and cytoskeletal structure for HT-1080 fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) in synthetic extracellular matrix (ECM).

<div><p>(<b>A</b>) Schematic representation of synthetic extracellular matrix (synthetic ECM) formed through “thiol-ene” photopolymerization chemistry to couple norbornene C=C bonds on 4-arm poly(ethylene glycol) (PEG) molecules with thiol (-SH) bonds of cysteine-containing peptides. Crosslinks were formed using matrix metalloproteinase (MMP)-degradable peptides with cysteine groups on each end while adhesion was promoted using pendant RGD-containing peptides (C<b>RGDS</b>) with a single cysteine (2.5 or 3 wt% by mass PEG-NB + MMP-degradable crosslinking peptide, shear moduli = 140 Pa or 220 Pa respectively). Constant total pendant peptide was maintained using non-bioactive C<i><b>RDGS</b></i> (1500 μM active C<b>RGDS</b> + non-active C<i><b>RDGS</b></i>). </p> <p>(<b>B</b>-<b>I</b>) Projected z-stack immunofluorescence (IF) images illustrating hDFs and HT-1080s seeded in synthetic ECM (220 Pa, 1000 μM CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). (<b>B</b>,<b>C</b>) Overview to illustrate morphological differences (F-actin, red; Nuclei, blue) for (<b>B</b>) hDFs and (<b>C</b>) HT-1080s. (<b>D</b>-<b>F</b>) IF images illustrating myosin IIb expression for hDFs: (<b>D</b>) Overlay image (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>E</b>) F-actin and (<b>F</b>) Myosin IIb. White arrows point to actomyosin filaments. (<b>G</b>-<b>I</b>) IF images illustrating myosin IIb expression for <b><i>HT-1080s</i></b>: (<b>G</b>) Overlay (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb. </p> <p>(<b>J</b>-<b>L</b>) Comparison of quantified mean (<b>J</b>) circularity and (<b>K</b>) elongation for hDFs and HT-1080s calculated using Nikon NIS Elements software (n > 150 cells, ≥ 6 hydrogels, at least two separate experiments; *** = p<0.001). (<b>L</b>) Fraction of elongated (Elongation ≥ 3.0), middle (2.0 ≤ Elongation < 3.0), and rounded (Elongation < 2.0) cells. Differences in fraction of elongated and rounded hDFs compared to HT-1080s were each statistically significant (N ≥ 6 total gels, at least two separate experiments; *** = p<0.001). </p></div

Matrix influences on migration and morphologies for HT-1080s in synthetic ECM.

<p>(<b>A</b>) Cell speed and (<b>B</b>) directionality (DTO/TD) for HT-1080s as a function of matrix conditions (≥ 6 gels, ≥ 40 cells, at least two separate experiments; * = p<0.05; ** = p<0.01). X-axis: Modulus in Pa / RGD concentration in μM. <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Error bars for DTO/TD represent standard error of the mean for individual cells. There is also a statistical difference in cell speed for the 140 Pa (1500 μM RGD) and 220 Pa (250 μM RGD) conditions (p<0.05, not shown on graph for clarity). (<b>C</b>) A comparison of the fraction of rounded HT-1080s (Elongation Index < 2.0) as a function of synthetic ECM conditions (x-axis: Modulus in Pa / RGD concentration in μM; white bar = 140 Pa; black bars = 220 Pa). Error bars represent standard deviation for fraction of rounded cells per gel (≥ 6 gels, at least two separate experiments; * = p<0.05; ** = p<0.01; *** = p<0.001). </p

Qualitative and quantitative migration for HT-1080s and hDFs.

<div><p>Time-lapse images (10 min./frame) illustrating migration for (<b>A</b>) an hDF and (<b>B</b>) an HT-1080 in synthetic ECM (220 Pa, 1000 μM CRGDS). Image sequences in (<b>A</b>,<b>B</b>) are contrast and brightness enhanced for better display (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s013" target="_blank">Movies S2, S3</a> for unaltered images, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s015" target="_blank">Movie S4</a> for overview comparison of many cells). (<b>A</b>) Migration movement for hDF is characterized by: 1. Front end extension, 2. Attachment, 3. Cell body contraction, and 4. Rear-end release. (<b>B</b>) HT-1080 movement illustrates cell body contraction (white dashed lines and arrow) and simultaneous front end extension (red dashed lines and arrow). </p> <p>(<b>C</b>-<b>E</b>) Comparison of quantified 3D migration for HT-1080s and hDFs in synthetic ECM (220 Pa and 1000 μM CRGDS): (<b>C</b>) Cell speed (adjusted by a factor of √3/2, which was a 3D correction for analysis on 2D minimum intensity z-projections), (<b>D</b>) directionality (DTO/TD), and (<b>E</b>) fraction migrating cells. DTO/TD is a dimensionless parameter that provides a measure of directional motility analogous to persistence time (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s001" target="_blank">Fig. S1A</a>) that is calculated as the distance-to-origin (DTO) after 6 hours divided by the total path length (total distance, TD) (shown schematically, panel to right of C). Migration was calculated from images collected in 15 min. increments for 6 hours, with dividing or interacting cells excluded (≥200 cells, ≥ 3 separate experiments, ≥ 9 total hydrogels). <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Values for DTO/TD represent the mean for all cells while fraction migrating represents the mean for replicate experiments (N ≥ 3). Significance was calculated for hDF relative to HT-1080 migration for each parameter (*** = p<0.001). </p></div

Contractile movement for HT-1080s in 3D culture.

<div><p>(<b>A</b>) Propagation of a constriction ring (arrow) for an HT-1080 migrating in synthetic ECM (220 Pa, 1000 μM CRGDS; 10 min / frame, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s014" target="_blank">Movie S3</a>). (<b>B</b>) Propagation of a constriction ring (arrow) for an HT-1080 migrating in collagen (1.7 mg/mL; 15 min./frame, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s020" target="_blank">Movie S9</a>). Scale bars = 25 μm.</p> <p>(<b>C</b>-<b>G</b>) Z-projected immunofluorescence images illustrating myosin IIb expression for an HT-1080 in synthetic ECM (220 Pa, 1000 μM CRGDS): (<b>C</b>) Overlay image illustrating myosin IIb (green), counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nucleus, blue). Single channel images (grayscale) illustrate (<b>D</b>) F-actin and (<b>E</b>) Myosin IIb. Profile plots generated using ImageJ “Interactive 3D Surface Plot” function (“Spectrum” intensity scale, projection for middle 3 planes) illustrate (<b>F</b>) F-actin and (<b>G</b>) Myosin IIb. Two consecutive single plane images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb.</p></div

Comparison of adhesion properties for HT-1080s and hDFs.

<div><p>(<b>A</b>-<b>M</b>) Projected z-stack immunofluorescence (IF) images for hDFs and HT-1080s in synthetic ECM (220 Pa, 1000 μM CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). </p> <p>(<b>A</b>-<b>D</b>) IF images illustrating vinculin expression for <i><b>hDFs</b></i> (boxed region from A shown in B-D): (<b>A</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). White arrows point to regions enriched with vinculin. (<b>B</b>) Overlay (Vinculin, green; F-actin, red); Single channel images (grayscale) illustrate (<b>C</b>) F-actin and (<b>D</b>) Vinculin. (<b>E</b>-<b>G</b>) IF images illustrating vinculin expression for <b><i>HT-1080s</i></b>: (<b>E</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). Single channel images (grayscale) illustrate (<b>F</b>) F-actin and (<b>G</b>) Vinculin. </p> <p>(<b>H</b>-<b>J</b>) IF images illustrating β1-integrin expression for <i><b>hDFs</b></i>: (<b>H</b>) Overlay (β1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>I</b>) F-actin and (<b>J</b>) β1-integrin (White arrows point to punctate β1-integrin features; Also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s003" target="_blank">Fig. S3</a>). (<b>K</b>-<b>M</b>) IF images illustrating β1-integrin expression for <b><i>HT-1080s</i></b>: (<b>K</b>) Overlay (β1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>L</b>) F-actin and (<b>M</b>) β1-integrin. </p> <p>(<b>N</b>) Comparison of attachment for hDFs and HT-1080s on RGD-SAMs as a function of RGD density. Attachment was statistically significant (cell number > 0 RGD control spots) for HT-1080s on surfaces with ≥ 0.19% mol fraction RGD and for hDFs on surfaces ≥ 0.007% mol fraction RGD. Error bars represent standard error of the mean (SEM) for array spots at given RGD density. Significance for cell attachment on individual spots was calculated for hDFs relative to HT-1080s (* = p<0.05; ** = p<0.01; *** = p<0.001). </p></div

A comparison of quantified migration and morphologies for HT-1080s in synthetic ECM and collagen.

<div><p>Migration and morphologies were compared for HT-1080s cultured in lower modulus synthetic ECM (140 Pa, 1500 μM CRGDS) and collagen (1.7 mg/mL, acid-extracted, rat tail collagen, BD Biosciences). Time-lapse microscopy (15 min / frame) illustrating HT-1080s migrating in (<b>A</b>) synthetic ECM (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s026" target="_blank">Movie S15</a>) and (<b>B</b>) collagen (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s020" target="_blank">Movie S9</a>). Scale bars = 25 μm.</p> <p>HT-1080s migrated in lower modulus synthetic ECM and collagen with statistically equivalent (<b>C</b>) speed and (<b>D</b>) directionality (DTO/TD). <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Differences in HT-1080 morphology were determined by comparing quantified (<b>E</b>) circularity and (<b>F</b>) fraction of rounded cells (Elongation < 2.0). Circularity was calculated for individual cells (3 separate experiments, N > 200); Fraction of rounded cells was calculated per gel (at least two separate experiments, N ≥ 3 gels), with error bars representing standard deviation (*** = p < 0.001). </p></div