Article Figures & Data
Figures
- Figure 1. Stress granule triggers inhibit macrophage necroptosis.
Primary BMDMs were stimulated as indicated. (A) Schematic for inducing necroptosis (zVAD + TNF) in unstressed or pre-stressed (thapsigargin [Thaps] treated) BMDMs. (B) Representative IncuCyte images collected at indicated time-points after addition of TNF, where necroptotic cells were quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (C) Quantification of necroptosis from automated image analysis of Sytox Green–positive nuclei at indicated time-points. (D) Schematic for acute pre-stressing of BMDMs with 1 h thapsigargin treatment before necroptosis induction with TNF. (E) Representative IncuCyte images of necroptosis quantification in acute thapsigargin pre-stressed BMDMs (green, with red analysis mask outline). (F) Quantification of necroptosis from automated analysis for acute thapsigargin pre-stressed BMDMs. Significance was determined (C, F) by two-way ANOVA followed by Dunnett’s multiple comparisons test (versus zVAD + TNF), ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bar (black) indicates 50 μm. Data are presented as mean ± SEM.
- Figure S1. Thapsigargin and arsenite pre-stress dose–response time course.
(A, B) Primary BMDMs were stimulated as indicated with or without thapsigargin (Thaps) (A) or with or without arsenite (Ars) (B). Quantification of necroptosis from automated image analysis of Sytox Green–positive nuclei at indicated time-points. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test, ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least two independent biological replicate experiments. Data are presented as mean ± SEM.
- Figure S2. Multiple stress granule inducers protect BMDMs from TNF-mediated necroptosis.
Primary BMDMs were stimulated with zVAD + TNF and pre-stressed as indicated. (A, F) Representative IncuCyte images collected at indicated time-points following addition of TNF, where necroptotic cells were quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (B, C, D, E, G) Quantification of necroptosis from automated image analysis of Sytox Green–positive nuclei at indicated time-points in cells pre-stressed with (B) brefeldin A, (C) tunicamycin (Tunica), (D) MG132, (E) arsenite (Ars), or (G) rocaglamide A (Roc A). Significance was determined (B, C, D, E, G) by two-way ANOVA followed by (B, C, D, E) Dunnett’s or (G) Sidak’s multiple comparisons test (versus zVAD + TNF), ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bar (black) indicates 50 μm. Data are presented as mean ± SEM.
- Figure S3. Primary MEF necroptosis is not inhibited by integrated stress response.
(A, B) Primary MEFs were stimulated with zVAD + TNF and pre-stressed with (A) thapsigargin (Thaps) or (B) arsenite (Ars). Quantification of necroptosis from automated image analysis of Sytox Green–positive nuclei at indicated time-points. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, and ***P < 0.001. Data are generated from three images per replicate well (n = 3) and are representative of at least two independent biological replicate experiments. Data are presented as mean ± SEM.
- Figure S4. TLR driven necroptosis is inhibited by stress granule triggers.
Primary BMDMs were stimulated as indicated and pre-stressed with thapsigargin (Thaps). (A, C) Representative IncuCyte images collected at indicated time-points following addition of (A) zVAD + LPS or (C) zVAD + poly I:C (pI:C), where necroptotic cells were quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (B, D) Quantification of necroptosis from automated image analysis of Sytox Green–positive nuclei at indicated time-points in cells pre-stressed with thapsigargin and stimulated with (B) zVAD + LPS or (D) zVAD + poly I:C. Significance was determined (B, D) by two-way ANOVA followed by Sidak’s multiple comparisons test, ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bar (black) indicates 50 μm. Data are presented as mean ± SEM.
- Figure S5. Pre-stressed BMDMs lacking TAK1 activity are protected from cell death.
Primary BMDMs were stimulated as indicated. (A) Schematic for inducing necroptosis (zVAD + TAK1i + TNF) in unstressed or pre-stressed (thapsigargin [Thaps] treated) wildtype BMDMs. (B) Representative IncuCyte images collected at indicated time-points following addition of zVAD + TAK1 inhibitor (TAK1i) + TNF with or without thapsigargin (Thaps) pretreatment, where necroptotic cells were quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (C) Quantification of cell death from automated image analysis of Sytox Green–positive nuclei at indicated time-points. (D, E, F, G) Primary BMDMs derived from control (Tak1fl/fl) or myeloid-specific Lyz2cre+Tak1fl/fl mice were treated as indicated, and representative images were obtained from automated IncuCyte analysis where cell death was quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). Significance was determined (C, E, G) by two-way ANOVA followed by (C) Sidak’s or (E, G) Tukey’s multiple comparisons test, *P < 0.05, ***P < 0.001, and ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bar (black) indicates 50 μm. Data are presented as mean ± SEM.
- Figure 2. Stress granule inducers inhibit RIPK1, RIPK3, and MLKL phosphorylation.
Necroptosis signaling was analyzed by immunoblotting at the indicated time after the induction of necroptosis by TNF treatment. (A, B, C, D, E) Lysates from primary BMDMs pre-stressed with (A) thapsigargin (Thaps), (B) brefeldin A, (C) tunicamycin (Tunica), (D) MG132, or (E) Thaps and treated with zVAD + TNF were collected at the indicated time-points. Necroptosis signaling was examined by immunostaining for phosphorylation of MLKL and RIPK3 (p-MLKL and p-RIPK3) and phosphorylation of RIPK1 (p-RIPK1) at serine-166 (S166) or serine-321 (S321) and activation of the integrated stress response by phosphorylation of eIF2α (p-eIF2α). Immunoblots are representative of at least two independent experiments.
- Figure S6. BMDMs form stress granules (SGs) in response to thapsigargin treatment.
Primary BMDMs were unstressed or treated with thapsigargin (Thaps) to induce SG formation. Cells were stained for G3BP1 (green), DDX3X (red), and DAPI (blue) to examine SG formation by confocal microscopy. Scale bars (white) indicate 20 μm. Images are representative from at least two independent experiments.
- Figure 3. DDX3X is not required for stress-dependent inhibition of necroptosis.
Primary BMDMs were stimulated as indicated. (A) Primary BMDMs derived from control (Ddx3xfl/fl) or myeloid-specific Lyz2creDdx3xfl/fl mice were treated as indicated, and representative images (6 h) were obtained from automated IncuCyte analysis where necroptotic cells were quantified by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (B) Necroptosis was quantified by automated analysis of Sytox Green–positive nuclei. (C) Immunoblots were performed from BMDM lysates at the indicated time-points to confirm DDX3X expression was reduced and assess necroptosis signaling (via phosphorylation of MLKL [p-MLKL]) in unstressed or thapsigargin-stressed BMDMs treated with zVAD + TNF. (D) Primary BMDMs derived from control (Ddx3xfl/fl) or myeloid-specific Lyz2creDdx3xfl/fl mice were treated as indicated, and necroptosis was quantified by automated analysis of Sytox Green–positive nuclei. Ddx3xfl/fl and Lyz2creDdx3xfl/fl quantifications overlap upon zVAD + Ars + TNF treatment. (E) Representative images (6 h) and IncuCyte quantification of NLRP3-dependent pyroptosis (via LPS + nigericin treatment) in BMDMs derived from the indicated genotypes. Significance was determined (B, D, E) by two-way ANOVA followed by (B, D) Dunnett’s or (E) Sidak’s multiple comparisons test, ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bar (black) indicates 50 μm. Data are presented as mean ± SEM.
- Figure 4. Disruption of SGs restores necroptosis in stressed BMDMs.
Disruption of SGs by anisomycin (Aniso) treatment in thapsigargin (Thaps) pre-stressed cells was examined. (A) Confocal microscopy images were obtained from primary BMDMs treated as indicated and stained for G3BP1 (red), DDX3X (green), and DAPI (blue), and SG signal intensities were compared. (B) Schematic for disrupting SGs in thapsigargin pre-stressed cells treated with Aniso before necroptosis induction. (C, D) Representative images of BMDMs (treated as indicated in panel B) and quantification of necroptosis (D) by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (E) Immunoblots were performed to determine the effect of SG disruption by Aniso on necroptosis signaling. Significance was determined (A) by Mann–Whitney test or (D) by two-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, ***P < 0.001, and ****P < 0.0001. Data are representative of (A) at least three images per condition or (C, D) from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Scale bars (black) indicate 50 μm or (white) 20 μm. Data are presented as mean ± SEM.
- Figure S7. Inhibitors of translation elongation restore necroptosis in thapsigargin-stressed BMDMs.
Primary BMDMs were stimulated as indicated. (A, B, C, D) Quantification of cell death from automated image analysis of Sytox Green–positive nuclei at indicated time-points in (A) WT BMDMs, (B) Ripk1KD/KD BMDMs, (C) Ripk3−/− BMDMs, or (D) Mlkl−/− BMDMs. (E) WT BMDM cell death was similarly quantified at indicated time-points in pre-stressed (thapsigargin [Thaps]-treated) cells subsequently treated with cycloheximide (CHX). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Data are presented as mean ± SEM.
- Figure 5. Knockdown of Perk restores necroptosis in thapsigargin pre-stressed BMDMs.
Primary BMDMs were stimulated as indicated. (A, B) Representative IncuCyte images of mock siRNA or Perk siRNA knockdown BMDMs treated as indicated (A) and necroptosis was quantified (B) by uptake of membrane-impermeant Sytox Green (green, with red analysis mask outline). (C) Confocal images of mock siRNA or Perk siRNA treated BMDMs treated as indicated and stained for G3BP1 (green), DDX3X (red), and DAPI (blue). (D) Immunoblots were performed to determine whether siRNA (mock or Perk) knockdown restored necroptosis signaling in pre-stressed BMDMs. Data are representative of a single independent biological replicate experiment with IncuCyte quantification performed on two replicate wells containing a total of eight image fields. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test (B), ****P < 0.0001. Data are presented as mean ± SEM. Scale bars indicate (black) 50 μm or (white) 10 μm.
