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: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Schematic of approach to generate NFκB Rela and p38 MAPK activity dual fluorescence reporter macrophages by lentiviral transduction of hMPs. ( B ) Signaling responses to three LPS doses over 2 h in mVenus-Rela p38-KTR-mCerulean hMPDMs compared to parent cell line by Western Blotting for phospho-p38, phospho-MK2, phospho-CREB, and IκBα protein levels. Band intensities were background corrected, normalized to tubulin control, and normalized across multiple membranes using an internal control sample. Data from one experiment are depicted (Western Blot membrane shown in Fig. ). ( C ) Fluorescence microscopy images of p38-KTR localization in hMPDMs upon stimulation with 100 ng/ml LPS over 6 h with and without p38 inhibitor pre-treatment, as well as nuclear dye (SiR-DNA) fluorescence and DIC image at baseline. Scale bar: 20 µm. ( D ) p38 activity dynamics in response to 100 ng/ml LPS with and without p38 inhibitor measured over 8 h by fluorescence microscopy, expressed as baseline-corrected p38-KTR fluorescence cytoplasmic/nuclear ratio, quantified by automated image analysis. Each row of the heatmap represents the p38 signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from one experiment are depicted. ( E ) Comparison of p38 activity over 4 h in hMPDMs measured by p38-KTR microscopy (mean of trajectories) and by bulk phospho-p38 levels measured by Western Blotting in response to indicated doses of P3C4, CpG, LPS, or TNF. Western Blotting quantification: band intensities were background corrected, normalized to tubulin control, normalized across multiple membranes using an internal control sample, and baseline-deducted; depicting data from a single experiment (Western Blot membrane shown in Fig. ). For microscopy, the mean of means of trajectories from two biological replicates is shown (Total # of cells: 923, 1171, 970, and 1055 cells for P3C4, CpG, LPS, and TNF). ( F ) Comparison of p38 activity over 1 h in hMPDMs measured by p38-KTR microscopy (mean of trajectories, as in Panel E ) and as intracellular p-p38 levels by flow cytometry in response to indicated doses of P3C4, CpG, LPS, or TNF. MFIs were baseline-deducted. Data from one flow cytometry experiment is shown. ( G ) Comparison of the fractions of cells with p38 activity in the hMPDM population as measured by p38-KTR microscopy or by intracellular p-p38 flow cytometry upon stimulation with P3C4, CpG, LPS, or TNF. Microscopy: A cell is considered p38 active if its KTR measurement passes a threshold of 3x STDV of baseline for 3 consecutive timepoints within 1 h of stimulation. Data from two pooled biological replicates are used. Flow cytometry: The fluorescence signal of an unstimulated, unstained sample is used to define a cutoff between p-p38 + and p-p38 − cells (Fig. ). For each dose, the fraction of cells passing the threshold at 30 min post stimulation (Fig. ) is plotted. Data from one experiment are displayed. DIC differential interference contrast, p38i p38 inhibitor, MFI mean fluorescence intensity. .

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activity Assay, Fluorescence, Transduction, Western Blot, Control, Membrane, Microscopy, Comparison, Flow Cytometry

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Gating strategy for FACS of mVenus-RelA+ p38-KTR-mCerulean+ hMPs. ( B ) mCerulean and mVenus expression by flow cytometry in WT hMPs, mVenus-Rela hMPs, and mVenus-Rela hMPs transduced with p38-KTR-mCerulean before FACS. ( C ) Signaling responses to three LPS doses over 2 h in mVenus-Rela p38-KTR-mCerulean hMPDMs compared to parent cell line by Western Blotting for phospho-p38, phospho -MK2, phospho-CREB, and IκBα protein levels. Quantification in Fig. . ( D ) Bulk p-p38 and p-MK2 protein levels in hMPDMs measured by Western Blotting in response to indicated stimulations over 4 h. Quantification in Figs. and EV1D. ( E ) Comparison of p38 activity over 4 h in hMPDMs measured by p38-KTR microscopy (mean of trajectories) and by bulk phospho-MK2 levels measured by Western Blotting in response to three doses of P3C4, CpG, LPS, or TNF (0.1, 1, 100 ng/ml for P3C4, LPS, TNF; 1, 10, 1000 nM for CpG). Western Blotting quantification: band intensities were background corrected, normalized to tubulin control, normalized across multiple membranes using an internal control sample, and baseline-deducted; depicting data from a single experiment (Western Blot membrane shown in Fig. EV1E). For microscopy, the mean of means of trajectories from two biological replicates is shown (in total: 923, 1171, 970, and 1055 cells included in the analysis for P3C4, CpG, LPS, and TNF, respectively; same data as in Fig. ). ( F ) Gating strategy for flow cytometry measurements of intracellular p-p38 levels in hMPDMs. ( G ) Example quantification of p-p38 + fraction of cells stimulated with indicated doses of LPS for 30 min (blue). The fluorescence signal of an unstimulated, unstained sample is used to define a cutoff between p-p38 + and p-p38 - cells (gray). Data from one experiment are displayed.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Expressing, Flow Cytometry, Transduction, Western Blot, Comparison, Activity Assay, Microscopy, Control, Membrane, Fluorescence

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) MAPK p38 activity dynamics in response to 100 ng/ml P3C4, 1000 nM CpG, 100 ng/ml LPS, and 100 ng/ml TNF stimulation over app. eight hours were measured by fluorescence microscopy of reporter hMPDMs. Each row of the heatmap represents the p38 signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from two pooled biological replicates are depicted. Total # of cells: 923, 1171, 970, and 1055 cells for P3C4, CpG, LPS, and TNF. ( B ) Schematic of 12 (out of 228) dynamic features derived from p38 activity trajectories used for quantitative analysis. ( C ) Mutual information between mock and high-dose P3C4, CpG, LPS, and TNF stimulations and dynamic features of p38 activity. Data from two pooled biological replicates are used. ( D ) Schematic of neural network machine learning classifier to test distinguishability of stimuli using p38 signaling time-series. ( E ) F1 scores by class of neural network classification of p38 time-series in response to the high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( F ) Schematic of decision tree ensemble machine learning classifier to test distinguishability of stimuli using p38 signaling dynamic features. ( G ) F1 scores by class of decision tree ensemble classification of p38 dynamic features in response to the high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( H , I ) Confusion matrices of neural network classification of p38 time-series ( H ) or decision tree ensemble classification of p38 dynamic features ( I ) in response to high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( J ) Violin plots of selected p38 dynamic features in response to the high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are depicted. Total # of cells: 923, 1171, 970, and 1055 cells for P3C4, CpG, LPS, and TNF. ( K ) Mutual information between mock and high-dose stimulation with P3C4, CpG, LPS, or TNF and dynamic features of p38 activity. Data from two pooled biological replicates are used.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activity Assay, Fluorescence, Microscopy, Derivative Assay

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A , B ) F1 scores by class and confusion matrix of decision tree ensemble classification of p38 dynamic features in response to mock and high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( C , D ) Confusion matrices of machine learning classification of NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38 (left) or NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) (right) time-series ( C ) or dynamic features ( D ) in response to high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( E ) Confusion matrices of machine learning classification of decision tree ensemble classification of NFκB only, NFκB + p38, NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38, or NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) dynamic features in response to mock and high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( F , G ) Overall classification accuracy ( F ) and F1 score for TNF class ( G ) in machine learning classifications using p38 only, NFκB only, NFκB + p38, NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38, NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) dynamic features in response to mock and high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( H – J ) Overall classification accuracy ( H ), F1 scores for individual classes ( I ), and confusion matrices ( J ) for machine learning classifications of time series using p38 only, NFκB only, NFκB + p38, NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38, or NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) in response to high-dose P3C4, CpG, and LPS stimulations. Data from two pooled biological replicates are used. ( K ) Confusion matrices of machine learning classification of NFκB (shuffled among all cells in all classes) + p38 (left) or NFκB + p38 (shuffled among all cells in all classes) (right) dynamic features in response to high-dose P3C4, CpG, and LPS stimulations. Data from two pooled biological replicates are used.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques:

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) p38 and NFκB activity dynamics in the same cells in response to 100 ng/ml P3C4, 1000 nM CpG, 100 ng/ml LPS, and 100 ng/ml TNF stimulation over app. eight hours were measured by fluorescence microscopy of reporter hMPDMs. Each row of the heatmap represents the p38 or NFκB signaling trajectory of one cell. Trajectories are sorted by the maximum amplitude of p38 activity. Example trajectories are shown below. Data from two pooled biological replicates are depicted. Total # of cells: 923, 1171, 970, and 1055 cells for P3C4, CpG, LPS, and TNF. p38 data is also shown in Fig. . ( B ) Confusion matrices of neural network classification of NFκB (left) or NFκB + p38 (right) time-series in response to high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( C ) Confusion matrices of decision tree ensemble classification of NFκB (left) or NFκB + p38 (right) dynamic features in response to high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( D , E ) Overall classification model accuracy of machine learning classifications using p38 only, NFκB only, NFκB + p38, NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38, NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) time-series ( D ) or dynamic features ( E ) in response to high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( F , G ) F1 score for TNF class in machine learning classifications using p38 only, NFκB only, NFκB + p38, NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38, NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) time-series ( F ) or dynamic features ( G ) in response to high-dose P3C4, CpG, LPS, and TNF stimulations. Data from two pooled biological replicates are used. ( H ) Mutual information (MI) between mock and high-dose stimulation with TNF and dynamic features of p38, NFκB, or p38 + NFκB activity. Data from two pooled biological replicates are used. ( I ) Mutual information between mock and high-dose P3C4, CpG, and LPS stimulations and dynamic features of p38, NFκB, or p38 + NFκB activity. Data from two pooled biological replicates are used. ( J , K ) Overall classification accuracy ( J ) and F1 scores for individual classes ( K ) of machine learning classifications using p38 only, NFκB only, NFκB + p38, NFκB (“shuffled”: incorrectly matched NFκB activities from cells randomly selected from all classes) + p38, NFκB + p38 (“shuffled”: incorrectly matched p38 activities from cells randomly selected from all classes) dynamic features in response to high-dose P3C4, CpG, and LPS stimulations. Data from two pooled biological replicates are used. ( L ) Confusion matrices for machine learning classifications using p38 only (left), NFκB only (center), and NFκB + p38 (right) dynamic features in response to high-dose P3C4, CpG, and LPS stimulations. Data from two pooled biological replicates are used. ( M ) Selected dynamic features of NFκB (top) and p38 (bottom) activity in response to P3C4, CpG, and LPS. Data from two pooled biological replicates are used. Total # of cells: 923, 1171, and 970 cells for P3C4, CpG, and LPS.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activity Assay, Fluorescence, Microscopy

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) p38 and NFκB activity dynamics in the same cells in response to 0, 0.01, 0.1, 1, 10, and 100 ng/ml P3C4 stimulation over app. eight hours were measured by fluorescence microscopy of reporter hMPDMs. Each row of the heatmap represents the p38 or NFκB signaling trajectory of one cell. Trajectories are sorted by the maximum amplitude of p38 activity. Data from two pooled biological replicates are depicted. Total # of cells: 898, 834, 827, 787, 778, and 923. Data for 100 ng/ml stimulation is also shown in Figs. A, . ( B ) Dose responses of NFκB and p38 activity, as measured by the fraction of responding cells among all cells, in response to six doses of P3C4 (0, 0.01–100 ng/ml), CpG (0, 0.1–1000 nM), LPS (0, 0.01–100 ng/ml), or TNF (0, 0.01–100 ng/ml), with Hill curve fits. Data from two pooled biological replicates are used. ( C ) Parameters of the Hill fits of dose-response curves (from Fig. 4B) and fold difference between concentrations of half maximum activity for p38 and NFκB. ( D ) Overall classification accuracy of decision tree ensemble classifications using p38 only, NFκB only, or NFκB + p38 total activities only (top) or all dynamic features (bottom) in response to 5 doses of P3C4 (0.01–100 ng/ml), CpG (0.1–1000 nM), LPS (0.01–100 ng/ml), or TNF (0.01–100 ng/ml). Data from two pooled biological replicates are used. ( E ) Mutual information between 6 doses of P3C4 (0, 0.01–100 ng/ml), CpG (0, 0.1–1000 nM), LPS (0, 0.01–100 ng/ml), or TNF (0, 0.01–100 ng/ml) and dynamic features of p38, NFκB, or p38 + NFκB activity. Data from two pooled biological replicates are used. ( F ) Overall classification accuracies of decision tree ensemble classifications using p38 only, NFκB only, or NFκB + p38 dynamic features in response to the respective two indicated adjacent doses of the indicated stimuli (P3C4, CpG, LPS, or TNF). Data from two pooled biological replicates are used.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activity Assay, Fluorescence, Microscopy

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Spearman correlation coefficients (CCs) between indicated p38 dynamic features and corresponding NFκB dynamic features upon stimulation of hMPDMs with high-dose P3C4, CpG, LPS, or TNF. Features are sorted by means of CCs across stimuli. Asterisk indicates statistically significant correlation ( p < 0.05) with |CC | > 0.15. Data from two pooled biological replicates are used. ( B ) Scatter plots of 30-min p38 activity integrals from 0 to 4 h and corresponding NFκB activity integrals upon stimulation with high-dose P3C4, CpG, LPS, or TNF. Number indicates the Spearman correlation coefficient. Data from two pooled biological replicates are depicted. ( C ) Spearman correlation coefficients (CCs) between p38 activity integrals from 0 to 4 h and all NFκB activity integrals from 0 to 4 h upon stimulation with high-dose P3C4, CpG, LPS, or TNF. Asterisk indicates a statistically significant correlation ( p < 0.05) with |CC | > 0.15. Data from two pooled biological replicates are used. ( D ) Coefficient of variation of indicated p38 and NFκB dynamic features upon stimulation with high-dose P3C4, CpG, LPS, or TNF. Data from two pooled biological replicates are used.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activity Assay

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Scatter plots of correlations of selected p38 vs. NFκB dynamic features (as in Fig. ) upon stimulation with high-dose P3C4, CpG, LPS, or TNF. Number indicates the Spearman correlation coefficient. Data from two pooled biological replicates are depicted. ( B ) Negative control: Spearman correlation coefficients (CCs) between indicated p38 dynamic features and corresponding NFκB dynamic feature using NFκB features randomly shuffled with respect to p38 features among cells within a stimulation condition for stimulation with high-dose P3C4, CpG, LPS, or TNF. The asterisk indicates a statistically significant correlation ( p < 0.05) with |CC | > 0.15. Data from two pooled biological replicates are used. ( C ) Spearman correlation coefficients (CC) between integrals of p38 activity over 30 min from 0 to 4 h and corresponding NFκB integrals upon stimulation with high-dose P3C4, CpG, LPS, or TNF. The asterisk indicates a statistically significant correlation ( p < 0.05) with |CC | > 0.15. Data from two pooled biological replicates are used. ( D ) Spearman correlation coefficients (CCs) between indicated p38 dynamic features and all indicated NFκB dynamic features upon stimulation with high-dose P3C4, CpG, LPS, or TNF. The asterisk indicates a statistically significant correlation ( p < 0.05) with |CC | > 0.15. Data from two pooled biological replicates are used. ( E ) Comparison of heterogeneity of p38 activity measured by flow cytometry (gray) and p38-KTR microscopy (blue) in unstimulated cells and upon 30 min 100 ng/ml LPS stimulation. For each assay, the mean of the unstimulated sample is deducted from each distribution. The flow cytometry distributions are scaled using the distance between the peaks of lognormal fits to the unstimulated and the stimulated distributions. Microscopy: Data from two pooled biological replicates are used. Flow cytometry: Data from a single experiment are displayed. ( F ) Coefficient of variation of indicated p38 and NFκB dynamic features upon stimulation across dose range of P3C4, CpG, LPS, or TNF. Data from two pooled biological replicates are used. ( G ) Coefficient of variation of 30-min p38 and NFκB activity integrals upon stimulation with high-dose P3C4, CpG, LPS, or TNF. Data from two pooled biological replicates are used.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Negative Control, Activity Assay, Comparison, Flow Cytometry, Microscopy

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Schematic of mathematical model structure. A model of p38 activation via IKK/Tpl2/MKK3/6 and TAK1/MKK4 (blue background) is integrated with established models of NFκB activation (yellow) downstream of TLR1/2 (orange), TLR9 (green), TLR4 (red), and TNF receptor (light blue) signaling (Adelaja et al, ). Within the p38 module, parameters in red indicate the parameters distributed to simulate heterogeneous single cell trajectories. All model modules except the p38 module are simplified. The full model schematic can be found in Fig. . ( B ) Simulation of ‘representative cell’ p38 and NFκB activity trajectories downstream of high-dose P3C4, CpG, LPS, and TNF stimulation after parameter fitting to representative p38 trajectories overlayed with the experimental p38 and NFκB trajectories from the representative cells. Example trajectories were scaled to model units as described in Materials and Methods. ( C ) Simulation of “representative cell” MKK4 and MKK6 activity trajectories downstream of P3C4, CpG, LPS, and TNF stimulation. ( D ) Assumed distributions of total MKK4, MKK6, and p38 concentrations and parameter values sampled from those distributions for simulation of cell-cell-heterogeneity of p38 activity in ( E ). ( E ) Simulated LPS-induced p38 and NFκB trajectories in corresponding cells compared to experimentally determined p38 and NFκB trajectories rescaled to model units as described in Materials and Methods. Cell-cell-heterogeneity is simulated by distributing starting concentrations of total p38, MKK4, and MKK6 (as in D ), in addition to the distribution of receptor- and NFκB-related parameters. Trajectories are sorted by the maximum amplitude of p38 activity. 500 cells are shown. ( F ) Spearman correlation coefficients between indicated p38 dynamic features and corresponding NFκB dynamic feature in simulated (sim.) or experimental (exp.) stimulation with LPS. For experimental data, two pooled biological replicates were used. Values are rounded to two decimals. ( G ) Spearman correlation coefficients between indicated p38 dynamic features and corresponding NFκB dynamic feature in simulated LPS stimulation with or without simulated molecular noise (i.e., using parameter distribution or single parameter) in the p38 and NFκB modules. An average of ten simulations is reported. ( H , I ) Coefficient of variation of 30-min activity integrals ( H ) or indicated dynamic features ( I ) of p38 and NFκB activity upon simulated LPS stimulation with or without simulated molecular noise (i.e., using parameter distributions or single parameters) in the p38 and NFκB modules. Results from one simulation are reported. .

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activation Assay, Activity Assay

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Detailed schematic of mathematical model structure including reaction numbers (see also Dataset ). A model of p38 activation via IKK/Tpl2/MKK3/6 and TAK1/MKK4 (blue background) is integrated with established models of NFκB activation (yellow) downstream of TLR1/2 (orange), TLR9 (green), TLR4 (red), and TNF receptor (light blue) signaling (Adelaja et al, ; Luecke et al, ). ( B ) Parameter sensitivity analysis probes the effect of kinase abundances within the p38 module. Effect of variations of the abundance of indicated kinase(s) on p38 activity of a representative cell (top), p38 dynamic features in a representative cell (middle), and heterogeneous p38 activity trajectories in single cells upon simulated LPS stimulation (bottom). ( C ) Scatter plots of p38 vs. NFκB integrated activity over indicated 30 min intervals in simulated LPS stimulation with or without simulated molecular noise (i.e., using parameter distributions or fixed parameter values) in p38 and NFκB modules.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activation Assay, Activity Assay

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Schematic of a mathematical model for simulating heterogeneous single cell gene expression controlled by NFκB or NFκB&p38 using experimental NFκB and p38 activity dynamics over 8 h as input and mRNA abundance over time as output; with NFκB enhancing transcription and p38 decreasing degradation of mRNA. ( B ) Distributions of simulated single-cell mRNA expression over 8 h when controlled by NFκB only or by NFκB&p38 using the model described in Panel ( A ). ( C ) Single cell gene expression distributions (log 2 (normalized counts+1)) of known NFκB&p38 or NFκB-only target genes in hMDPMs upon stimulation with 100 ng/ml P3C4, 100 nM CpG, 100 ng/ml LPS, or 10 ng/ml TNF over 8 h, measured by BD Rhapsody. Data from one experiment are depicted. Number of cells analyzed at each timepoint: Unstimulated: 1415 cells; P3C4: 835, 1840, 1251, 1153 cells; CpG: 992, 1229, 2235, 1236 cells; LPS: 941, 1045, 663, 1338 cells; TNF: 980, 776, 994, 2412 cells. ( D ) Fano factor (left, variance/mean) and bimodality coefficient (right) of single cell expression of indicated genes over 8 h (as in C ). The dashed lines indicate a Fano factor of 1 (left) and a bimodality coefficient of 0.5 (right). ( E ) Fano factor of all immune response genes regulated by NFκB-only (132 genes) or NFκB&p38 (59 genes) upon stimulation with P3C4, CpG, LPS, or TNF at 1 h and 8 h. Purple line indicates the mean of Fano factors. Statistical significance was determined using a permutation test for differences in means using 10,000 permutations. ( F ) Bimodality coefficient of all immune response genes regulated by NFκB&p38 (top, 59 genes total) or NFκB-only (bottom, 132 genes total) categorized by absence or presence of ARE-element(s) upon stimulation with P3C4, CpG, LPS, or TNF at 1 h. The purple line indicates the mean. Statistical significance was determined using a permutation test for difference in means using 10000 permutations. ( G ) Gene ontology results of NFκB-only or NFκB&p38 regulated genes. Benjamini–Hochberg adjustment was applied to p values. ( H ) Heatmap of Fano factors of single cell expression of NFκB-only or NFκB&p38 regulated genes upon stimulation with P3C4, CpG, LPS, or TNF at 8 h, sorted by average Fano factor across the four stimuli. Cytokine vs. non-cytokine identity and NFκB-only vs NFκB&p38 regulation are indicated by a color map. Positions of selected genes of interest are labeled. GRS gene regulatory strategy.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Gene Expression, Activity Assay, Expressing, Labeling

Journal: Molecular Systems Biology

Article Title: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages

doi: 10.1038/s44320-024-00047-4

Figure Lengend Snippet: ( A ) Effect of NFκB and p38 activity levels on simulated mRNA transcription and mRNA-degradation, respectively. ( B ) Simulated mRNA expression under control of NFκB only or NFκB&p38 over 8 h using experimentally determined NFκB and p38 activity upon high-dose P3C4, CpG, LPS, and TNF stimulation as input to the mathematical model. ( C ) Bimodality coefficient of all immune response genes regulated by NFκB-only (132 genes) or NFκB&p38 (59 genes) in single cells upon stimulation with P3C4, CpG, LPS, or TNF at 1 and 8 h. Purple line indicates mean. Statistical significance was determined using a permutation test for difference in means using 10000 permutations. Data from one experiment are depicted. ( D , E ) Bimodality coefficient ( D ) and Fano Factor ( E ) of all immune response genes in single cells categorized by absence or presence of ARE-element(s) upon stimulation with P3C4, CpG, LPS, or TNF at 1 and 8 h. Purple line indicates mean. Statistical significance was determined using a permutation test for difference in means using 10000 permutations. Data from one experiment are depicted. ( F ) Bimodality Coefficients of 30-min p38 and NFκB activity integrals upon stimulation with high-dose P3C4, CpG, LPS, or TNF determined by dual reporter macrophage imaging. Data from two pooled biological replicates are used.

Article Snippet: p38-KTR-mCerulean3 insert was amplified by Q5 polymerase PCR from pENTR-p38-KTR-mCerulean3 (Addgene #59149) (Regot et al, ) using primers containing restriction sites for AgeI and SalI (Fwd: 5’-gatccaccggtcgccaccATGCGTAAGCCAGATCTC, Rev: 5’-gttgattgtcgacgcggccgctttacttgtacagctcgtccatgc).

Techniques: Activity Assay, Expressing, Control, Imaging

Journal: bioRxiv

Article Title: Temporally multiplexed imaging of dynamic signaling networks in living cells

doi: 10.1101/2022.08.22.504781

Figure Lengend Snippet: ( A ) Diagram of cell cycle indicators (known as fluorescent, ubiquitination-based cell cycle indicator 4, abbreviated FUCCI4). Dronpa, YFP, rsGreenF-E and Skylan62A were fused to cell cycle-regulated proteins Cdt1 30-120 , SLBP 18-126 , Geminin 1-110 , and histone H1.0, respectively. Analogous to the original FUCCI4, the G1-S transition is reported by the emergence of rsGreenF-E fluorescence while YFP fluorescence persists, and the S-G2 transition is marked by the fluorescence loss of YFP and stable rsGreenF-E fluorescence. Chromosome condensation, labeled by Skylan62A, indicates the M phase; finally, loss of rsGreenF-E fluorescence and the appearance of Dronpa and YFP fluorescence means the beginning of the G1 phase. ( B ) Tracking of cell cycle phase in NIH/3T3 cells via time-lapse imaging (i.e., taking brief movies at different points in time, each of which becomes a distinct datapoint in the time lapse) using TMI-based single-color FUCCI4. A mother cell dividing into two daughter cells was captured during a 11-hour imaging session, with brief movies acquired at each of the indicated times. Scale bar, 20 µm. ( C ) Fluorescence traces of Dronpa-Cdt1 30-120 (orange), rsGreenF-E-Geminin 1-110 (green), and YFP-SLBP 18-126 (Cyan) of 3 cells over their cell divisions, tracing one arbitrary daughter cell from each mother cell after M phase. Fluorescence was normalized to maximum value over the time-lapse period. Magenta bars indicate time of observation of chromosome condensation. Cell-cycle phases were assigned as depicted in A . ( D ) Diagram of TMI-based kinase translocation reporters (KTRs). A KTR contains a kinase docking site, a phospho-inhibited bipartite nuclear localization signal (bNLS) containing phosphorylation sites (P sites), a phospho-enhanced nuclear export signal (NES) containing P sites, and an rsFP. When the corresponding kinase is inactive, the KTR protein is unphosphorylated and is nuclear enriched. When the corresponding kinase is active, the KTR protein is phosphorylated and excluded from the nucleus. ( E ) Four kinase sensors were made using four green FPs: an rsFastLime-based ERK sensor, an rsGreenF-E-based JNK sensor, a Dronpa-based P38 sensor, and a Clover-based PKA sensor. NIH/3T3 cells expressing all four KTRs and H2B-TagBFP as a nuclear marker were imaged and stimulated with mouse basic fibroblast growth factor 2 (bFGF2, 20 ng/ml), which is known to drive JNK, P38, ERK, and PKA. A representative cell (out of 16 cells taken from two cell culture batches) is shown (four pseudo-channels unmixed from the green channel, and one blue channel) at the indicated time points. Scale bar, 20 µm. ( F ) Activity traces of the four kinases from the representative cell in E . R, ratio of cytoplasmic intensity to nuclear intensity. Change in fluorescence of the sensors is plotted as ΔR/R 0 (R 0 was the averaged value from t = −6 min to t= −4 min). ( G ) Averaged traces of four kinase activities recorded from NIH/3T3 cells, with 20 ng/ml bFGF2 stimulation at t = 0 min; n = 16 cells from two culture batches. Data are shown as mean ± standard error of the mean (SEM). Wilcoxon rank sum tests were run between the averaged values from t = −6 min to t = 0 min and the averaged values from t = 12 min to t = 18 min for PKA KTR and JNK KTR. Wilcoxon rank sum tests were run between the averaged values from t = −6 min to t = 0 min and the averaged values from t = 48 min to t = 54 min for ERK KTR and P38 KTR. Traces of individual cells are shown in . ( H ) Averaged traces of four kinase activities recorded from NIH/3T3 cells, with stimulation with 50 µM forskolin at t = 0 min; n = 10 cells from two culture batches. Data are shown as mean ± SEM. Wilcoxon rank sum tests were run between the averaged values from t = −6 min to t = 0 min and t = 30 min to t = 36 min for all KTRs. Traces of individual cells are shown in . Throughout the figure: *p< 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Article Snippet: The constructs of JNK KTR-rsGreenFast-E, P38 KTR-Dronpa, ERK KTR-rsFastLime were built based on the original KTRs constructs JNK KTR-Clover (Addgene plasmid 59151), P38 KTR-mCerulean3 (Addgene plasmid 59155), ERK KTR-Clover (Addgene plasmid 59150), all gift of Markus W. Covert ( ).

Techniques: Ubiquitin Proteomics, Fluorescence, Labeling, Imaging, Translocation Assay, Phospho-proteomics, Expressing, Marker, Cell Culture, Activity Assay

Journal: bioRxiv

Article Title: Temporally multiplexed imaging of dynamic signaling networks in living cells

doi: 10.1101/2022.08.22.504781

Figure Lengend Snippet: ( A ) Represented NIH/3T3 cell expressing all four KTR sensors and H2B-TagBFP was stimulated with 50 µM forskolin and imaged at the indicated time points. Forskolin was added at t = 0 min. Scale bar, 20 µm. ( B ) Fluorescence traces of ERK KTR, JNK KTR, P38 KTR, and PKA KTR from the cell in A . R: cytoplasmic intensity to nuclear intensity ratio. Change in fluorescence of the sensors is plotted as ΔR/R 0 (R 0 was the averaged value from t = −6 min to t= −4 min). ( C ) Additional kinase activity traces from individual cells. Forskolin was added at t = 0 min. The same color code is used for all the traces throughout the figure.

Article Snippet: The constructs of JNK KTR-rsGreenFast-E, P38 KTR-Dronpa, ERK KTR-rsFastLime were built based on the original KTRs constructs JNK KTR-Clover (Addgene plasmid 59151), P38 KTR-mCerulean3 (Addgene plasmid 59155), ERK KTR-Clover (Addgene plasmid 59150), all gift of Markus W. Covert ( ).

Techniques: Expressing, Fluorescence, Activity Assay

Journal: bioRxiv

Article Title: Temporally multiplexed imaging of dynamic signaling networks in living cells

doi: 10.1101/2022.08.22.504781

Figure Lengend Snippet: ( A - D ) Simultaneous observation of cell cycle phase changes and kinase activity. ( A ) Left, diagram of combined use of green rsFP-based FUCCI4 with a TagBFP2-based CDK2 reporter and an mCherry-based CDK4/6 reporter. Right, schematic of CDK activity in different cell cycle phases. CDK activity is higher at the end of G2 phase than that at the beginning of S phase, but the detailed CDK activity progression during S and G2 phases was unknown (dashed line) previously . ( B ) Representative traces of four fluorescence signals from FUCCI4, and two CDK signals from their respective reporters, over the cell cycle. Traces of four dividing NIH/3T3 cells are shown. Cells had brief movies acquired every 30 min, without stimulation. Fluorescence of Dronpa-Cdt1 30-120 (orange), rsGreenF-E-Geminin 1-110 (green), and YFP-SLBP 18-126 (Cyan) was normalized to their maximum values. Cell cycle phases were determined by FUCCI4 signals using the principles described in ; magenta bars indicate observation of chromosome condensation. CDK2 activity (blue) was calculated as cytoplasm to nucleus fluorescence ratio; CDK4/6 activity (purple) was calculated as was CDK2 activity, followed by signal processing as in the previous paper . Both CDK2 and CDK4/6 traces were normalized to their maximum value for comparison. More traces from individual cells are shown in . ( C, D ) Plots of CDK2 ( C ) and CDK4/6 ( D ) activity as a function of stage (early, middle, and late stage) of each cell cycle phase. CDK2 and CDK4/6 activities were calculated as in B, without normalization. Each cell cycle phase was divided into early, middle and late stages evenly, and CDK activity for a given stage was obtained by averaging CDK values across that stage (see Methods). Data are shown as mean ± SD (blue, CDK2; purple, CDK4/6), with all individual values plotted as dots (grey; dots of the same cell cycle phase of the same cell are connected); n = 25 cells from 5 culture batches (note well, not all cells exhibited all complete phases). Wilcoxon signed-rank test with Holm-Bonferroni correction, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. ( E ) Superimposed plots of CDK2 and CDK4/6 activity. Data points were from the mean values of the plots in C and D , and then normalized between 0 and 1 for visual comparison. ( F - H ) Simultaneous imaging of 7 signals within single NIH/3T3 cells. ( F ) Diagram of combined use of green rsFP-based KTRs with BlueCKAR, Pink Flamindo and NIR-GECO2G. ( G ) Signals of PKA (cyan), P38 (orange), ERK (black), JNK (green), Ca 2+ (magenta), cAMP (purple), and PKC (blue) under stimulation with 50 µM forskolin (added at t = 8 min), and 100ng/ml PMA (added at t = 44 min); n = 7 cells from 2 culture batches, values are shown as mean ± SEM. Signals from individual cells are shown in . ( I ) Superimposed traces of H, for visual comparison.

Article Snippet: The constructs of JNK KTR-rsGreenFast-E, P38 KTR-Dronpa, ERK KTR-rsFastLime were built based on the original KTRs constructs JNK KTR-Clover (Addgene plasmid 59151), P38 KTR-mCerulean3 (Addgene plasmid 59155), ERK KTR-Clover (Addgene plasmid 59150), all gift of Markus W. Covert ( ).

Techniques: Activity Assay, Fluorescence, Comparison, Imaging

Journal: bioRxiv

Article Title: Temporally multiplexed imaging of dynamic signaling networks in living cells

doi: 10.1101/2022.08.22.504781

Figure Lengend Snippet: Activity traces of P38 (orange), PKA (cyan), ERK (black), JNK (green), Ca 2+ (magenta), cAMP (purple), and PKC (blue) from single cells.

Article Snippet: The constructs of JNK KTR-rsGreenFast-E, P38 KTR-Dronpa, ERK KTR-rsFastLime were built based on the original KTRs constructs JNK KTR-Clover (Addgene plasmid 59151), P38 KTR-mCerulean3 (Addgene plasmid 59155), ERK KTR-Clover (Addgene plasmid 59150), all gift of Markus W. Covert ( ).

Techniques: Activity Assay