![Figure 3. Bid modulates Ripk1 signaling in MPCs in a caspase-8–dependent manner. (A) Examination of death in MPCs. MPCs were treated with 25 ng/mL TNF-α + 50 ng/mL ActD. Viability was determined by Annexin V/propidium iodide staining. The experiment was performed three independent times. Statistics indicate differences between Bid+/+ vs Bid−/−, DKO, and TKO. (B) Bid+/+ and TKO MPCs treated with TNF-α/ActD were examined by TEM. A total of 50 cells with a nucleus were examined and characterized as being apoptotic or necrotic. Arrows indicate apoptotic cells, and asterisks indicate necrotic cells (scale bars, 2 μm). Quantitation of cells is to the right of images. (C) MPCs unstimulated or stimulated with 250 ng/mL LPS for 10 minutes and 1 hour followed by immunoblot for Ripk1. pRipk1, phospho-Ripk1. The experiment was performed 4 times. (D) Immunoblot of Bid+/+ MPCs treated with or without TNF-α in the presence or absence of calf intestinal alkaline phosphatase (CIAP). pRipk1 is lost in the presence of CIAP. (E) Immunoblot of Ripk1 in Bid+/+, DKO, TKO, and TKO+ FLAG-Bid MPCs following LPS stimulation. The experiment was performed 2 independent times. (F) Immunoblot of MLKL trimerization in Bid+/+, DKO, and TKO MPCs following stimulation with LPS. MLKL trimer is ∼150 kDa. The experiment was performed 3 times. (G) Bone marrow from Bid+/+ and TKO mice treated with or without LPS (250 ng/mL) for 4 hours followed by bismaleimidohexane (BMH) crosslinking and probed for MLKL. Control lane indicates Bid+/+ bone marrow pretreated with zVAD (25 μM) followed by Smac mimetic (Birinapant [100 nM]) and TNF-α (25 ng/mL) for 4 hours as a positive control. The experiment was performed 2 independent times. NT, not treated. (H) Immunoblot of Ripk1 in Bid+/+, DKO, and TKO MPCs following stimulation with 250 ng/mL LPS and pretreatment with 20 μM Z-IETD-FMK (an inhibitor of caspase-8). The experiment was performed 3 independent times. (I) Ripk1 levels by immunoblot after knockdown of caspase-8 utilizing the CRISPR-Cas9 system in Bid+/+ MPCs. The experiment was performed 2 times. (J) Immunoblot of bone marrow from Bid+/+, DKO, and TKO mice treated with LPS for the indicated times. The experiment was performed 3 independent times. **P < .01, ***P < .001. Data represent mean ± SEM.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/133/2/10.1182_blood-2018-05-847335/6/blood847335f3.png?Expires=1722565157&Signature=aNWMkgMMw0kUO-iUgFnxIaV7jP~D6CDd9B3ZSIF2a5cqR4mi4zg0510KgLbN152iXJ3rtva46YR9MzEncY2UGqCf1KTY2GeXmR-K5oQGy-tCVCnmB7cLCdBWXBz3QyZ98rmX4snQB1hAnh-vabbL5yOtuD7R4ETX8ZIdxhaODMCd8rbIv3w95nJwJZ-NMoP9efeqImXS2DlHQo~kC1pfBvXAnoTtXA~VNfL3Qp94TPAlKIxTUkTQ6kFAgWWt7JS62Muu-~7JM2FsN1t-jYSN-0AjiKFx4ZnCjtAl5BUk18eNREX4Ep0GKEiwjshOyvteLeQPPdxYGUje8t6TF~JyHQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Bid modulates Ripk1 signaling in MPCs in a caspase-8–dependent manner. (A) Examination of death in MPCs. MPCs were treated with 25 ng/mL TNF-α + 50 ng/mL ActD. Viability was determined by Annexin V/propidium iodide staining. The experiment was performed three independent times. Statistics indicate differences between Bid+/+ vs Bid−/−, DKO, and TKO. (B) Bid+/+ and TKO MPCs treated with TNF-α/ActD were examined by TEM. A total of 50 cells with a nucleus were examined and characterized as being apoptotic or necrotic. Arrows indicate apoptotic cells, and asterisks indicate necrotic cells (scale bars, 2 μm). Quantitation of cells is to the right of images. (C) MPCs unstimulated or stimulated with 250 ng/mL LPS for 10 minutes and 1 hour followed by immunoblot for Ripk1. pRipk1, phospho-Ripk1. The experiment was performed 4 times. (D) Immunoblot of Bid+/+ MPCs treated with or without TNF-α in the presence or absence of calf intestinal alkaline phosphatase (CIAP). pRipk1 is lost in the presence of CIAP. (E) Immunoblot of Ripk1 in Bid+/+, DKO, TKO, and TKO+ FLAG-Bid MPCs following LPS stimulation. The experiment was performed 2 independent times. (F) Immunoblot of MLKL trimerization in Bid+/+, DKO, and TKO MPCs following stimulation with LPS. MLKL trimer is ∼150 kDa. The experiment was performed 3 times. (G) Bone marrow from Bid+/+ and TKO mice treated with or without LPS (250 ng/mL) for 4 hours followed by bismaleimidohexane (BMH) crosslinking and probed for MLKL. Control lane indicates Bid+/+ bone marrow pretreated with zVAD (25 μM) followed by Smac mimetic (Birinapant [100 nM]) and TNF-α (25 ng/mL) for 4 hours as a positive control. The experiment was performed 2 independent times. NT, not treated. (H) Immunoblot of Ripk1 in Bid+/+, DKO, and TKO MPCs following stimulation with 250 ng/mL LPS and pretreatment with 20 μM Z-IETD-FMK (an inhibitor of caspase-8). The experiment was performed 3 independent times. (I) Ripk1 levels by immunoblot after knockdown of caspase-8 utilizing the CRISPR-Cas9 system in Bid+/+ MPCs. The experiment was performed 2 times. (J) Immunoblot of bone marrow from Bid+/+, DKO, and TKO mice treated with LPS for the indicated times. The experiment was performed 3 independent times. **P < .01, ***P < .001. Data represent mean ± SEM.