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: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 1: GFP-Rab17 localization in B16 cells. B16 cells were transiently transfected with GFP-Rab17 for 24 h. Panel D was imaged using the Delta Vision microscope, panel A and B were imaged via confocal microscopy. Brightness and contrast was adjusted for each colour channel individually in photoshop. Scale bars on full-size images represent 10 μm and scale bars on the zoom images represent 5 μm. A) Transfected cells were incubated with mouse Tfn-Alexa-546 (Tfn-546). Left to right: GFP-Rab17 (green), Tfn-536 (red) and an overlay of the coloured images also including DAPI (blue). White arrows indicate peripheral GFP-Rab17 that does not colocalize with Tfn, yellow arrows indicate GFP-Rab17 colocalization with Tfn. B) Transfected cells were stained with the HMB45 Ab. Left to right: The distribution of GFP-Rab17 (green), the HMB45 melanosome marker (red), and a merge of the coloured images. Yellow indicates colocalization between Rab17 and the melanosome. C) Pearson’s correlation coefficients for colocalization between GFP-Rab17 and melanosome marker (HMB45) (white bar) or GFP-Rab17 and Golgi marker GM130 Ab in B16 cells (black bar). Error bars represent ±SD from 6 to 12 individual cells. D) Transfected cells were stimulated with FSK and stained with the TRP1 Ab to the Tyrp1 protein. FSK was used to increase the number of cells with a high number of mature, Tyrp1-positive melanosomes. From left to right: The distribution of the GFP-Rab17 fusion protein (green), Tyrp1 (red), a merge of the coloured images and a differential interference contrast (DIC) image of melanin distribution. The yellow arrow indicates colocalization between GFP-Rab17, Tyrp1 and melanin-filled melanosomes. The white arrow indicates colocalization between GFP-Rab17 and a melanin-filled melanosome. Manual counting using IMAGEJ cell counter revealed approximately 66% of the dark melanosomes in DIC colocalized with GFP-Rab17.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Transfection, Microscopy, Confocal Microscopy, Incubation, Staining, Marker

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 2: GFP-Rab17 localizes to the RE and melanosomes in primary melanocytes. Scale bars represent 10 μm. QF863 primary melanocytes were transiently transfected with GFP-Rab17 for 24 h. A) Cells were incubated with human Tfn-Alexa647 before fixing. From left to right: The distribution of GFP-Rab17 (green), Tfn-647 (red) and a merge of the coloured images also including DAPI (blue). Arrow indicates colocalization of GFP-Rab17 with the RE. B) Cells were fixed, permeabilized and stained with the HMB45 Ab. From left to right: The distribution of the GFP-Rab17 fusion protein (green), HMB45 (red) and a merge of the coloured images. This image is the tip of a melanocyte dendrite.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Transfection, Incubation, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 3: Rab17 expression in melanocytic cells and regula- tion by MITF. The level of Rab17 mRNA was determined by Q-RT-PCR and normalized to B2M expression. A) Two differenti- ated melanocyte strains (QF1177 and QF1185) were stimulated with 20 μM FSK or dimethyl sulphoxide (DMSO) (control) for 12 h in the absence of cholera toxin (CT). Rab17 mRNA was expressed relative to the control. Error bars indicate ±SEM (n = 3). B) West- ern blot of QF863 melanocytes stimulated with 10 μM FSK or DMSO (control) for 4 days in the absence of CT. Bands were cropped from the same blot at the same exposure. This result was replicated in the QF1177 and QF1185 strains. C) Rab17 mRNA in MM96L cells or differentiated QF1160 melanocytes transfected with negative control or MITF siRNA for 48 h. Rab17 mRNA was expressed as fold relative to the appropriate control cells (negative siRNA). Error bars represent ±SEM (n = 3). D) Western blot of differentiated QF1177 melanocytes transfected with negative control or MITF siRNA for 48 h. This result was replicated in the QF1185 strain. E) Rab17 mRNA in A06MLC cells transfected with either negative control or MITF siRNA (Ambion) as indicated. Twenty-four hours after transfection, cells were stimulated with normal media plus 0.25% BSA (control), 10 nM NDP-MSH in the presence of 100 μM IBMX (MSH) or 10 μM FSK for a further 24 h. A06MLC cells were used as they were known to be responsive to both FSK and MSH (37). Rab17 mRNA was expressed as fold over the control cells. Error bars indicate ±SEM (n = 3–4). MITF siRNA transfected cells (control, MSH and FSK) are not significantly different to the negative siRNA (control).

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Negative Control, Western Blot

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 4: B16 Rab17 siRNA treatment: imaging of morphology and melanosome distribution. B16 cells were transiently transfected with negative control siRNA, Rab17 siRNA (17.1 and 17.2) or Rab27a siRNA. Some negative control cells were also treated with 10 nM NDP-MSH (MSH) for 24 h. Transfected cells were fixed after 48 h, permeabilized and stained with HMB45 Ab, Texas-red phalloidin and DAPI. All cells were imaged via confocal microscopy. Scale bars represent 20 μm. Arrows indicate accumulation of mature melanosomes. A) Upper panel: Merged images are of HMB45 Ab staining (melanosome – green) and DAPI (blue). Lower panel: Transmitted light images. The outline of the Rab27a siRNA cells was drawn with coloured lines using Texas-red phalloidin staining as a guide. B) Q-RT-PCR analysis of mouse Rab17 mRNA in B16 cells transiently transfected with negative control siRNA or Rab17 siRNA. Rab17 mRNA was normalized to GAPDH expression then expressed as fold relative to the control cells. Error bars indicate ±SEM (n = 3). C) Rab17 western blot of B16 cells transiently transfected with negative control or Rab17 siRNA. This blot is representative of three independent experiments. D) Rab27a western blot of B16 cells transiently transfected with negative control siRNA or Rab27a siRNA. This result is representative of three independent experiments.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Imaging, Transfection, Negative Control, Staining, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 5: Representation of melanosome marker intensity and distribution, quantification of melanosome localization and numbers per cell. B16 cells were transiently transfected with negative control siRNA, Rab17.1 siRNA or Rab27a siRNA for 48 h as indicated. A) HMB45 fluorescence intensity is represented by the raised surfaces and colour indicates proximity to the cell membrane or nucleus. B) Quantification of melanosome localization in B16 siRNA knockdown cells. The boundary of individual cells is defined by actin staining, the nucleus is defined by DAPI staining and the intensity of the HMB45 Ab staining is used to determine melanosome distribution. The intensity ratio is calculated by the average intensity of HMB45 pixels proximal to the cell boundary divided by the average intensity of pixels further from the cell boundary (closer to the nucleus). Therefore, a cell with a relatively high intensity ratio has a relatively high proportion of melanosomes near the periphery of the cell. Error bars represent ±SEM (n ≥9 individual cells). C) Quantification of the numbers of HMB45-positive melanosomes per cell in negative siRNA- or Rab17 siRNA-treated cells as indicated. Error bars represent ±SEM (n ≥18 cells per treatment).

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Marker, Transfection, Negative Control, Membrane, Knockdown, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 6: B16 Rab17 knockdown and dendrite length, melanosome velocity, melanin content and melanosome imaging by EM. A) B16 dendrite length (distance in micrometers of the longest dendrite from the nucleus) was quantified using IMAGEJ. Lengths were normalized to the control cells, error bars represent ±SEM (n = 4). B) Melanosome velocity in live B16 cells transfected with negative control siRNA or Rab17 siRNA imaged via bright-field time-lapse microscopy. Error bars represent ±SEM of melanosome velocity from 2 to 3 cells, with at least four individual melanosomes tracked per cell. Movements >0.1 μm/second but <0.3 μm/second are thought to be actin movements and movements >0.3 μm/second are thought be microtubule movements (38). C) Total melanin content in B16 cells was measured 3 days after transient siRNA transfection. Values were normalized to the negative control. Error bars indicate ±SEM (n ≥4). D) Representative digital photo of cell pellets of transient siRNA knockdown B16 cells, or cells treated with 10 nM NDP-MSH (MSH). E) Transmission electron micrographs of control, Rab17.1 knockdown or Rab27a knockdown cells. Control B16 cells show low numbers of melanosomes near the cell body and in dendrites, while Rab17 knockdown cells show an increase in melanosomes which were mainly in peripheral regions of the cell body and in the dendrites. Rab27a knockdown cells showed an even larger increase in melanosomes, which were concentrated throughout the cell body/perinuclear region. Yellow arrows indicate mature, stage IV melanosomes and black arrows indicate stage II–III melanosomes. N = Nucleus, G = Golgi, mc = mitochondria. Scale bars = 2 μm.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Knockdown, Imaging, Control, Transfection, Negative Control, Time-lapse Microscopy, Transmission Assay

Journal: bioRxiv

Article Title: Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma

doi: 10.1101/388553

Figure Lengend Snippet: A .Total cellular proteins (left) and EGFR mRNA levels (right) of control (CTR) and RAB5A-MCF10A seeded at jamming density and detected by immunoblotting or qRT-PCR, respectively. At the bottom, quantification of total EGFR and of the ratio of phosphorylated/total EGFR from the immunoblotting is shown. Data are the relative ratio (mean±SD, n=5 independent experiments) with respect to control, obtained after normalizing each intensity values to Vinculin. The levels of EGFR mRNA are expressed relative to control (mean±SD, n=5 independent experiments) after normalizing to GAPDH. B .Control and RAB5A-MCF10A cells seeded at a jamming density were fixed and either permeabilized or non-permeabilized with 0.1% Triton X100 before staining with anti-EGFR ab. Data are the relative ratio (mean ± SD) with respect to control of total cell surface or internalized EGFR signals (n = 100 cells out of at least 3 independent experiments) normalized to cell number. Scale Bar, 20 μm.**p< 0.01, ***p<0.005. P values were calculated using each-pair Student’s t-test. C .Representative images of control and RAB5A-MCF10A cells seeded at a jamming density, fixed and stained with the indicated abs. Data are the mean±SD of positive EEA1and EGFR vesicles/cells (n>150 out of 3 independent experiments). ** p< 0.01, Student’s t-test. Scale Bar, 20 μm D .Number of EGFR/cell of control and RAB5-MCF10A seeded at jamming density measured by 125 I-EGF saturation binding after subtracting unspecific background counts (see methods for details). Data are the mean±SD of triplicate measurements of a representative experiment. *** p<0.005. P values were calculated using each-pair Student’s t test. E-G . Representative images ( E ) of control and RAB5A-MCF10A cells seeded at a jamming density, which were either deprived of EGF for 24 h or treated with AG1478 before fixation and staining as described in B. Scale Bar, 20 μm. Data ( F ) are the relative ratio (mean ± SD) with respect to control of total cell surface EGFR (n>120 cells in 2 independent experiments) normalized to cell number. ** p<0.05, each-pair Student’s t - test versus control. Immunoblots ( G ) showing the efficacy of EGF deprivation and EGFR inhibition on total and phosphorylated EGFR levels using the indicated abs. Each band of total EGFR was quantified and its mean intensity value is shown. The experiment is representative of at least 5 independent ones with similar outcome. H. Number of EGFR/cell of control and RAB5-MCF10A seeded at jamming density measured by 125 I-EGF saturation binding after various time of EGF starvation as described in D. Data are the mean±SD of triplicate measurements of representatives experiments. * p<0.05. P values were calculated using each-pair Student’s t-test.

Article Snippet: Primer assay IDs were: GAPDH, Hs99999905_m1; RAB5A, Hs00702360_s1; RAB5B, Hs00161184_m1 and RAB5C, Hs00428044_m1, Dynamin2 (DNM2) Hs00974698_m1, MP1 (LAMTOR3) Hs00179753_m1, P14 (LAMTOR2) Hs00203981_m1, Reticulon3 (RTN3) Hs01581965_m1, Reticulon4 (RTN4) Hs01103689_m1.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma

doi: 10.1101/388553

Figure Lengend Snippet: A-B . Control and RAB5A-MCF10A-expressing mCherry-H2B cells were grown overlaid on top of Matrigel plugs. Between 14 and 21 days, cells formed fully differentiated hollow acini. At this stage, Doxycycline was added and the kinematic of the 3D acini was monitored by confocal times lapse for 24 h (Movies S12). Representative images of single Z planes are shown. RAB5A induction was verified by QRT-PCR, expressed relative to control after normalizing to GAPDH. The data are the relative level of gene expression compared to control expressed as mean ± SD (n=3 independent experiments). Scale Bar, 50 μm. In B , snapshots of the tangential velocity field at t = 10 h (indicated by yellow arrows) obtained from PIV analysis are shown, overlaid on radial projection of the acini onto a unit spherical surface (see also Movies S13). The direction of the red arrow shown in the middle panel is parallel to the instantaneous total angular momentum l and provides the orientation of the instantaneous axis of rotation, while its length is equal to the instantaneous order parameter Ψ. On the right: time evolution of the root mean square velocity v RMS and of the rotational order parameter Ψ (see text and Methods for details). The data are representative of 4 movies in 3 independent experiments. C . Doxycycline-treated control and RAB5A-MCF-10A mcherry-H2B-expressing MCF10A acini were treated with the indicated vehicle or the indicated inhibitor and monitored by confocal time-lapse microscopy for 24 hr (Movie S14). Average values of v RMS and of Ψ, calculated over the time window comprised between 4 and 12 h, are reported. Values are from 5 movies form 3 independent experiments. D-E . Doxycycline-treated control and RAB5A-MCF10A cells were grown overlaid on top of Matrigel plugs for up to 21 days. Acini were fixed and processed for phase contrast imaging to monitor acini shape and size (left images) or, at various time point, for immunofluorescence to detect apoptotic caspase+ and proliferating, Ki67+ cells (see Fig. S4). Exemplar, phase contrast images are shown, (see also Movie S15). Scale Bar, 100 μm. In ( E) , The average size of acini was quantified and expressed as mean±SD (n=100 acini/conditions in 5 independent experiments. *p<0.05, ** p<0.01. P value were calculated using each-pair Student’s t - test. F .Control and RAB5A-MCF10A cells were grown overlaid on top of Matrigel plugs for 14 days to allow full differentiation into hollow acini. Doxycycline was then added (time schedule of drug administration is on the top) to induce RAB5A expression in the presence or absence of PD0325901 and after 6 days acini were fixed and stained as indicated. Scale Bar, 80 μm. The size of acini was calculated by measuring their diameter. The number of KI67+ acini is also reported. Data are mean±SD (n=25 acini/conditions in 3 independent experiments). ** p<0.01. P value were calculated using each-pair Student’s t-test. G .Doxycycline-treated control and RAB5A-MCF10A cells were grown overlaid on top of mixed Matrigel:Collagene Type I (1:1) plugs for 21 days. Acini were fixed and processed for phase contrast imaging to monitor acini shape and size (left images). Exemplar phase contrast images are shown. Scale bar, 100 μm. Area of acini and acini roundness was quantified and expressed as mean±SD (n=40 acini/conditions in 5 independent experiments). ** p<0.01, paired Student’s t - test.

Article Snippet: Primer assay IDs were: GAPDH, Hs99999905_m1; RAB5A, Hs00702360_s1; RAB5B, Hs00161184_m1 and RAB5C, Hs00428044_m1, Dynamin2 (DNM2) Hs00974698_m1, MP1 (LAMTOR3) Hs00179753_m1, P14 (LAMTOR2) Hs00203981_m1, Reticulon3 (RTN3) Hs01581965_m1, Reticulon4 (RTN4) Hs01103689_m1.

Techniques: Expressing, Quantitative RT-PCR, Time-lapse Microscopy, Imaging, Immunofluorescence, Staining