Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet:

Article Snippet: Total RNA was extracted from E19 male Lz using RNeasy Midi Kits (Qiagen – 75144) and quantity and quality were verified using RNA 6000 Nano Kit (Agilent – 5067-1511) and an Agilent 2100 Bioanalyzer.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, BIA-KA, Western Blot, Stripping Membranes, Reverse Transcription, SYBR Green Assay, Red Blood Cell Lysis, Staining, Enzyme-linked Immunosorbent Assay, In Situ, TUNEL Assay, Imaging, Flow Cytometry, Transfection, Gene Expression, Expressing, Microarray, Isolation, Software

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) A flowchart of the chemical screen demonstrating the major steps. ( B ) Raw data of cell size and cell cycle stage measured from a single control well in the screen. The scatter plot represents single-cell measurements of cell size and three markers of cell cycle stage; Cdt1 (mKO2-hCdt1), Geminin (mAG-hGem) and DNA (DAPI). Every point in the plot represents measurements on one single cell. Cell size is represented by a color scheme depicted by the colorbar on the right. White arrows are added to guide the reader along the cell cycle trajectory. ( C ) Average size of early G1 cells is negatively correlated with the fraction of cells in early G1. The scatterplot displays the result from one example 384-well plate. Each point on the plot corresponds to one particular screened condition (screened compound or control), and represents the average size of early G1 cells in that condition versus the proportion of cells in G1. Red circles highlight the conditions that significantly affect the size of early G1 cells and/or the proportion of cells in G1. The arrows designate examples of on-axis and off-axis compounds (also see ). ( D ) Distribution of correlation coefficients between average size of early G1 cells and the fraction of cells in G1, calculated for all screened plates (as described in Materials and methods - Analysis of the compound screen), demonstrating that the two variables are significantly negatively correlated (p<10 −16 ). ( E, F ) Ranked p-values from the target enrichment analysis of on-axis and off-axis compounds, respectively (Fisher’s exact test). Components of the mTOR pathway and p38 MAPK pathway, which are highlighted, are among the top-ranked hits of on-axis and off-axis phenotypes, respectively. The Matlab script used to perform the target enrichment analysis is presented in . 10.7554/eLife.26947.009 Figure 1—source data 1. The screen metadata used to identify on-axis and off-axis outliers. 10.7554/eLife.26947.010 Figure 1—source data 2. The analysis script to visualize on-axis and off-axis outliers using . 10.7554/eLife.26947.011 Figure 1—source code 1. The Matlab script used to perform the target enrichment analysis.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Components from the p38 pathway (highlighted) were highly enriched. Specifically, MK2/MAPKAPK2, a direct downstream substrate of p38 is the top-ranking genes that associate with increased cell size variability.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques:

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: To estimate the cell size variability that results from inhibition of a specific protein, z-scores were averaged from all screen compounds targeting that protein. An average cell size variability was calculated for each of the target proteins and ranked from small to large. Components of the p38 MAPK pathway (highlighted in red) are ranked among the top proteins with increased z-scores in cell size variability.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Quantifying the coordination of cell size and G1 length. Samples of unsynchronized cells were treated with increasing concentrations of rapamycin (a rapamycin concentration series: 0, 0.03, 0.3, 3 and 30 nM) for a period of 24 hr, and then stained and imaged to quantify cell size and cell cycle stage on a single-cell basis. Each data point (circle) corresponds to a different concentration of rapamycin and shows the average size of early G1 cells and the proportion of cells in G1 resulting from that treatment. Populations treated with higher concentrations of rapamycin had smaller cells and higher fractions of cells in G1, resulting in a robust negative correlation. Rapamycin concentrations are redundantly represented by both the size of the circles and their color, as shown in the colorbar. The small white circles represent control populations that were treated with DMSO, rather than rapamycin. Calculation of the average size and the proportion of G1 cells, in each of the represented samples, was performed by classifying single cells into cell cycle stage as depicted in . Each data point was measured from an unsynchronized population with a minimum of 7000 cells. Additional details on the experiment and analysis is provided in the Materials and methods section. ( B ) The experiment described in panel A is repeated with (red) or without (blue) a chemical inhibitor of p38 (SB203580, 5 μM). The negative correlation between the size of early G1 cells and the proportion of cells in G1 is apparent in populations not treated with SB203580 (blue) but not in the populations that are treated with SB203580. The blue and red trend lines represent linear regressions. ( C ) Western-blots of whole cell lysates from populations that were treated with different combinations of SB203580, rapamycin and Torin-2. The experimental procedure used here are the same as those used to generate the data shown in panel A and B. The increased levels of phopho-p38 in the population that is treated with SB203580 (a p38 inhibitor) should not be interpreted as a lack of efficacy of SB203580. Rather, these higher levels of phopho-p38 are explained by a negative feedback in the p38 pathway , and the fact that while p38 inhibitors prevent p-p38 from phosphorylating its downstream substrates, these inhibitors do not block phosphorylation of p38 itself by upstream regulators . ( D ) Inhibition of the p38 MAPK pathway, but not the MAPK/ERK or SAPK/JNK pathways, disrupts the correlation between the average size of early G1 cells and the proportion of cells in G1. Results were obtained with the same assay used to create panel A and B. Larger circle size indicates higher rapamycin concentration. The rapamycin concentration series includes: 0, 0.03, 0.1, 0.3, 3 and 30 nM. The results shown here are representative of three independent experiments. ( E ) Fitted slopes corresponding to the trends shown in . Error bars represent 90% confidence intervals. For each compound treatment, its fitted slope is compared with the slope of the control (DMSO) from the same experiment. Significance was calculated with one-tailed Student’s t -test (H 0 : slope drug <= slope control ). The meta data and source code used for this analysis and visualization of results is presented in . 10.7554/eLife.26947.017 Figure 2—source data 1. Measurements of cell size and cell cycle stages from the chemical inhibitor experiments as shown in , and .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Staining, Control, Western Blot, Blocking Assay, Phospho-proteomics, Inhibition, One-tailed Test

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A–C ) Scatterplots displaying relationship between average growth rate in G1 stage with G1 duration for individual cells in DMSO control, p38 inhibition and mTOR inhibition. ( D–F ) Scatterplots displaying relationship between average growth rate over cell cycle with cell cycle duration for individual cells in the three conditions. r indicates Spearman’s correlation coefficient. ( G–I ) Distribution of average growth rate in G1 (before Geminin rise), S/G2 (after Geminin rise) and over the entire cell cycle for the three tested conditions. While mTORC1 inhibition by Rapamycin decreases growth rate, p38 inhibition by SB203580 does not significantly affect growth rate in G1. The meta data and source code used in this analysis is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Live cells subject to p38 inhibition (SB203580) or to mTORC1 inhibition (rapamycin) were followed with time-lapse microscopy to monitor proliferation over a period of 50 hr. mTOR inhibition significantly slowed rates of proliferation, while p38 inhibition increased rates of proliferation. ( B ) As an alternative method to assay cell cycle lengths, populations of cells were treated with p38 inhibitors and samples were fixed every 20 hr over a period of 3 days (see Materials and methods -Estimation of cell proliferation durations and growth rate from bulk measurements). Proportion of cells in the different cell cycle stages, in each of the collected samples, were calculated based on the cell cycle indicators depicted in . Consistently, p38 inhibitors accelerate proliferation by shortening the duration of G1 but not the durations of S or G2 (also see ). The meta data and source code used for this cell cycle analysis is presented in . ( C–F ) Live cells were imaged by time-lapse microscopy for a period of 50 hr to obtain growth trajectories of single cells over the course of their entire cell cycle. Computer generated image processing and cell tracking were performed, as described in Materials and methods - Automated lineage tracking and analysis, to obtain single cell growth curves. Nuclear size was used as a proxy of cell size, as has been validated in . Cells that were successfully tracked throughout their entire cell cycle were collected to calculate the cell cycle durations and cell size dynamics. ( G–I ) Scatterplots displaying relationship between nuclear size at birth and G1 duration for individual cells that are subject to chemical inhibition of p38 ( H ), chemical inhibition of mTOR ( I ), and a control population treated with DMSO ( G ). Every single point corresponds to the birth size and G1 length of a single live cell that was followed by time-lapse microscopy. Also shown are means and errorbars (SEM) of average G1 length calculated for different cell size bins. The dashed line shows the result of linear regression with the binned data. ( J ) Slopes obtained by the linear regression shown in ( G–I ) Error bars indicate 95% confidence bounds. The results shown here are representative of two independent experiments. The single-cell tracking data from the live-cell imaging experiments and the source code for analysis and visualization of the results is presented in . 10.7554/eLife.26947.022 Figure 3—source data 1. Estimation of cell cycle duration and growth rate from bulk measurements of fixed cell populations. 10.7554/eLife.26947.023 Figure 3—source data 2. Measurements of single-cell dynamics of cell size captured by live-cell imaging.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Inhibition, Time-lapse Microscopy, Cell Cycle Assay, Generated, Cell Tracking Assay, Control, Single Cell Tracking, Live Cell Imaging

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Cells were treated with indicated inhibitors for 24 hr before collecting lysates. Anisomycin was added to select wells 1 hr prior to making lysates, to activate MAPK pathways. All inhibitors were used at the ‘high dose’ indicated in and . ( A ) Cells treated with p38 inhibitors display a lower level of p-HSP27 (downstream of p38). The p38 inhibitors induce a higher level of p-p38. This is due to negative feedback in the p-p38 pathway, and the fact that p38 inhibitors prevent p-p38 from phosphorylating downstream substrates, but do not block phosphorylation of p38 itself by upstream regulators. ( B, C ) Cells treated with JNK or MEK I/II inhibitor inactivate the corresponding pathway under Anisomycin induction. The influence of the inhibitor is not obvious under control condition probably due to low basal activation of the pathways.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Blocking Assay, Phospho-proteomics, Control, Activation Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Measurements collected in the same experiment as . ( A ) Scatterplot comparing cells of negative control (DMSO) with cells under p38 inhibition (treated with indicated inhibitor and concentration). Each data point was measured from a cell population with a minimum of 7000 cells. The rapamycin concentration range is as follows: 0, 0.03, 0.1, 0.3, 3 and 30 nM. The results are representative of three independent experiments. ( B ) The slope between size and proportion of cells in G1 is either disturbed or weakened. p-Values were calculated with one-tailed Student t -test (H 0 : slope of control >= slope of compound treatment). The meta data and source code used for the analysis and visualization of the results is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Negative Control, Inhibition, Concentration Assay, One-tailed Test, Control

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: The p38 inhibitors and three higher concentrations shown here are also included in and . ( A ) Cells treated with only rapamycin concentration series (blue) display negative correlation between cell size and proportion of cells in G1. However, the negative correlation between cell size and proportion of cells in G1 disappears or weakens when cells are co-treated with p38 inhibitors and the rapamycin concentration series (red). The blue and red lines show the result of linear regression. The rapamycin concentration range is as follows: 0, 0.03, 0.1, 0.3, 3 and 30 nM. ( B ) The fitted slope of measurements shown in ( A ) For each compound treatment, its fitted slope is compared with the slope of the control (DMSO) from the same experiment. Significance was calculated with one-tailed Student t -test (H 0 : slope drug <= slope control ). The meta data and source code used for the analysis and visualization of the results is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Control, One-tailed Test

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A–C ) Scatterplots displaying relationship between nuclear size at birth with cell cycle duration for individual cells in DMSO control, p38 inhibition and mTOR inhibition. The points with error bar shows mean and SEM by binning cells with similar size. The dashed line shows the result of linear regression with the binned data. Measurements were obtained from time-lapse experiments as indicated in . (D) Bar plot comparing the slopes of the linear regression between size and G1 duration as shown in panel A–C. Error bar indicates 95% confidence bounds. The meta data and source code used to in this analysis is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Cells were transfected with siRNA as indicated and subsequently assayed with a rapamycin concentration series (0, 0.03, 0.1, 0.3, 3 and 30 nM) as described in to assay the correlation of size and G1 length. Larger circle size indicates higher concentrations of rapamycin. ( A ) Knocking down p38α/βpartially weakens the negative correlation between cell size and proportion of cells in G1, while knockdown of p38γ/δ drastically disturbs the correlation. ( C ) The negative correlation between cell size and proportion of cells in G1 is disturbed when cells are transfected with siRNA against MKK3/4/6 but not MKK7. Each data point in is measured on an unsynchronized population with a minimum of 3000 cells. The results shown in are representative of two and three independent experiments with duplicates or triplicates. ( B, D ) Fitted slopes of the trends shown in . Error bars indicate 90% confidence intervals. Analysis is performed with the same method as indicated in . The meta data and source code to analyze and visualize the genetic knock down results is presented in . 10.7554/eLife.26947.027 Figure 4—source data 1. Binding activity (Kd’s in nM) of the p38 inhibitors used in the study against each of the p38 isoforms. Kd values in the table were extracted from ). As marked in that paper, blank fields indicate combinations that were tested, but for which binding was weak (Kd >10 μM), or not detected in a 10 μM primary screen. 10.7554/eLife.26947.028 Figure 4—source data 2. Measurements of cell size and cell cycle stage from the knockdown experiments as shown in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Transfection, Concentration Assay, Knockdown, Binding Assay, Activity Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Western-blot of cell lysates from conditions shown in confirms efficiency of knockdown of MKKs ( A ) or p38 isoforms ( B ).

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Western Blot, Knockdown

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: The bar plot and error bar display mean and SEM across three replicate Western-blot experiments. Treatment of rapamycin or Torin-2 increases both p-p38 and p-CREB, confirming that activity in the p38 pathway is upregulated under mTORC1 inhibition. SB203580, a p38 inhibitor, significantly reduces the phosphorylation of CREB (downstream of p38), confirming that p38 activity is inhibited. p27 is a negative regulator of G1 progression. mTOR inhibition by rapamycin or Torin-2 upregulates p27 activity, which promotes a longer G1. Strikingly, cells co-treated with p38 inhibitor and mTOR inhibitor have lower p27 compared to treatment of p38 inhibitor alone. This may be the mechanism by which p38 inhibition disturbs the cells’ ability to compensate their small size with longer G1.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Western Blot, Activity Assay, Inhibition, Phospho-proteomics

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Cells were treated with either 50 nM of Torin-2 or DMSO (control) for 20 hr, followed by drug wash-out and media replacement. Cells undergoing mTOR inhibition, on average, decrease in size and slow their proliferation rate. Following release from mTOR inhibition, cells grow but maintain a low proliferation rate until their normal size is reached. Cells resume a wild type rate of proliferation only when their size reaches the size of the untreated population. ( B ) Western blots of whole cell lysates collected at time points ranging from 0 to 20 hr post release from mTOR inhibition. Levels of mTOR pathway activity recover within 1 hr after Torin-2 wash-out. By contrast, activity of p38 remains upregulated in the Torin-treated cells compared with controls, and gradually fades away only as cells recover their wild-type size. ( C ) Cells simultaneously expressing reporters of both p38 MAPK and JNK were treated with a series of rapamycin concentrations, as in . Each data point (circle) corresponds to the average G1 cell size and the average level of MAPK activity (JNK and p38) that corresponds to a given concentration of rapamycin. As positive controls, we include populations that were co-treated with the p38 inhibitor, SB203580 (orange circles). Higher concentrations of rapamycin (bigger circle size) result in smaller cells with higher activity of p38 (top left panel). Unlike p38, activity of JNK was not upregulated in proportion to cell size (left bottom panel). Also shown are the correlations of MAPK activity (JNK and p38) and cell size at 6 hr post release from mTOR inhibition (right panels) (also see ). Each data point represents average values of cell size and MAPK activity of the G1 cells subpopulation from an unsynchronized population with a minimum of 3000 cells. Results shown here are representative of three independent experiments. The meta data and source code used to analyze and visualize the correlation between cell size and KTR readout is presented in . ( D ) Western-blots of whole cell lysates from samples collected at 0 or 6 hr post release from a 22 hr treatment with either 50 nM Torin-2, 1 μM cycloheximide or DMSO (control). ( E ) Western-blots of whole cell lysates from samples collected at 0 or 6 hr post release from a 30-min treatment with either 25 ng/mL anisomycin, hyperosmotic shocks (NaCl and Sucrose) or DMSO (control). 10.7554/eLife.26947.035 Figure 5—source data 1. Measurements of cell size and p38 KTR as shown in and .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Inhibition, Western Blot, Activity Assay, Expressing, Concentration Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: The p38 KTR functions by translocating to the cytoplasm once p38 is activated. Cells were imaged after a 1 day treatment with DMSO (control), 3 nM rapamycin or 5 μM SB203580 (p38 inhibitor), or a 30 min treatment with 25 ng/mL Anisomycin (a stimulator of the p38 pathway). Anisomycin treatment results in reduced fluorescence in the nucleus, and a less defined nuclear boundary as compared to control. By contrast, treatment with SB203580 results in elevated fluorescence in the nucleus, implying a lower p38 activity. Cells subject to rapamycin treatment are smaller in size as compared to control. While these cells display a spectrum of cytoplasmic-to-nuclear localization, rapamycin treatment increases the fraction of cells that display p38 activity, as indicated by the blurry nuclear boundary and increased cytoplasmic localization of the KTR (highlighted by arrows).

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Fluorescence, Activity Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Cells treated with either DMSO (control), 30 nM rapamycin or 1 μM cycloheximide for 1 day, or with 25 ng/mL Anisomycin (a stimulator of the p38 pathway) for 30 min were fixed and imaged. While Anisomycin treatment leads to hyperactivation of the p38 pathway, rapamycin or cycloheximide treatment result in a weak but visible elevation in p-p38 level in the nucleus (also see ).

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Measurements were obtained from the same experiment as indicated in . Cells express dual reporters of both p38 MAPK and JNK were treated with a concentration series of rapamycin, decreasing cell size to varying extents. For each condition/replicate, cells were partitioned into G1, S and G2 stage according to the cellular DNA readout (see Materials and methods – Cell cycle stages). The activity of p38 MAPK negatively correlates with cell size after mTOR inhibition for cells that are in G1. Cells that are in S and G2 also display a negative correlation, but with a lower correlation coefficient. Interestingly, after cells are released from mTOR inhibition, p38 activity negatively correlates with cell size only among cells that are in G1, but not S or G2. This result supports the hypothesis that cell-size-dependent regulation of p38 activity is exclusive to the G1 phase of cell cycle, which is consistent with its assumed role in regulating G1 duration. The meta data and source code used to analyze and visualize the correlation between cell size and KTR readout among different cell cycle stages is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Activity Assay, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Workflow of the experiment. Cells were treated with 50 nM Torin-2 with or without the indicated MAPK inhibitors for 22 hr, and then released from Torin-2 while still being subject to the indicated MAPK inhibitors (red) or DMSO (blue). MAPK inhibitors were administrated at a concentration consistent with the highest corresponding concentration used in and . At 0, 6, 24, 30 and 48 hr post release from Torin-2 treatment, samples were measured for both average cell size (B) and cell count (C). ( B ) Cells treated with DMSO (control) recovered in size within 24 hr and remained at a constant average size thereafter. Cells treated with p38 inhibitors, but not ERK or JNK inhibitors, failed to recover their size, even 48 hr post Torin-2 wash-out. This suggests that p38 inhibitors suppressed the recovery in cell size rather than slowing the kinetics associated with this process. ( C ) Cells treated with inhibitors of p38, but not inhibitors of ERK or JNK, show increased rates of proliferation as compared to control conditions, after being released from mTOR inhibition. Results shown in this figure are representative of two replicate experiments. The measurements and source code for visualization of the results is presented in . 10.7554/eLife.26947.038 Figure 6—source data 1. Cell size dynamics after released from mTOR inhibition.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Cell Counting, Control, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Workflow of the experiment. Similarly as indicated in , Cells were co-treated with both Torin-2 (50 nM) with or without the indicated MAPK inhibitor for 22 hr. The cells were then released from both inhibitors and grown in regular medium. At 0, 6, 24, 30, and 48 hr post release from the inhibitors, the cells were measured for both cell size ( B ) and cell number ( C ) by Coulter counter. ( B ) Cells in ‘DMSO’ condition recovered in size within 24 hr and stays at this size. Cells with a history of p38 inhibition, but not Erk or JNK inhibition, displayed a delayed size-recovery dynamics. ( C ) Cell proliferation were followed after release from the inhibitors. The results shown in this figure are representative of two replicate experiments. The measurements and source code used to visualize the results shown in this figure is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Inhibition

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer, pSi-shUSP22-1 or pSi-shUSP22-2 plasmid. ( A ) Western blot analysis of USP22 expression; β-actin was used as endogenous control. ( B ) Cell viability was measured using MTT assay at 1, 2, 3, and 4 d after transfection. ( C ) EdU staining used to examine the proliferation. Scale bar: 5 μm. ( D ) Percentage of EdU-positive staining in (C). ( E ) Effect of USP22 knockdown on cell proliferation evaluated by colony formation assay. After 21 d of transfection, cells were stained with Giemsa solution. ( F ) Histograms representing the colony formation number in (E). ( G ) Western blot conducted to analyze the expression of USP22 and proliferation-related proteins including Rb, p-Rb, Akt, p-Akt, and cyclin D2. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B, D, and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, MTT Assay, Staining, Knockdown, Colony Assay

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A and B ) After 48 h of transfection, the scratch wound-healing assay was performed to evaluate the effect of USP22 on cell migration. The representative images of cell migration were shown in (A), and the width of wounds at the indicated times was shown in (B). ( C ) Invasion of CAL-62 and 8505C cells analyzed by transwell assay. The invaded cells were stained with DAPI. Scale bar: 5 μm. ( D ) Number of DAPI-positive cells per field counted under a fluorescent microscopy. ( E ) Immunofluorescence staining was performed to analyze the effect of USP22 on the expression of E-cadherin and vimentin in 8505C cells. Scale bar: 5 μm. ( F and G ) Expression of invasion-related proteins containing BMI-1, E-cadherin, vimentin, and snail in ATC cells was measured by qPCR (F) and western blot (G). GAPDH and β-actin were used as endogenous controls, respectively. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with the group at 0 h after transfection (B). ## P < 0.01, as compared with mock or pSilencer group at 24 h after transfection (B). * P < 0.05, as compared with mock or pSilencer group (D and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Migration, Transwell Assay, Staining, Microscopy, Immunofluorescence, Expressing, Western Blot

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A ) Apoptosis of 8505C cells analyzed by flow cytometry. ( B ) Apoptosis rate in (A) was calculated. ( C ) Fluorescent TUNEL assay conducted to determine the apoptosis of 8505C cells. Scale: 10 μm. ( D ) Percentage of TUNEL-positive cells in (C). ( E ) CAL-62 and 8505C cell apoptosis evaluated by nucleosomal fragmentation assay. ( F ) Quantification of caspase-3 activity in CAL-62 and 8505C cells. ( G ) Western blot analyses of apoptosis-related protein (Bid, Bax, cl-caspase-3, caspase-3, and Bcl-2) expressions in CAL-62 and 8505C cells. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B and D–F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, TUNEL Assay, Activity Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: Female six-week-old SCID mice were inoculated subcutaneously into right hind flanks or injected via tail vein with stably expressed pLKO.1 or pLKO.1-shUSP22 8505C-luc cells. Mock-treatment was used as control. ( A ) Tumor growth progression was measured by in vivo luciferase imaging of the xenografts at days 1, 5, 9, and 12 d after inoculation. ( B ) Representative gross photos of tumors 22 d after subcutaneous xenografting ( n = 6). ( C ) Tumor volumes of subcutaneous implantation models of ATC were monitored and calculated after 0, 7, 11, 13, 17, 20, and 22 d of inoculation. D–F. After 28 d of injection through tail vein, the lungs were removed and photographed ( D ), the number of metastatic nodules in lungs was counted ( E ), and the weight of lungs was measured ( F ). ( G ) Fluorescence TUNEL assay was carried out to determine cell apoptosis in the same tumor tissues as indicated above. The rate of TUNEL-positive cells was calculated. Scale bar: 10 μm. ( H ) qPCR assay was performed to detect the mRNA expression of USP22 in tumor tissues from (B). ( I ) Protein expression of USP22 in tumor tissues was analyzed by western blot. ( J ) Representative results of western blot analyses of cyclin D2, Akt, p-Akt, E-cadherin, vimentin, Bax, Bcl-2, cl-caspase-3, and caspase-3 in tumor tissues. (I and J) β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pLKO.1 group (C and E–H).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Injection, Stable Transfection, Control, In Vivo, Luciferase, Imaging, Fluorescence, TUNEL Assay, Expressing, Western Blot

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Cytokinetic phenotype in Kif5b knockdown ATDC5 cells. a Western blot of protein extracts from single cell clones with stable expression of sh-ctl or sh-kif5b constructs. b Immunofluorescence of KIF5B (green) in sh-Kif5b clone #4. Scale bar: 10 μm. c sh-ctl and sh-Kif5b cells stained with α-tubulin (red) and DAPI (blue). Yellow asterisks denote typical binucleated cells. Scale bar: 20 μm. d Quantification of the bi- and multi-nucleation rate in control cell clones ( n = 3, sh-ctl clone #1–3) and Kif5b-knockdown cell clones ( n = 5, sh-Kif5b clone #4–8). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Re-introduced GFP tagged full length KIF5B reduced the bi- and multi-nucleation rate in Kif5b knockdown ATDC5 cells clone #4 ( n = 10 independent experiments) and #5 ( n = 5 independent experiments). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. f Time-lapse images of sh-ctl and sh-Kif5b cells in mitosis. Scale bar: 10 μm. g Quantification of duration of cytokinesis in sh-ctl cells ( n = 84 cells from sh-ctl clone #1–3) and sh-Kif5b cells ( n = 68 cells from sh-Kif5b clone #4, #5 and #8). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 5% to 95% percentile (whiskers). Duration of cytokinesis was calculated from the furrow ingression to the final separation of the two daughter cells. Cells fusing back were not included in this analysis

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Knockdown, Western Blot, Clone Assay, Expressing, Construct, Immunofluorescence, Staining, Control, Two Tailed Test, MANN-WHITNEY, Whisker Assay

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Midbody microtubule organization is impaired in Kif5b deficient cells. a Immunofluorescence of α-tubulin in primary chondrocytes in late cytokinesis. Scale bar: 10 μm. b Quantification of tubulin intensity in midbody in primary chondrocytes ( Kif5b fl/ + cells: n = 24; Col2cre ; Kif5b fl/ − cells: n = 27). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 10% to 90% percentile (whiskers). c Electron micrographs of midbody regions from sh-ctl and sh-Kif5b ATDC5 cells. Red lines denote the region of Flemming body. Yellow arrows and boxes denote the broken regions in Flemming body. Scale bar: 0.5 μm. d Quantification of the length of Flemming body in sh-ctl and sh-Kif5b cells (sh-ctl cells: n = 12; sh-Kif5b cells: n = 9). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Live imaging of sh-ctl and sh-Kif5b cells expressing GFP-tubulin in cytokinesis. Scale bar: 10 μm. f Quantification of the bi- and multi-nucleation rate in sh-Kif5b cells transiently expressing GFP, GFP-Kif5b and GFP-Kif5bΔMT ( n = 5 independent experiments). *** P < 0.0001; NS, P = 0.4039; unpaired two-tailed t-test. Data are mean ± S.D. g A model showing the function of KIF5B in cytokinesis. In late cytokinesis, KIF5B alone or together with other unknown molecules cross-links microtubules in the midbody, and therefore the structure of midbody can be stably maintained

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Imaging, Expressing, Stable Transfection

Journal: PLOS Pathogens

Article Title: Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion

doi: 10.1371/journal.ppat.1011203

Figure Lengend Snippet: (A) Schematic of the timeline of Single-Molecule DNA Fiber Assays during MVM infection. Mouse A9 fibroblasts are synchronized in Isoleucine deficient media for 42 hours before being released into complete DMEM and concurrently infected with MVMp at an MOI of 25 or treated with hydroxyurea (HU) at a concentration of 2μM. At the indicated timepoints post-infection (12, 18, and 24 hours post infection), cells were sequentially pulsed with IdU and CldU and processed for DFA. (B, C) Each datapoint represents the length of a single IdU and/or CldU labelled DNA fiber. The median length of many measurements of IdU and CldU labelled fibers are represented by red and green horizontal bars respectively. The experiment was performed as described by the schematic depicted in (A). At least 150 individual fibers were measured for each condition. Similar results were obtained for three independent biological replicates of MVM infection. Statistical significance was determined by Mann Whitney Wilcoxon test, **** represents P ≤ 0.0001, ns represents not statistically significant. (D) Representative DFA images of single fibers in Mock infected A9 cells (top panel) compared with MVM infected A9 cells at 24 hpi (bottom panel) with the respective measurements of the IdU and CldU lengths indicated in white text. (E) Western blot showing the levels of NS1 and γH2AX for viral replication and DNA damage, respectively, over the time-course of MVM infection. The respective band intensities are shown below the sample. The top row of γH2AX is taken with a 10 minute exposure (short) and the bottom with a 20 minute exposure (long). Cells pulsed with Hydroxyurea at 2μM concentration is a positive control for DNA damage, as has been previously described . Tubulin serves as the loading control for the Western blots. (F) Categorization of DNA fiber types as percentages out of a total of 100% as determined by presence of IdU or CldU, divided into percentages that are progressing replication forks (black), stalled replication forks (red) and new origins (green with black stripes). (G,H) Non-synchronous U2OS cells were infected with wild-type MVMp or the NS2-deficient (MVM ΔNS2 ) mutant of MVM at an MOI of 25 for 24 hours before being pulsed with IdU/CldU as described in the schematic in (A), and processed for imaging. At least 150 individual fibers were measured for each condition. Similar results were obtained for three independent biological replicates of MVM infection. Statistical significance was determined by Mann Whitney Wilcoxon test, **** represents P ≤ 0.0001.

Article Snippet: Male murine A9 fibroblasts and female human U2OS osteosarcoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Gibco) supplemented with 5% Serum Plus (Sigma Aldrich) and 50 μg/ml gentamicin (Gibco).

Techniques: Infection, Concentration Assay, MANN-WHITNEY, Western Blot, Positive Control, Mutagenesis, Imaging

Journal: PLOS Pathogens

Article Title: Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion

doi: 10.1371/journal.ppat.1011203

Figure Lengend Snippet: (A) Schematic of the Single-Molecule DNA Fiber Assays using the CDC7 inhibitor PHA 767491 (Millipore Sigma). Mouse A9 fibroblasts were synchronized in Isoleucine deficient media for 42 hours before being released into complete DMEM media and concurrently infected with MVMp at an MOI of 25. At the indicated 20 hpi timepoint, 5μM of iCDC7 was added to the cells for 4 hours. At 24 hpi, MVM infected iCDC7 pulsed cells were sequentially pulsed with IdU and CldU for 20 minutes each before being processed for DFA analysis. (B, C) Each datapoint represents the length of a single IdU and/or CldU labelled DNA fiber. The median length of many measurements of IdU and CldU labelled fibers are represented by red and green horizontal bars respectively. The experiment was performed as described by the schematic depicted in (A). At least 150 individual fibers were measured for each condition. Similar results were obtained for three independent biological replicates of MVM infection. Statistical significance was determined by Mann Whitney Wilcoxon test, **** represents P ≤ 0.0001. (D) Categorization of DNA fiber types as percentages out of a total of 100% as determined by presence of IdU or CldU, divided into percentages that are progressing replication forks (black), stalled replication forks (red) and new origins (green with black stripes). (E) Western blot showing the levels of NS1 and γH2AX for viral replication and cellular DNA damage, respectively, upon pulsing with iCDC7 as shown in the schematic in (A) prior to MVM infection. Activity of the CDC7 inhibitor activity was verified by phospho-MCM2 levels and Tubulin was served as loading control for the western blots. The respective band intensities are shown below the sample. (F) Samples from lanes 3 and 4 in panel (E) were processed for formation of viral replication centers by NS1 staining (red) and phospho MCM2 levels (green) measured for CDC7 inhibitor activity. Cells infected with MVM at 24 hpi under mock-treated conditional (top panel) were compared to those pulsed with iCDC7 (bottom panel) by NS1 staining (red). White broken line demarcates the nuclear border, which was identified by DAPI staining (blue). The white scalebar represents 10 microns.

Article Snippet: Male murine A9 fibroblasts and female human U2OS osteosarcoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Gibco) supplemented with 5% Serum Plus (Sigma Aldrich) and 50 μg/ml gentamicin (Gibco).

Techniques: Infection, MANN-WHITNEY, Western Blot, Activity Assay, Staining