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: Acta Neuropathologica Communications

Article Title: MRC1 and LYVE1 expressing macrophages in vascular beds of GNAQ p.R183Q driven capillary malformations in Sturge Weber syndrome

doi: 10.1186/s40478-024-01757-4

Figure Lengend Snippet: EC-R183Q promote significant THP1 cell adhesion under static and laminar flow-induced condition. a Fluorescence-labeled THP1 cells were incubated with EC-WT and EC-R183Q under static conditions (N = 10). Adherent cells were quantified after 1 h. P -value was calculated by two-tailed t -test. Phase-contrast images of EC-WT (top) or EC-R183Q (bottom) incubated with THP1 cells (green) at 1 h incubation. Scale bar = 50 µm. b Schematic of live-cell imaging set up. A flow rate of 0.5 ml/min was setup using a tabletop syringe pump with a 20 ml syringe Luer-Lock tip. After 5 min of recording, a switch system was used to deliver pre-stained THP1 cells under continuous uninterrupted flow for 30 min. c Time-lapse imaging of THP1 cells (yellow) adhesion to EC-WT (top), and EC-R183Q (bottom) under laminar flow. Images are at time point = 0, 10, 20, and 30 min. Scale bar = 200 µm. N = 6 independent experiments were performed. d Quantification of THP1 cell adhesion under flow over 10, 20, and 30 min. Mann Whitney test was performed to calculate p-value at each time point. e Proteome profiler cytokine array on conditioned media from EC-WT (top) and EC-R183Q (bottom) after incubation in 2% fetal bovine serum EBM2 media for 24 h. Altered protein levels between EC-WT and EC-R183Q are boxed. Protein levels were quantified by measuring dot intensity using FIJI (right). Three independent experiments were performed. f Intercellular adhesion molecule 1 (ICAM1, grey), UEAI (red), and nuclei counterstaining for DAPI (blue) in the SWS brain sections (n = 4). Scale bar = 50 µm. g Time-lapse imaging of THP1 cell (yellow) adhesion to EC-R183Q treated with IgG2A isotype control (top), and EC-R183Q treated with anti-ICAM1 antibody (bottom) under laminar flow. Images are at time point = 0, 10, 20, and 30 min. Scale bar = 200 µm. N = 5 independent experiments were performed. h Quantification of single cell tracking of THP1 cells under flow over 10, 20, and 30 min. Mann Whitney test was performed to calculate p-value at each time point

Article Snippet: The cytokine array was performed on conditioned media using Proteome Profiler human cytokine array (Cat# ARY005B, R&D Systems) in accordance with the manufacturer protocol.

Techniques: Fluorescence, Labeling, Incubation, Two Tailed Test, Live Cell Imaging, Staining, Imaging, MANN-WHITNEY, Control, Single Cell Tracking

Journal: Bioinformatics advances

Article Title: Different approaches to Imaging Mass Cytometry data analysis.

doi: 10.1093/bioadv/vbad046

Figure Lengend Snippet: Fig. 2. A schematic overview of the Imaging Mass Cytometry workflow (created with BioRender.com)

Article Snippet: IMC was developed in 2014 based on earlier available suspensionbased, single-cell mass cytometry [cytometry time of flight (CyTOF)] technology [described in Bandura et al. (2009)] but combined with an additional platform for UV ablation (Hyperion Tissue Imager, Standard BioTools, South San Francisco, USA) and performed on stained tissue sections, giving the spatial resolution of the data (Fig. 1A) (Bandura et al., 2009).

Techniques: Imaging, Mass Cytometry

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Schematic representation and immunostaining images of blastocysts and ICMs at stages E3.5 and E4.5. GATA6 and GATA4 are used as markers for PrE fate (green), and NANOG and SOX2 are used as markers for EPI fate (magenta) at stages E3.5 and E4.5, respectively. B. Quantification of total cell numbers in the ICM from blastocysts and isolated ICMs at stage E3.5, blastocysts and isolated ICMs at stage E4.5, and isolated ICMs cultured in vitro for 24 hours from stage E3.5 to E4.5. n=33, 30, 40, 21, 31 embryos for the different groups, respectively. Independent samples t-test between E3.5 blastocysts and E3.5 ICMs, p =0.106. One-way ANOVA between E4.5 Blastocysts, E4.5 ICMs, and E3.5 ICMs+24hr, p =0.145. C. Representative time-lapse imaging of ICMs isolated from E3.5 blastocysts expressing PrE-specific H2B-GFP ( Pdgfrα H2B-GFP , green) and ubiquitous H2B-mCherry ( R26-H2B mCherry , magenta), out of total 8 datasets from 3 independent experiments. Time is indicated in hh:mm, t=00:00 corresponds to start of live-imaging at stage E3.5+3hours, following completion of immunosurgery. D. Schematic representation of single-cell tracking of EPI and PrE cells from isolated ICMs from (C). Line plots indicating radial distances of all cells from one representative ICM until E4.0 stage. Colour of the line indicates cell fate – PrE, green and EPI, magenta. Shaded regions show spatial dispersion as mean ±SD of cell position along ICM radial axis. The geometric centroid of the ICM is considered as d=0.0 and ICM outer surface is considered as d=1.0 to normalise cell position across samples. Time-series plots for cell position were smoothed using a rolling average. E. Quantification of sorting score for isolated ICMs between stage E3.5 and E4.0. Data from n=8 ICMs. F. Line plots for radial cell position versus time from tracking of PrE and EPI cell movements in isolated ICMs. Time-series plots for cell position were smoothed using a rolling average. Cell tracking data pooled from n=160 PrE cells and n=133 EPI cells from 8 ICMs. G. Schematic diagram for analysis of PrE and EPI cell movements. Cell displacement is measured along the radial axis between consecutive timepoints and classified as inward or outward movement depending on the direction of displacement. H. Polar plots indicating preferential direction of cell movements among PrE and EPI. Cell position is plotted along radial axis, time is plotted along angular axis. Measurements are binned according to initial radial cell position and time. The mean displacement of each interval is plotted, colour indicates direction of movement. Scale bars 20μm. ns, non-significant

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunostaining, Isolation, Cell Culture, In Vitro, Imaging, Expressing, Single Cell Tracking, Dispersion, Cell Tracking Assay

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing laminin distribution around PrE cells. White dotted line, ICM-cavity interface. White arrowhead marks GATA6-expressing nucleus of a PrE cell enriched for laminin expression. B. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing PKCλ+ζ distribution in PrE cells. White arrowhead marks leading edge of a PrE cell with PKCλ+ζ localisation. C. Immunofluorescence image of an E3.75 ICM showing PKCλ+ζ localisation in PrE and EPI cells. White dotted lines mark cell boundaries. Yellow line indicates the line segments from cell inner edge (towards ICM centroid) to cell outer edge (towards ICM-fluid interface) along which fluorescence intensity is measured. D. Line plots for normalised fluorescence intensity of PKCλ+ζ in individual inside cells from E3.75 isolated ICMs. Colour of the line indicates GATA6-expression level of the cell. n=260 cells from 32 ICMs. Each of the thin lines corresponds to measurement from one cell. Bold line and shaded region indicate mean±SD of aPKC intensity for GATA6-high and GATA6-low cells. E. Schematic description of polarisation index. Polarisation index is calculated as the ratio between mean aPKC intensity at 1/4 th distance from outer edge and mean aPKC intensity at 1/4 th distance from inner edge. Boxplots for comparison of the polarisation index in PrE (GATA6 high) versus EPI cells (GATA6 low). GATA6 expression level is categorised as high or low by thresholding the bimodal distribution of GATA6 fluorescence intensity. Colour of the line indicates GATA6-expression level of the cell. n=136 GATA6-high and 124 GATA6-low cells from 32 ICMs. One-way ANOVA, p =6.03e -20 . F. Scatterplot of polarisation index of cells versus radial distance of the cell from the ICM centroid. Colour of the datapoint indicates GATA6-expression level of the cell. Black dotted line, linear regression with Pearson’s R=0.079, p =0.205. n=260 cells from 32 ICMs. G. Immunofluorescence images of control and Gö6983-treated E4.0 isolated ICMs and quantification of sorting score. n=16,24 ICMs for control and Gö6983-treated ICMs respectively. Independent samples t-test, p = 8.01e -04 H. Immunofluorescence images of representative WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- E4.5 blastocysts and quantification of number of ectopic PrE cells in E4.5 blastocysts from each group. n= 25, 17, 12 blastocysts for WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- respectively. Mann-Whitney U test, p = 2.43e -04 , 6.36e -04 Scale bars 20μm. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunofluorescence, Expressing, Fluorescence, Isolation, Comparison, Control, MANN-WHITNEY

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Distribution of CD44 + cells within intestinal epithelium. a , b Immunohistochemical (IHC) staining for Lgr5 + ISCs ( black dotted lines ) in vivo. c , d IHC staining for CD44 + cells ( black dotted lines ) in vivo. a , c Magnification ×400. Bars 50 μm. b , d Magnification ×1000. Bars 20 μm. e–n Immunocytochemical (ICC) staining for CD44 + cells in vitro. e , j Differential interference contrast (DIC) imaging. f , k Propidium iodide (PI) staining for nuclei. g , l Fluorescein isothiocyanate (FITC) for CD44 + cells ( white arrowheads crypt cells strongly positive for CD44). h , m Overlay of PI image and FITC image. i , n Overlay of FITC image and DIC image. e–i Magnification ×200. Bars 200 μm. j–n Magnification ×630. Bars 100 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Immunohistochemical staining, Immunohistochemistry, In Vivo, Staining, In Vitro, Imaging

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Identification of CD44 + cells. a–a’’’’’ Fluorescence-activated cell sorting (FACS) analysis for cellular phenotype. a Isotype control, IgG2a-phycoerythrin (PE). a’ CD31-PE. a’’ CD34-PE. a’’’ Isotype control, IgG2b-allophycocyanin (APC). a’’’’ CD44-APC ( lo low-positive for CD44, hi high-positive for CD44). a’’’’’ CD45-APC. b Development of CD44 + ISC in 3D-culture system. Numbers represent days. Top Magnification ×400. Bar 50 μm. Bottom Magnification ×200. Bars 100 μm. c–c’’ Colony-forming efficacies of CD44 - cells and CD44 + cells. c CD44 - cells in 3D-culture system for 14 days. c’ CD44 + cells in 3D-culture system for 14 days. c , c’ Magnification ×40. Bars 500 μm. c’’ Comparision of colony-forming efficacy per 100 sorted cells seeded in one well of a 96-well plate. CD44 - group in 48 wells; CD44 + group in 48 wells. Data represent means ± SD of 48 independent measurements ( n = 48). Bars SD values. Paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 + group versus CD44 - group). All experimental procedures were repeated twice. d–d’’’’ Transmission electron microscope imaging of CD44 + ISC differentiation. CD44 + ISC were cultured in the 3D-system for 6 days and formed a cystic structure. d Cystic structure of a single CD44 + ISC-derived organoid at 6 days. Boxed areas are shown at higher magnification in d’–d’’’’ ( Lu lumen). Magnification ×400. Bar 50 μm. d’ Absorptive cell ( Ab ). d’’ Endocrine cell ( En ). d’’’ Goblet cell ( Go ). d’’’’ Paneth cell ( Pa ). Black arrowhead in d’ indicates brush border. Black arrowheads in d’’–d’’’’ indicates granules. d’–d’’’’ Magnification ×1500. Bars 5 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Fluorescence, FACS, Control, Transmission Assay, Microscopy, Imaging, Cell Culture, Derivative Assay

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: CD44 + ISCs resemble CBC stem cells. a–a’’ Strategy for sorting CD44 - , CD44 low+ and CD44 hi+ subpopulations by using the FACS technique ( R1 determining the cell-zone, R2 determining viable cells, PI propidium iodide, APC allophycocyanin). b Semi-quantitative reverse transcription (RT) followed by the polymerase chain reaction (PCR) for ISC-related gene-expression in sorted cells. Fold expression values were normalized to the CD44 - group. Data represent means ± SD of six independent measurements ( n = 6). Bars indicate value of SD. The paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 hi+ group versus CD44 low+ group); $ P ≤ 0.05 represents low significance (CD44 hi+ group versus CD44 low+ group); ns represents no statistic differences between the CD44 hi+ group and CD44 low+ group. P -values for Lgr5 , Bmi1 , Hopx , mTERT , Ascl2 , Smoc2 , Lrig1 , Rnf43 and Prominin-1 are respectively 0.016, 0.036, 0.010, 0.844, 0.007, 0.001, 0.041, 0.005 and 0.859. c , c’ Colony-forming efficacy of CD44 low+ and CD44 hi+ cells in 3D-culture system for 14 days. c CD44 low+ group. c’ CD44 hi+ group. Magnification ×40. Bars 500 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Expressing

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Epithelial homeostasis in CD44 + ISC-derived organoid. a , b TUNEL staining for apoptotic cells in normal epithelium. c , d IHC staining of Ki67 for proliferative cells within normal epithelium. a , c Magnification ×200. Bars 100 μm. b , d Magnification ×400. Bars 50 μm. e–j Epithelial homeostasis in vitro. e–g TUNEL staining for apoptotic cells in CD44 + ISC-derived organoid. e DAPI staining ( blue ) for nuclei. f dUTP-FITC ( green ) for apoptotic cells. g DAPI image merged with dUTP-FITC image. Magnification ×100. Bars 200 μm. h–j ICC staining for proliferative cells in CD44 + ISC-derived organoid. h DAPI staining for nuclei. i Ki67-FITC for proliferative cells. j DAPI image merged with Ki67-FITC image. Magnification ×200. Bar 100 μm. k Representation of an organoid

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, In Vitro

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: Overview of the high-content screening and analysis. (A) Summary of RNAi screening workflow. Reverse transfection of HeLa cells was performed in 384-well format for 72 h, followed by 48-h infection with GFP-expressing B. abortus , PFA fixation, and staining of HeLa cells with DAPI and DY-547−phalloidin before automated imaging. GW, genomewide. (B) Image analysis was performed with CellProfiler to segment nuclei and bacteria and to extract measurements. (C) Accurate association of segmented bacteria to nuclei enables quantitative single-cell measurements. The naive association (middle image) of segmented pathogen can be affected by oversplitting in dense cell populations (left image). Our proposed solution (right image) based on a nucleus attraction score. (D) The plate histogram shows the bimodal distribution of integrated GFP intensity corresponding to Brucella replication. Intensity on the x axes is log 2 scaled to account for exponential growth. The normal distribution fitted (red curve) to the kernel density estimation of the histogram allows us to compute a robust binary infection threshold (dashed line) separating HeLa cells with (right) and without (left) replicating Brucella . Associated are samples of single-cell images corresponding to the intervals of the intensity distribution (for more details, see Materials and Methods).

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: High Content Screening, Transfection, Infection, Expressing, Staining, Imaging, Bacteria, Quantitative Single Cell

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: Entry assay identifies new components required for postentry processes during Brucella infection. (A) Representative images from the entry assay showing nuclei (DAPI) of HeLa cells and intracellular Brucella abortus (GFP) for control condition (mock) and cells treated with siRNAs against CDC42 or VPS35. HeLa cells were infected with B. abortus expressing GFP under a tetracycline-inducible system for 8 h (see Materials and Methods). Bars = 100 μm. (B) Scatter plot in double logarithmic scale showing infection scores measured for the entry assay (8 hpi) versus endpoint assay (48 hpi), normalized to the respective mock data set ( Table S3 ). For the entry assay, cells containing single bacteria were considered infected, and the final readout is proportional to the median number of bacteria per infected cells. For the endpoint assay, only cells containing replicating bacteria were considered infected ( Fig. 1 and Materials and Methods). Each data point corresponds to the average of all siRNAs or esiRNAs targeted against the gene of interest ( n = 3). The straight fit (oblique line, r 2 = 0.763) indicates a globally high correlation between both assays. The blue region shows all points within 1 standard deviation (SD) of the fitted data. The genes falling out of this range are marked in red. For ease of visualization, only the averaged values over all RNAi products targeting a given gene are displayed.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: Infection, Control, Expressing, End Point Assay, Bacteria, Standard Deviation

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: The VPS retromer is a key component of Brucella intracellular trafficking. (A) Schematic representation of the retromer components and their prominent interactors. (B) Schematic representation of the shRNA constructs used in panel C. The gray box on the shVPS35/rescue construct indicates the silent mutations that prevent base-pairing with the coexpressed shRNA . (C) Infection index from transfected cells. Displayed are the averaged infection index and associated standard deviation after 48 h of Brucella infection. Data were normalized to the YFP-only condition ( n = 3). Values that are statistically significantly different from the value for the scrambled YPF-only condition as determined by paired t test are indicated by an asterisk (*, P value of ≤0.01; ns, not significant). (D) Dot box representation of the z-scored infection score for components of the retromer and interactors, including the positive-control ARPC3. Asterisks indicate statistically significant differences from the values for the scrambled siRNA-treated bacteria (control [Ctrl]) as determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple-comparison test (**, P value of ≤0.001; ns, not significant). (E) Normalized CFU recovered from siRNA-treated cells at 6, 20, or 44 hpi. The presented data correspond to CFU count normalized to control, siRNA-treated cells ( n = 3). Significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test (*, P ≤ 0.01; **, P ≤ 0.001; ns, not significant). (F) Western blot analysis of the indicated proteins in total lysate of HeLa cells treated with siRNA targeting the designated genes, 72 h posttransfection. Displayed is a representative example of an experiment performed in biological triplicate ( n = 3). See Table S4 for the matching averaged intensity quantification.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: shRNA, Construct, Infection, Transfection, Standard Deviation, Positive Control, Bacteria, Control, Comparison, Western Blot

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: VPS35 is required for Brucella to escape the lysosomal pathway. (A) Immunofluorescence approach used to quantify localization of Brucella within LAMP-1-positive vesicles, illustrated with a representative example of control-treated cells 6 hpi. Individual channels and merged picture are presented. Arrows indicate examples of colocalization of bacteria with LAMP-1-positive compartments. Staining of Brucella LPS (anti-LPS [αLPS]) was used to confirm the presence of LAMP-1 in direct proximity of the bacterial surface. Asterisks indicate examples of LAMP-1-negative Brucella . (B) Representative images from Brucella infected cells either mock transfected (ctrl) or after VPS35 knockdown (siVPS35). Samples were fixed 6 and 18 hpi. For clarity, only the LAMP-1 (cyan) and dsRed (magenta) channels are presented. Bars, 5 μm. (C) Global quantification of LAMP-1-negative Brucella. Displayed are the average and associated standard deviation for more than 500 bacteria and more than 50 HeLa cells per time point and condition ( n = 3). (D) Single-cell data representation of the data presented in panel C. Displayed is the distribution of LAMP-1-positive Brucella per cell as a function of the total number of bacteria counted in that given cell.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: Immunofluorescence, Control, Bacteria, Staining, Infection, Transfection, Knockdown, Standard Deviation