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: Frontiers in Cell and Developmental Biology

Article Title: Cellular Senescence in Sarcopenia: Possible Mechanisms and Therapeutic Potential

doi: 10.3389/fcell.2021.793088

Figure Lengend Snippet: Therapeutic interventions based on specific molecules on the signaling pathway of cellular senescence accounting for sarcopenia.

Article Snippet: In this regard, pharmacological inhibition of the p38 MAPK pathway by SB203580 (Tocris) or SB202190 (EMD Chemicals) ameliorates age-associated sarcopenia and reveals a potential therapy for the treatment of progressive muscle wasting ( ; ).

Techniques: Inhibition

Journal: bioRxiv

Article Title: ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow

doi: 10.1101/2022.09.10.507283

Figure Lengend Snippet: A . Quantification of p38 phosphorylation by Western blot in HUVECs expressing a non-targeting shRNA (shNT) or a shRNA targeting Arhgef18 (shA18.1), under static condition or exposed to physiological SS (PSS). Static: N=3; PSS: N=8. **p<0.01; ***p<0.001 2-way ANOVA. B . Quantification of cell orientation and elongation with the flow in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR), exposed to physiological SS. 24h of SS. Each dot represents a cell. N=4 with in-between 200 and 450 cells analyzed by experiment. ***p<0.001; Mann-Whitney T-test. C . Quantification of Actin localized at junction in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR), exposed to physiological SS. 24h of SS. N=2, 10 to 14 areas analyzed by experiment. **p<0.01; Unpaired T-test. D . Representative images of HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR) exposed to physiological SS. 24 and stained for ZO-1 (green), VE-cadherin (magenta) and actin (red). Flow direction is indicated by the arrow. E . Quantification of ZO-1 localized at junction in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR) exposed to physiological SS. 24h of SS. N=2, 10 to 15 areas analyzed by experiment. ***p<0.001; Unpaired T-test. F . Representative images of Paxillin staining in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR), exposed to physiological SS. Flow direction is indicated by the arrow. Green: paxillin; magenta: VE-cadherin; red: Actin. G . Quantification of focal adhesion type by hand classification of paxillin staining within each cell based on the representative images for each category on the left (white box: paxillin high = numerous, elongated and aligned focal adhesions; black box: paxillin low = few, short and misaligned focal adhesions). N=2. Quantified blindly.

Article Snippet: For p38 MAPK inhibition studies, confluent HUVEC were pretreated with p38 MAPK specific inhibitor (100nM, SB239063; TOCRIS) for at least 1hr prior to physiological SS, treatment was maintained during the flow stimulation.

Techniques: Phospho-proteomics, Western Blot, Expressing, shRNA, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow

doi: 10.1101/2022.09.10.507283

Figure Lengend Snippet: A . Representative blot of ARHGEF18 binding on nucleotide-free RhoA (GST-RhoA G17A ) by pull-down assay with HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18. B . Quantification of p38 phosphorylation by Western blot in HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18. N=4. Paired T-test. C . Representative images of HUVECs HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18 and stained VE-cadherin. Flow direction is indicated by the arrow. D . Quantification of cell orientation and elongation with the flow in HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18. Each dot represents a cell. N=1 with in-between 1000 and 1350 cells analyzed by experiment. **p<0.01; Unpaired T-test. E. Representative images of ZO-1 staining in a mix population of WT cells (FLAG negative, non-red) and cells over-expressing the mutant form (A18 Y260A ) of ARHGEF18 (FLAG positive, red), exposed to physiological SS. Green: ZO-1; red: FLAG; Magenta: VE-cadherin. F. Quantification of the ZO-1 staining at junction using plot profile analysis. N=1, 25 couples of cells analyzed per experiment.

Article Snippet: For p38 MAPK inhibition studies, confluent HUVEC were pretreated with p38 MAPK specific inhibitor (100nM, SB239063; TOCRIS) for at least 1hr prior to physiological SS, treatment was maintained during the flow stimulation.

Techniques: Binding Assay, Pull Down Assay, Expressing, Mutagenesis, Phospho-proteomics, Western Blot, Staining

Journal: Frontiers in Immunology

Article Title: Macrophage-derived macrophage migration inhibitory factor mediates renal injury in anti-glomerular basement membrane glomerulonephritis

doi: 10.3389/fimmu.2024.1361343

Figure Lengend Snippet: Selective MIF depletion in macrophages ameliorates experimental anti- GBM GN by inactivating NF-kB and p38/MAPK signaling and inhibiting M1 macrophages activation. (A) Western blots. (B–E) Statistics data of the protein expression (iNOS, CD74, pp65/p65 and pp38/p38). Each bar represents mean ± SEM. Each dot represents one mouse. *p <0.05, ***p<0.001 versus corresponding control; # p<0.05, ## p < 0.01, ### p< 0.001 versus corresponding MIF f/f .

Article Snippet: After blocking nonspecific binding with 5% BSA, membranes were incubated overnight at 4 ° C with the primary antibodies against rabbit anti-iNOS (Abcam ab-15323), rabbit anti-iNOS (Abcam ab-178945), goat anti-CD74 (Santa Cruz, sc-5438), mouse anti-CD74 (Santa Cruz, sc-6267), goat anti-CD74 (Santa Cruz, sc-5438), rabbit anti-phosphorylated NF-kB p65 (Cell Signaling, #3031), rabbit anti-phosphorylated NF-kB p65 (Cell Signaling, #3033s), mouse anti-NF-kB p65 (Cell Signaling, #6965), rabbit anti-NF-kB p65 (Cell Signaling, # 8242S), rabbit anti-pp38 (Cell signaling, #9211), rabbit anti-pp38 (Cell signaling, # 4631L), rabbit anti-p38 (Cell signaling, #9212), rabbit anti-p38 (Cell signaling, # 8690S), mouse anti-ß-actin (Santa Cruz, sc-69879), mouse anti-GAPDH(proteintech,#60004–1-Ig).Then the membranes were incubated with IRDye800-conjugated secondary antibody (Rockland Immun- chemicals).

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Macrophage-derived macrophage migration inhibitory factor mediates renal injury in anti-glomerular basement membrane glomerulonephritis

doi: 10.3389/fimmu.2024.1361343

Figure Lengend Snippet: Bone marrow-derived macrophages (BMDM) lacking MIF show inhibition of MIF signaling and M1 macrophage activation in response to TNFa in vitro . (A) Western blots; (B–E) Statistics data of the protein expression (CD74, pp65/p65, pp38/p38, INOS). Results show that bone marrow-derived macrophages (BMDMs) isolated MIF KO mice inhibit TNF-α (10ng/ml)-induced upregulation of MIF signaling by downregulating CD74 expression and activation of NF-kB/p65 and p38/MAPK, there by inhibiting M1 macrophage activation by suppressing iNOS expression. Each bar represents mean±SEM. Each dot represents one mouse. ***P < 0.001 compared with MIF WT mice; ## P < 0.01, ### p< 0.001 compared with MIF WT mice treated with TNF-a 10ng/ml.

Article Snippet: After blocking nonspecific binding with 5% BSA, membranes were incubated overnight at 4 ° C with the primary antibodies against rabbit anti-iNOS (Abcam ab-15323), rabbit anti-iNOS (Abcam ab-178945), goat anti-CD74 (Santa Cruz, sc-5438), mouse anti-CD74 (Santa Cruz, sc-6267), goat anti-CD74 (Santa Cruz, sc-5438), rabbit anti-phosphorylated NF-kB p65 (Cell Signaling, #3031), rabbit anti-phosphorylated NF-kB p65 (Cell Signaling, #3033s), mouse anti-NF-kB p65 (Cell Signaling, #6965), rabbit anti-NF-kB p65 (Cell Signaling, # 8242S), rabbit anti-pp38 (Cell signaling, #9211), rabbit anti-pp38 (Cell signaling, # 4631L), rabbit anti-p38 (Cell signaling, #9212), rabbit anti-p38 (Cell signaling, # 8690S), mouse anti-ß-actin (Santa Cruz, sc-69879), mouse anti-GAPDH(proteintech,#60004–1-Ig).Then the membranes were incubated with IRDye800-conjugated secondary antibody (Rockland Immun- chemicals).

Techniques: Derivative Assay, Inhibition, Activation Assay, In Vitro, Western Blot, Expressing, Isolation

Journal: Journal of biomolecular screening

Article Title: Discovery and Characterization of a Biologically Active Non–ATP-Competitive p38 MAP Kinase Inhibitor

doi: 10.1177/1087057115615518

Figure Lengend Snippet: Novel p38 kinase inhibitors discovered during the screening campaign. Numbered as referenced within the article text: chemical structures, IUPAC name, and measured p38 IC50 value.

Article Snippet: Gels were then transferred to nitrocellulose membrane by electrophoresis in Novex transfer buffer for 1 h at 100 V. Membranes were then blocked for 1 h in Licor blocking buffer (Licor Technologies, Omaha, NE) and incubated overnight with the following antibody combinations: (1) phospho-MNK1 (CST, rabbit polyclonal No. 2111), (2) total p38 (CST, rabbit polyclonal No. 9212)/tubulin (CST, mouse monoclonal No. 3873), (3) phospho-ERK (CST, rabbit polyclonal No. 9101)/tubulin.

Techniques:

Journal: Journal of biomolecular screening

Article Title: Discovery and Characterization of a Biologically Active Non–ATP-Competitive p38 MAP Kinase Inhibitor

doi: 10.1177/1087057115615518

Figure Lengend Snippet: Compound 1 inhibits p38-dependent phosphorylation of MNK1. Anisomycin was used to stimulate p38 activity in the presence of compound 1. Compound 1 was able to achieve intracellular p38 inhibition without reducing ERK phosphorylation or phosphorylation of p38 itself.

Article Snippet: Gels were then transferred to nitrocellulose membrane by electrophoresis in Novex transfer buffer for 1 h at 100 V. Membranes were then blocked for 1 h in Licor blocking buffer (Licor Technologies, Omaha, NE) and incubated overnight with the following antibody combinations: (1) phospho-MNK1 (CST, rabbit polyclonal No. 2111), (2) total p38 (CST, rabbit polyclonal No. 9212)/tubulin (CST, mouse monoclonal No. 3873), (3) phospho-ERK (CST, rabbit polyclonal No. 9101)/tubulin.

Techniques: Activity Assay, Inhibition

Journal: Journal of biomolecular screening

Article Title: Discovery and Characterization of a Biologically Active Non–ATP-Competitive p38 MAP Kinase Inhibitor

doi: 10.1177/1087057115615518

Figure Lengend Snippet: Compound 1 reduces p38-dependent T-cell proliferation and cytokine secretion. (A) Effect of compound 1 treatment on T-cell receptor (anti-CD3/CD28) and stress-stimulated (PMA + ionomycin [P+I]) T-cell proliferation, as measured by [3H] thymidine incorporation. (B) Effect of compound 1 treatment on T-cell receptor or stressor-mediated tumor necrosis factor–α production. N.D. indicates analyte was not detected.

Article Snippet: Gels were then transferred to nitrocellulose membrane by electrophoresis in Novex transfer buffer for 1 h at 100 V. Membranes were then blocked for 1 h in Licor blocking buffer (Licor Technologies, Omaha, NE) and incubated overnight with the following antibody combinations: (1) phospho-MNK1 (CST, rabbit polyclonal No. 2111), (2) total p38 (CST, rabbit polyclonal No. 9212)/tubulin (CST, mouse monoclonal No. 3873), (3) phospho-ERK (CST, rabbit polyclonal No. 9101)/tubulin.

Techniques:

Journal: Journal of biomolecular screening

Article Title: Discovery and Characterization of a Biologically Active Non–ATP-Competitive p38 MAP Kinase Inhibitor

doi: 10.1177/1087057115615518

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) indicates direct binding of compound 1 to p38. (Upper) Isothermal titration of compound 1 with p38. (Lower) Normalized binding isotherm of compound 1 interacting with p38 indicating a 1:1 molar relationship between p38 and compound 1.

Article Snippet: Gels were then transferred to nitrocellulose membrane by electrophoresis in Novex transfer buffer for 1 h at 100 V. Membranes were then blocked for 1 h in Licor blocking buffer (Licor Technologies, Omaha, NE) and incubated overnight with the following antibody combinations: (1) phospho-MNK1 (CST, rabbit polyclonal No. 2111), (2) total p38 (CST, rabbit polyclonal No. 9212)/tubulin (CST, mouse monoclonal No. 3873), (3) phospho-ERK (CST, rabbit polyclonal No. 9101)/tubulin.

Techniques: Isothermal Titration Calorimetry, Binding Assay, Titration

Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 2A, a Negative Regulator of the ERK Signaling Pathway, Is Activated by Tyrosine Phosphorylation of Putative HLA Class II-associated Protein I (PHAPI)/pp32 in Response to the Antiproliferative Lectin, Jacalin

doi: 10.1074/jbc.m400017200

Figure Lengend Snippet: FIG. 2. Effect of jacalin (JAC) on MAPK activity. HT29 cells treated with 30 g/ml jacalin for various times were lysed, and 50 g/lane of proteins were separated by 10% SDS-PAGE followed by immunoblotting with antibodies against phospho-MEK1/2 (p-MEK1/2) (A, top panel), phospho-ERK1/2 (p-ERK1/2) (A, third panel), phospho- JNK (p-JNK) (A, fifth panel), phospho-p38 (p-p38) (A, seventh panel). Each blot was subsequently stripped and reblotted with antibodies against MEK1/2 (A, second panel), ERK2 (A, fourth panel), JNK1 (A, sixth panel), or p38 (A, eighth panel). Jacalin induced inhibition of p-MEK1/2 and p-ERK1/2 but not p-JNK and p-p38. On the figure, the circled p indicates phosphorylation. In B, the cells were incubated with or without 30 g/ml jacalin in the presence or absence of 100 g/ml asialo-fetuin (ASF) or fetuin for 10 min (B, top two panels) or 60 min (B, bottom two panels) before lysis of the cells and immunoblotted with antibodies against p-MEK1/2 (B, first panel) or p-ERK1/2 (B, third panel). Each blot was subsequently stripped and reprobed with anti- bodies against MEK1/2 (B, second panel) or ERK2 (B, fourth panel). The presence of asialo-fetuin or fetuin almost completely abolished jacalin- induced decrease of p-MEK1/2 and p-ERK1/2.

Article Snippet: Polyclonal antibodies against phospho-p38 (Thr180/Tyr182), phospho-MEK1/2 (Ser217/Ser221), and MEK1/2 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay, SDS Page, Western Blot, Inhibition, Phospho-proteomics, Incubation, Lysis

Journal: The FASEB Journal

Article Title: Fructose induces inflammatory activation in macrophages and microglia through the nutrient‐sensing ghrelin receptor

doi: 10.1096/fj.202402531R

Figure Lengend Snippet: Ghsr Inhibition suppresses fructose‐induced CREB–AKT, NF‐κB p65, and p38 MAPK signaling cascades in macrophages and microglia. WT & Ghsr ‐mutant ( Ghsr mutant ) RAW 264.7 macrophages and IMG microglia were treated with fructose (+Fru) for 24 h. (A, B) Representative immunoblot images and quantification results of p‐CREB (Ser133), CREB, p‐AKT (Ser473), p‐AKT (Thr308), AKT, p‐NF‐κB p65 (Ser536), and NF‐κB p65 in RAW 264.7 (A) and IMG (B). (C, D) Representative immunoblot images and quantification results of p‐p38 MAPK (Thr180/Tyr182) and p38 MAPK in RAW 264.7 (C) or IMG (D). Data were presented as mean ± SEM, # p < .05; ## p < .01; ### p < .001, +Fru vs. −Fru; * p < .05; ** p < .01; *** p < .001, Ghsr mutant vs. WT. (E) The schematic diagram illustrates the effect of GHSR on fructose‐induced inflammatory pathways of RAW 264.7 macrophages and IMG microglia. The dashed arrow from GHSR to p38 indicates that the regulation of p38 by GHSR is likely to involve multiple steps.

Article Snippet: Primary antibodies for AKT (Cat# 4691), AMPKα (Cat# 2603), β‐Actin (Cat# 4970), CREB (Cat# 9197), NF‐κB p65 (Cat# 8242), p38 MAPK (Cat# 9212), phospho‐AKT‐Ser473 (Cat# 9271), phospho‐AKT‐Thr308 (Cat# 4056), phospho‐AMPKα‐Thr172 (Cat# 2535), phospho‐CREB‐Ser133 (Cat# 9198), phospho‐NF‐κB p65‐Ser536 (Cat# 3033), and phospho‐p38 MAPK‐Thr180/Tyr182 (Cat# 4511) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Mutagenesis, Western Blot

Journal: The FASEB Journal

Article Title: Fructose induces inflammatory activation in macrophages and microglia through the nutrient‐sensing ghrelin receptor

doi: 10.1096/fj.202402531R

Figure Lengend Snippet: The proposed model illustrating the role of GHSR in fructose‐induced pro‐inflammatory activation in macrophages and microglia. In this study, we demonstrated that fructose exposure promotes inflammatory activation in macrophages and microglia. GHSR is involved in three aspects of the regulation: (1) GHSR stimulates the CREB–AKT and NF‐κB pro‐inflammatory signaling pathways, which are essential for fructose‐induced inflammation (noted as number 1 in the diagram); (2) GHSR expression directly or indirectly regulates fructose uptake and metabolism by promoting GLUT5 and KHK. This regulation alters the intracellular energy balance and AMP/ATP ratio, and the upregulation of AMP can subsequently promote inflammation via the AMPK–AKT and p38 pathways (noted as number 2 in the diagram); (3) Fructose increases Ghsr gene expression in macrophages and microglia potentially through CREB, resulting in a positive feedback loop that further exacerbates the inflammatory state (noted as number 3 in the diagram).

Article Snippet: Primary antibodies for AKT (Cat# 4691), AMPKα (Cat# 2603), β‐Actin (Cat# 4970), CREB (Cat# 9197), NF‐κB p65 (Cat# 8242), p38 MAPK (Cat# 9212), phospho‐AKT‐Ser473 (Cat# 9271), phospho‐AKT‐Thr308 (Cat# 4056), phospho‐AMPKα‐Thr172 (Cat# 2535), phospho‐CREB‐Ser133 (Cat# 9198), phospho‐NF‐κB p65‐Ser536 (Cat# 3033), and phospho‐p38 MAPK‐Thr180/Tyr182 (Cat# 4511) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Platelet-derived microparticles stimulate the invasiveness of colorectal cancer cells via the p38MAPK-MMP-2/MMP-9 axis

doi: 10.1186/s12964-023-01066-8

Figure Lengend Snippet: Effect of PMPs on p38MAPK phosphorylation. A Representative Western blots of phosphorylated p38MAPK (phospho-p38) and total p38MAPK (p38) in CRC cells before and after 10 min of incubation with PMPs. B Normalized densitometric values of phospho-p38 and the total p38MAPK ratio. Quantified data are presented and statistically analysed as in Fig. . *** P < 0.001, * P < 0.05, N = 5

Article Snippet: The lysates (25 μg of protein) were then separated on an SDS‒PAGE 10% polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with rabbit anti-human antibodies against nonphosphorylated p38MAPK (Cell Signaling) and ERK1/2 (Thermo Fisher Scientific) and phosphorylated p38MAPK (Cell Signaling) or mouse anti-human antibodies against CXCR4 (Thermo Fisher Scientific), MMP-2, MMP-9 (Thermo Fisher Scientific) and phosphorylated ERK1/2 (Thermo Fisher Scientific), followed by incubation with secondary goat anti-mouse or goat-anti-rabbit IgG (Santa Cruz Biotechnology) conjugated with horseradish peroxidase (HRP) (Additional file : Supplementary Methods).

Techniques: Phospho-proteomics, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Platelet-derived microparticles stimulate the invasiveness of colorectal cancer cells via the p38MAPK-MMP-2/MMP-9 axis

doi: 10.1186/s12964-023-01066-8

Figure Lengend Snippet: Inhibition of p38MAPK phosphorylation and its effect on CRC cell migration and MMP-2/MMP-9 level/release after PMP uptake. A Representative Western blots of phospho-p38MAPK in CRC cells after 10 min of incubation with PMPs in the absence or presence of the inhibitor SB202190. B Migration of CRC cells through gelatine-coated Boyden chambers after 4 h of incubation with the inhibitor SB202190. Quantified data (presented and statistically analysed as in Fig. ) of the percentage of gelatine-migrated cells compared to the control (nontreated cells, 100%, marked with dashed horizontal bars). * P < 0.05, N = 5. C Representative Western blots of MMP-2 and MMP-9 in CRC cells after 4 h of incubation with the inhibitor SB202190. D Amount of MMP-9 released from CRC into conditioned medium after 4 h of incubation with PMPs in the absence or presence of the inhibitor SB202190, as measured by ELISA. The data are presented and statistically analysed as in Fig. . ** P < 0.01, * P < 0.05. N = 6

Article Snippet: The lysates (25 μg of protein) were then separated on an SDS‒PAGE 10% polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with rabbit anti-human antibodies against nonphosphorylated p38MAPK (Cell Signaling) and ERK1/2 (Thermo Fisher Scientific) and phosphorylated p38MAPK (Cell Signaling) or mouse anti-human antibodies against CXCR4 (Thermo Fisher Scientific), MMP-2, MMP-9 (Thermo Fisher Scientific) and phosphorylated ERK1/2 (Thermo Fisher Scientific), followed by incubation with secondary goat anti-mouse or goat-anti-rabbit IgG (Santa Cruz Biotechnology) conjugated with horseradish peroxidase (HRP) (Additional file : Supplementary Methods).

Techniques: Inhibition, Phospho-proteomics, Migration, Western Blot, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p < 0.05 compared to the negative control group.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Inhibition, Western Blot, Phospho-proteomics, In Vitro, Migration, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon up-regulates p38β expression in hepatic cells. Pokemon was delivered by expression plasmid pcDNA3.1(−)-Pokemon with pcDNA3.1(−) as a negative control. Pokemon silencing was triggered by si-RNA. ( a ) Targeted expression or silencing of Pokemon in HepG2 cells. Cells were collected at 60 h after transfection or silencing (Left panel). And the quantification of western blot data was displayed on the Right panel; ( b ) Real-time quantitative polymerase chain reaction (qPCR) at 48 h after transfection in HepG2 cells. Upper panel: Ectopic expression of Pokemon; lower panel: Silencing of Pokemon; ( c ) Ectopic expression of Pokemon in HL7702 cells. Upper panel: Western blot in which cells were lysed and total proteins were collected at 36, 48, 60 and 72 h, respectively. Lower panel: Real-time qPCR at 48 and 60 h after transfection; ( d ) Silencing of Pokemon in BEL7404 cells. Upper panel: Western blot; lower panel: Real-time qPCR. * p < 0.05 compared to the negative control. N.C. means negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon stimulates p38β promoter activity. ( a ) ChIP assays in HepG2 and BEL7404 cells. Protein-DNA complexes are immunoprecipitated either with anti-Pokemon antibody or anti-IgG as negative control, followed by PCR with primers specific to p38β promoter sequence and agarose-gel electrophoresis for visualization. Total lysates were used as the input samples and positive control; ( b ) Dual luciferase reporter assay. Luciferase activities were normalized to Renilla activity. Y axis stands for the relative fold changes of activity as the pcDNA3.1(−)-Pokemon plasmid increases. * p < 0.05 compared to the negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Activity Assay, Immunoprecipitation, Negative Control, Sequencing, Agarose Gel Electrophoresis, Positive Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Arthritis and rheumatism

Article Title: Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.

doi: 10.1002/art.21009

Figure Lengend Snippet: Figure 4. Effects of IgG aPL on A, the phosphorylation of p38 MAPK and B, the induction of inducible nitric oxide synthase (iNOS). HUVECs were grown to confluence and treated for A, 120 minutes (p38 MAPK experiments; IgG aPL samples 1–6 and IgG NHS samples 1 and 2) or B, 60 minutes (iNOS experiments; IgG aPL samples 1–4 and IgG NHS sample 1) with 100 g/ml of IgG aPL, 100 g/ml of IgG NHS, or 2 g/ml of LPS. Western blot analyses to determine the phosphorylation of p38 MAPK and the expression of iNOS were performed as described in Materials and Methods. Actin was used as the housekeeping gene. Bands were quantified by densitometric analysis as described in Materials and Methods. See Figure 1 for other definitions.

Article Snippet: The membranes were blocked in 5% dry milk, incubated with a 1:2,000 dilution of mouse monoclonal anti-human phosphorylated p38 MAPK (Thr180/Tyr182) (antibody 28b10; Cell Signaling Technology, Beverly, MA) or with rabbit polyclonal anti-human NOS2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by a 1-hour incubation in a 1:2,000 dilution of horseradish peroxidase– conjugated goat anti-mouse or goat anti-rabbit IgG.

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Arthritis and rheumatism

Article Title: Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.

doi: 10.1002/art.21009

Figure Lengend Snippet: Figure 6. Proposed intracellular events in antiphospholipid antibody (aPL)–activated endothelial cells (ECs). The diagram shows some intracellular pathways mediated by aPL in ECs and the effects of specific inhibitors. ? uncertain; activation; – inhibition; P phosphorylation; broken arrows unconfirmed pathway; solid ar- rows confirmed pathway; IL-1 interleukin-1; p38 MAPK p38 mitogen-activated protein kinase; iNOS inducible nitric oxide synthase; NO nitric oxide; ICAM-1 intercellular adhesion mole- cule 1; VCAM-1 vascular cell adhesion molecule 1.

Article Snippet: The membranes were blocked in 5% dry milk, incubated with a 1:2,000 dilution of mouse monoclonal anti-human phosphorylated p38 MAPK (Thr180/Tyr182) (antibody 28b10; Cell Signaling Technology, Beverly, MA) or with rabbit polyclonal anti-human NOS2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by a 1-hour incubation in a 1:2,000 dilution of horseradish peroxidase– conjugated goat anti-mouse or goat anti-rabbit IgG.

Techniques: Activation Assay, Inhibition, Phospho-proteomics

Journal: Cell Reports Medicine

Article Title: Drug screening in 3D microtumors reveals DDR1/2-MAPK12-GLI1 as a vulnerability in cancer-associated fibroblasts

doi: 10.1016/j.xcrm.2025.102357

Figure Lengend Snippet: Doramapimod unexpectedly targets DDR1/2 and MAPK12, regulating extracellular matrix gene expression in cancer-associated fibroblasts (A) Kinome profiling of doramapimod . Left: residual kinase activity for 370 kinases treated with 500 nM doramapimod using a radioactive ATP assay. Kinases with <20% residual activity are indicated in red. Right: bar chart highlighting top inhibited kinases. (B) CAF gene expression after kinase knockdown . Heatmap showing changes in ACTA2 and CXCL12 expression in breast cancer-derived CAFs following siRNA knockdown of doramapimod target kinases. (C) Plot showing qPCR analysis of CXCL12 expression in breast CAFs following siRNA-mediated knockdown of DDR1, DDR2, and MAPK12, with or without doramapimod treatment. Data represent mean ± SEM; n = 3 per group. Student’s t test, compared with DMSO control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (D) Transcriptional changes in CAFs upon DDR1 , DDR2 , or MAPK12 depletion . Volcano plots showing DEGs (>2-fold, p < 0.05) after siRNA knockdown of DDR1 (left), DDR2 (middle), or MAPK12 (right) in primary breast CAFs. Downregulated genes are shown in blue and upregulated in red. n = 3 per group. (E) Pathway enrichment of downregulated genes following combined DDR1/2 and MAPK12 knockdown . Bar graph showing enrichment across Gene Ontology, Reactome, and KEGG pathways, with ECM-related processes highlighted in red. (F) Neutralization of CAFs’ growth stimulatory effect through depletion of DDR1/2 and MAPK12 kinase expression . Top: schematic of the experimental design showing breast CAFs treated with siRNAs against the indicated kinases, then co-cultured with 4T1 cancer cells labeled with nuclear GFP. Bottom: growth curve of 4T1 cells co-cultured with kinase depleted CAFs, displayed as Mean ± SEM. n = >3 in each group. (G) DDR1/2 enhances p38 phosphorylation . Western blot analysis showing elevated levels of phospho-p38 in CAFs overexpressing DDR1 or DDR2 compared to GFP control. Total p38 and β-actin are shown as loading controls. (H) Proposed model of doramapimod action . Doramapimod inhibits the DDR1/2–MAPK12 signaling axis, which drives ECM production in CAFs.

Article Snippet: Rabbit monoclonal Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Antibody , Cell Signaling Technology , Cat#4631; RRID:AB_331765.

Techniques: Gene Expression, Activity Assay, ATP Assay, Knockdown, Expressing, Derivative Assay, Control, Neutralization, Cell Culture, Labeling, Phospho-proteomics, Western Blot

Journal: Cell Reports Medicine

Article Title: Drug screening in 3D microtumors reveals DDR1/2-MAPK12-GLI1 as a vulnerability in cancer-associated fibroblasts

doi: 10.1016/j.xcrm.2025.102357

Figure Lengend Snippet: DDR1/2 and MAPK12 converge at GLI to regulate ECM production and support cancer cell growth (A) Gene overlap and expression analysis . Left: a Venn diagram showing the overlap of genes downregulated in response to knockdown of DDR1/2 and MAPK12 in CAFs. Right: a heatmap displaying changes in the expression of 7 genes commonly downregulated by depletion of DDR1/2 and MAPK12. The transcriptional factors, ECM proteins, and immune regulatory roles of these genes are highlighted. (B) Doramapimod reduces GLI1 nuclear localization in CAFs . Left: representative immunofluorescence images showing reduced nuclear GLI1 signal in CAFs following doramapimod treatment (1 μM for 48 h). Right: quantification of nuclear GLI1 intensity from at least 20 cells per group, indicating significantly reduced nuclear localization. Scale bars, 20 μm; ∗∗ p < 0.01; Student’s t test. (C) GLI1 transcriptional activity is inhibited by doramapimod , DDR1/2 , and MAPK12 knockdown . Left: luciferase reporter assay in pancreatic CAFs shows decreased GLI1 transcriptional activity upon treatment with doramapimod (1 μM) or GANT61 (1 μM). Right: similar reduction in GLI1 activity observed upon knockdown of DDR1, DDR2, or MAPK12. Data represent mean ± SEM from two-three biological replicates; ∗ p < 0.05 and ∗∗ p < 0.01; unpaired Student’s t test. (D) GLI1 inhibition downregulates ECM-associated pathways . Pathway enrichment plots based on RNA-seq of breast and pancreatic CAFs treated with GANT61. Genes involved in ECM regulation, including integrin signaling, collagen formation, and matrix remodeling, are significantly downregulated (adjusted p values indicated by color scale). (E) DDR1/2 promotes nuclear localization of GLI . Representative images showing that overexpression of DDR1/2 in normal human pancreatic fibroblasts promotes nuclear localization of phosphorylated p38 MAPK and GLI1. Scale bars, 20 μm. (F) Neutralization of CAFs' growth stimulatory effect by GLI depletion . Left: plot showing growth of GFP-labeled 4T1 cancer cells on CAFs with intact or depleted levels of GLI1, displayed as mean ± SEM. N = >3 in each group. Student’s t tests with Holm-Sidak correction. ∗∗ p < 0.01. Right: Gant61 treatment does not affect 4T1 cancer cell growth directly in serum-supported monoculture, but significantly reduces 4T1 cell growth when co-cultured with CAFs in serum-free condition, displayed as mean ± SEM. n indicates at least 3 replicates in each group. (G) M odel of the non-canonical hedgehog pathway in CAFs . Schematic illustrating DDR1/2-mediated activation of p38/MAPK12 and GLI drives ECM production in CAFs and supports cancer cell growth.

Article Snippet: Rabbit monoclonal Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Antibody , Cell Signaling Technology , Cat#4631; RRID:AB_331765.

Techniques: Expressing, Knockdown, Immunofluorescence, Activity Assay, Luciferase, Reporter Assay, Inhibition, RNA Sequencing, Over Expression, Neutralization, Labeling, Cell Culture, Activation Assay

Journal: Developmental biology

Article Title: Regulation of vertebrate myotome development by the p38 MAP kinase-MEF2 signaling pathway.

doi: 10.1016/j.ydbio.2005.04.009

Figure Lengend Snippet: Fig. 2. Expression and activity of p38 MAP kinase in the 9.5 dpc mouse somite. Upper panel shows the expression of p38 MAPK by Western immunoblotting in muscle (M), brain (B), heart (H), somites vi–x (S vi–x), somites (i–v), and in the unsegmented paraxial mesoderm (upm). Lower panel shows p38 MAP kinase activity indicated by phosphorylation of a bacterially produced ATF2 substrate by p38 MAPK immunoprecipitated from the indicated tissues. UPM — unsegmented paraxial mesoderm, UPM NT—UPM with neural tube, S i–v—somites i–v, SNT i–v—somites i–v with neural tube, S vi–x—somites vi–x, SNT somites vi–x with neural tube, C2 ANI—positive control of C2C12 myoblasts treated with anisomycin (a known activator of p38 MAPK).

Article Snippet: A kinase assay was performed using the p38 MAP Kinase Assay Kit (Cell Signaling).

Techniques: Expressing, Activity Assay, Western Blot, Phospho-proteomics, Produced, Immunoprecipitation, Positive Control

Journal: Developmental biology

Article Title: Regulation of vertebrate myotome development by the p38 MAP kinase-MEF2 signaling pathway.

doi: 10.1016/j.ydbio.2005.04.009

Figure Lengend Snippet: Fig. 4. P38 MAPK inhibition blocks the induction of a myotomal myogenic marker gene (MLC3F). The mouse picture indicates the expression of the MLC3F LacZ transgene in the 9.5 dpc embryo (previously reported in Kelly et al., 1995). Monolayer cultures derived from tissue dissected from the interlimb region were incubated with the diluent (Control) or with the SB203580 inhibitor for 3–5 days in culture and then stained with X gal (representative data from 6 independent experiments). Panel B shows the results of placental injection of the p38 MAPK inhibitor (SB203580). In some cases, we observed the SB203580-injected embryos to be smaller than the controls. We have normalized the size to allow details of the embryo to be properly observed.

Article Snippet: A kinase assay was performed using the p38 MAP Kinase Assay Kit (Cell Signaling).

Techniques: Inhibition, Marker, Expressing, Derivative Assay, Incubation, Control, Staining, Injection

Journal: Developmental biology

Article Title: Regulation of vertebrate myotome development by the p38 MAP kinase-MEF2 signaling pathway.

doi: 10.1016/j.ydbio.2005.04.009

Figure Lengend Snippet: Fig. 3. A p38 MAPK inhibition abrogates MEF2 activity in the MEF2 sensor mouse. The mouse picture indicates the expression of the MEF2 LacZ transgene in the 9.5 dpc embryo (previously reported in Naya et al., 1999). Explants dissected from the interlimb region were incubated with the diluent (Control) or with the SB203580 inhibitor for 2 days and then stained with X gal (representative data from 3 independent experiments). Panel B shows representative data from a 48-h whole embryo incubation with or without the p38 MAPK inhibitor (10 AM). This experiment was repeated three times. H indicates the heart field, S indicates the somite region.

Article Snippet: A kinase assay was performed using the p38 MAP Kinase Assay Kit (Cell Signaling).

Techniques: Inhibition, Activity Assay, Expressing, Incubation, Control, Staining

Journal: Developmental biology

Article Title: Regulation of vertebrate myotome development by the p38 MAP kinase-MEF2 signaling pathway.

doi: 10.1016/j.ydbio.2005.04.009

Figure Lengend Snippet: Fig. 5. Activation of the Myf 5 locus is unaffected by P38 MAPK inhibition. The mouse picture indicates the expression of the Myf 5nLacZ transgene in the 9.5 dpc embryo (previously reported in Tajbakhsh et al., 1996a). Explants dissected from the interlimb region were incubated with the diluent (Control) or with the SB203580 inhibitor for 2 days and then stained with X gal. Two independent experiments are indicated. Panel B indicates the effect of incubating Myf 5 nLacZ somite cultures with the inactive analog of SB203580 (SB203474). Panel C shows representative results of placental injection of the p38 MAPK inhibitor on the expression of the Myf 5nLacZ transgene.

Article Snippet: A kinase assay was performed using the p38 MAP Kinase Assay Kit (Cell Signaling).

Techniques: Activation Assay, Inhibition, Expressing, Incubation, Control, Staining, Injection