Cell-to-Cell Heterogeneity in p38-Mediated Cross-Inhibition of JNK Causes Stochastic Cell Death  Haruko Miura, Yohei Kondo, Michiyuki Matsuda, Kazuhiro Aoki  Cell Reports  Volume 24, Issue 10, Pages 2658-2668 (September 2018) DOI: 10.1016/j.celrep.2018.08.020 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Establishment of a Multiplexed Imaging System for Monitoring JNK and p38 Kinase Activities (A) Schematic of the phosphorylation-mediated translocation of a KTR. (B) HeLa cells stably expressing p38 KTR-mEGFP and mEGFP-MK2, respectively, were stimulated with 1 μg/mL anisomycin. Representative images are shown at left. Scale bar: 20 μm. Mean C:N ratios with SDs are plotted as a function of time (right). n = 38 cells for p38 KTR-mEGFP, n = 34 cells for mEGFP-MK2 from two independent experiments. (C) Clonal HeLa cells stably expressing JNK KTR-mCherry, mKO-MK2, and NLS-iRFP-NLS were stimulated with 1 μg/mL anisomycin and imaged over time. Scale bar: 20 μm. (D and E) Clonal HeLa reporter cells were stimulated with 1 μg/mL anisomycin and treated with 10 μM JNK inhibitor VIII (JNKiVIII), 10 μM of the p38 inhibitor SB203580 (SB), or 0.1% DMSO at the indicated time point (arrow). Mean JNK KTR-mCherry (D) and mKO-MK2 (E) C:N ratios with SDs are shown. n = 40 cells for each condition from two independent experiments. (F and G) Dose-response curves of the JNK KTR-mCherry (F) and mKO-MK2 (G) C:N ratios after 1 hr of anisomycin stimulation are shown. Experimental data were fitted with the Hill function and the derived EC50 values and Hill coefficients (nHs) are indicated. Data are presented as the mean with SD n ≥ 100 cells for each condition, from at least three independent experiments. (H and I) JNK KTR-mCherry (H) and mKO-MK2 (I) C:N ratios linearly correlate with normalized p-JNK/JNK and p-p38/p38 ratios, respectively. Imaging and western blot data were obtained after 1 hr of treatment with various doses of anisomycin and/or SB203580. Scatterplots were fitted to a linear regression, and the obtained Pearson correlation values are shown. Data are presented as the mean with SD n = 3 independent experiments for western blot data and n ≥ 60 cells for imaging data from three independent experiments. See also Figure S1. Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Transcriptional and Post-translational Cross-Inhibition of JNK Activity by p38 under Various Stress Conditions (A) Clonal HeLa cells stably expressing JNK KTR-mCherry, mKO-MK2, and NLS-iRFP-NLS were pretreated with 0.1% DMSO and 10 μM SB203580, respectively, and then stimulated with 200 mM sorbitol, 10 ng/mL TNF-α, 10 ng/mL IL-1β, 100 J/m2 UV-C, 10 ng/mL anisomycin, or imaging medium as a control. Representative images of JNK KTR-mCherry and mKO-MK2 after 30 min of treatment with TNF-α and 1 hr of treatment with the other stimuli are shown. Scale bar: 20 μm. (B) Schematic representation of cross-inhibition mechanisms of JNK by p38. (C) Clonal HeLa reporter cells were pretreated with 0.1% DMSO, 1 μg/mL actinomycin D, or 10 μM SB203580, then stimulated with 200 mM sorbitol, 10 ng/mL TNF-α, 10 ng/mL IL-1β, 100 J/m2 UV-C, or 10 ng/mL anisomycin, and imaged over time. Mean JNK KTR-mCherry and mKO-MK2 C:N ratios with SDs are shown. n = 70 cells, from at least two independent experiments. (D) JNK and p38 activation and post-translational and transcriptional cross-inhibition of JNK by p38 are indicated for 200 mM sorbitol at 120 min, 10 ng/mL TNF-α at 30 min, 10 ng/mL IL-1β at 42 min, 100 J/m2 UV-C at 120 min, and 10 ng/mL anisomycin stimulation at 120 min. Edge thickness is proportional to the strength of pathway activities. (E) HeLa DUSP1 KO and RLuc KO control cells stably expressing NJP were stimulated with 100 J/m2 UV-C. The time courses of the mean JNK KTR-mCherry and mKO-MK2 C:N ratios with SDs are shown. n = 50 cells for each condition, from two independent experiments. (F) Schematic of post-translational and transcriptional cross-inhibition of JNK by p38. The TAK1/TAB1 complex may mediate cross-inhibition of JNK by p38, when stimulated with TNF-α and IL-1β. See also Figures S2 and S3. Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Cell-to-Cell Variability of Stress- and Cytokine-Induced JNK Activities Arises from Cross-Inhibition by p38 (A) Histograms of the JNK KTR-mCherry and mKO-MK2 C:N ratio distribution in 0.1% DMSO or 10 μM SB203580-pretreated cells at 60 min of 10 ng/mL anisomycin stimulation. The coefficient of variation (CV) is indicated. n ≥ 350 cells per condition, from four independent experiments. (B) The time course of the CV upon 10 ng/mL anisomycin stimulation is shown for the JNK KTR-mCherry and mKO-MK2 C:N ratios in 0.1% DMSO or 10 μM SB203580-pretreated cells. n = 50 cells per condition, from two independent experiments. (C) The CV of the JNK KTR-mCherry and mKO-MK2 C:N ratios at 30 min of 10 ng/mL TNF-α, 60 min of 10 ng/mL IL-1β, 60 min of 10 ng/mL anisomycin, 60 min of 200 mM sorbitol, or 60 min of 100 J/m2 UV-C treatment are shown for 0.1% DMSO or 10 μM SB203580-pretreated clonal reporter cells. n = 50 cells per condition, from two independent experiments. (D) HeLa DUSP1 KO and RLuc KO control cells stably expressing NJP were stimulated with 100 J/m2 UV-C. Representative images of JNK KTR-mCherry and mKO-MK2 are shown. Scale bar: 20 μm. (E) The time course of the CV upon 100 J/m2 UV-C stimulation is shown for the JNK KTR-mCherry and mKO-MK2 C:N ratios in DUSP1 KO and RLuc KO control cells. n = 50 cells per condition, from two independent experiments. See also Figure S4. Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Cell-to-Cell Variation in JNK Activity Leads to Fractional Killing upon UV-C Treatment (A) Clonal HeLa cells stably expressing NLS-iRFP-NLS, JNK KTR-mCherry, and mKO-MK2 were exposed to 100 J/m2 UV-C and imaged over time. Representative images are shown. Yellow asterisks indicate apoptotic cells. Scale bar: 20 μm. (B) C:N ratios of JNK KTR-mCherry and mKO-MK2 were quantified for dying (red lines) and surviving (blue lines) cells after 100 J/m2 UV-C stimulation. n = 10 representative cells. Black dots indicate apoptosis. (C) JNK KTR-mCherry and mKO-MK2 C:N ratios are displayed as heatmaps for cells from (B). White traces indicate cells that underwent cell death. n = 70 cells per condition, from four independent experiments. (D) Histograms of average JNK and p38 activity in the initial phase (<2 hr) and later phase (>4 hr) of UV-C stimulation are shown in dying (red) and surviving (blue) cells. The green dashed line shows the estimated probability of cell death as a function of JNK activity, which is produced by the logistic regression. See also Figure S5. Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 DUSP1 Generates Cell-to-Cell Heterogeneity of JNK Activation and Protects from Cell Death (A) HeLa DUSP1 KO and RLuc KO control cells stably expressing NJP were exposed to 100 J/m2 UV-C and imaged over time. JNK KTR-mCherry and mKO-MK2 C:N ratios are displayed as heatmaps. n = 49 cells for RLuc KO and n = 100 cells for DUSP1 KO. White traces indicate cells that underwent cell death. (B) Apoptosis of DMSO, 10 μM SB203580, or 10 μM JNK inhibitor VIII-pretreated DUSP1 KO cells and RLuc KO control cells 12 hr after 100 J/m2 UV-C or mock treatment. Data are shown as the mean with SD. n = 3 independent experiments. (C) Histogram of the death time of DUSP1 KO and RLuc KO HeLa cells upon 100 J/m2 UV-C treatment, determined by live-cell microscopy. n = 255 cells for RLuc KO and n = 347 cells for DUSP1 KO. (D) HeLa DUSP1-mScarlet knockin cells stably expressing JNK KTR-Clover were stimulated with 100 J/m2 UV-C. Representative images are shown at 8 hr 20 min after UV-C. Scale bar: 30 μm. (E) JNK KTR-Clover C:N ratios of single cells negatively correlate with DUSP1-mScarlet levels. Data were obtained 8 hr 20 min after 100 J/m2 UV-C exposure. n = 200 cells, from two independent experiments. The scatterplot was fitted to a linear regression, and the obtained Pearson correlation value is shown. (F) Representative DUSP1-mScarlet time courses are shown for dying (red lines) and surviving (blue lines) cells after 100 J/m2 UV-C stimulation. n = 54 cells, from two independent experiments. Black dots indicate apoptosis. See also Figure S6. Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Cross-Inhibition by p38 Causes Cell-to-Cell Variability of Stress- and Cytokine-Induced JNK Activities DUSP1-mediated cross-inhibition of JNK by p38 generates heterogeneity in JNK activity upon UV-C stress and leads to fractional killing (left). Depletion of DUSP1 reduces cell-to-cell variability of JNK activity, thereby inducing complete cell death (right). Cell Reports 2018 24, 2658-2668DOI: (10.1016/j.celrep.2018.08.020) Copyright © 2018 The Author(s) Terms and Conditions