Fig 2: MK2 deficiency confers sensitivity of PML-null cells to necroptosis A, BWT and Mk2 -/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 µM) (A) or with zVAD (20 µM) + AT-406 (1 µM) (B), and then, the contents of p-RIPK1[S321], RIPK1, p-p38, and p38 were determined at the indicated time-points.CWT, Pml -/-, Mk2 -/-, and Pml -/- Mk2 -/- BMDMs were treated with zVAD (20 µM), AT-406 (1 µM), Nec-1 (40 µM), and GSK872 (10 µM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm–Sidak method.DWT, Pml -/-, Mk2 -/-, and Pml -/- Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 µM), Nec-1 (40 µM), and GSK872 (10 µM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm–Sidak method.EWT, Pml -/-, Mk2 -/-, and Pml -/- Mk2 -/- BMDMs were treated with zVAD (20 µM) and AT-406 (1 µM) for the indicated timeframes. The levels of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates were analyzed by immunoblots. Results are representative of three biological replicates.FWT, Pml -/-, Mk2 -/-, and Pml -/- Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n = 10). *P < 0.05 for log-rank (Mantel–Cox) test.G, HWT, Pml -/-, Mk2 -/-, and Pml -/- Mk2 -/- mice (n = 5) were injected i.v. with TNF (1.5 mg/kg), before recording body temperature for the next 6 h (G), and collecting serum samples at 3 h to quantitate LDH release (H). *P < 0.05 as determined by two-way ANOVA (G), or P-values calculated by unpaired t-test with Welch's correction (H). Source data are available online for this figure.

Fig 3: Interaction and co-localization of MK2 with PML HEK293T cells were transfected with HA-PML-I and/or FLAG-MK2. Total cell lysates (TCL) were immunoprecipitated with anti-HA or anti-FLAG, and the contents of HA-PML-I and FLAG-MK2 in precipitates and TCL were analyzed by immunoblotting.Co-localization of Myc-MK2 with PML. HEK293T cells were transfected with Myc-MK2 and PML-I, as indicated, and then stained with anti-PML, anti-Myc, and DAPI. Scale bars indicate 10 µm.Interaction of MK2 with PML. Myc-WT MK2 (1–386), Myc-MK2 (1–354), Myc-MK2 (1–311), or Myc-MK2 (50–386) (constructs depicted in bottom panel) was co-transfected with PML-I into HEK293T cells. TCL were immunoprecipitated with anti-Myc, and the association of PML-I was determined.Interaction of PML with MK2. Myc-WT MK2 was co-transfected with HA-PML-I, HA-PML-I?NLS, or HA-PML-I exon 1–4 into HEK293T cells. TCL were immunoprecipitated with anti-Myc, and the presence of PML-I and Myc-MK2 in precipitates and TCL was analyzed.Co-localization of MK2 with PML NBs in macrophages. The specificity of anti-PML and anti-MK2 was confirmed in WT, Pml -/- and Mk2 -/- BMDMs (left panel). WT macrophages before (-) and after TNF treatment (100 ng/ml, 15 min) were stained with anti-PML, anti-MK2, and DAPI. White scale bars indicate 10 µm, and pink scale bars indicate 2 µm (last column). Arrows indicate co-localization. The rightmost diagram shows quantitation of PML-MK2 co-localization. Data information: The experiments were independently repeated (biological replicates) three (A, B) or two times (C, D, E) with similar results. Source data are available online for this figure.

Fig 4: PML deficiency enhances phosphorylation of p38 MAPK, MK2, and RIPK1[S321], and a MK2 inhibitor restores necroptosis A Pml +/+ and Pml -/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 µM) for the indicated timeframes. The contents of p-IKKa/ß, IKKa/ß, I?Ba, p-MK2, and MK2 were analyzed by immunoblotting.B Pml +/+and Pml -/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 µM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time-points were then determined.C–FWT (Pml +/+) and Pml -/- mice were treated with DMSO or Nec-1s (6 µg/g) or MK2 inhibitor PF-3644022 (75 µg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 µg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± SEM is shown (C, E). P-values were determined by two-way ANOVA for multiple comparisons (C, E) or by log-rank (Mantel–Cox) test (D, F). Source data are available online for this figure.

Fig 5: An MK2 inhibitor increases necroptotic phosphorylation of RIPK1[S166] and MLKL AWT and Pml -/- BMDMs were pre-treated with DMSO or PF-3644022 (2 µM) for 30 min, followed by TNF (100 ng/ml) + zVAD (20 µM). Cell viability was then determined at 6 h. Data show mean ± SD from three technical repeats and are representative of two biological replicates.B, CWT and Pml -/- BMDMs were pre-treated with DMSO or PF-3644022 (2 µM) for 30 min, followed by TNF (100 ng/ml) + zVAD (20 µM) (B) or zVAD (20 µM) + AT-406 (1 µM) (C) at the indicated time-points. The contents of p-RIPK1[S166], RIPK1, p-MLKL, and MLKL were determined by immunoblotting. Due to extensive cell death following PF-3644022 treatment (A), experimental timeframes were shorter when deploying this MK2 inhibitor. Data shown in (B, C) are representative of three biological replicates.DWT and Mk2 -/- BMDMs were treated with zVAD (20 µM), AT-406 (1 µM), Nec-1 (40 µM), and GSK872 (10 µM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD from three technical repeats and are representative of three biological replicates.EWT and Mk2 -/- BMDMs were treated with zVAD (20 µM) +AT-406 (1 µM) for 18 h, and autocrine TNF production was analyzed by ELISA. Data show mean ± SD of three biological replicates. Source data are available online for this figure.