Fig 2: Ripoptosome and mitochondrial stress. (A) Viability of WT or Ripk1−/−Casp8−/−Ripk3−/− BMDM infected with K181 or ∆M38.5/M41.1 (MOI = 10) assessed by the loss of ATP at 48 hpi. Each bar represents the pooled data from at least three experiments. Horizontal lines indicate the two groups being compared by Wilcoxon matched-pairs signed rank test. Only significant relevant comparisons are indicated in panels. Error bars indicate standard error and mean for each dataset. *** is p < 0.001. (B) Representative light microscopy images (lower left scale bar = 200 mM) of WT or Ripk1−/−Casp8−/−Ripk3−/− BMDM infected with indicated viruses at indicated times. Time stamp from the microscope is included in the lower-right corner of each image. (C) Immunoblot assessment to detect the appearance of cleaved forms of CASP9 (Cl-C9; ~37 kDa), CASP8 (Cl-C8; 43 and 18 kDa), CASP7 (Cl-C7; 18 kDa), and CASP3 (Cl-C3; 17 kDa) in cellular protein lysates from Casp8−/−Ripk3−/− BMDM treated with medium (mock) or infected with the indicated viruses. β-actin (38.5 kDa) serves as the loading control.

Fig 3: Microbiota from Tph1-/-mice attenuates DSS-induced colitis. (A) Schematic of cecal microbiota transfer, followed by colitis induction with 5% DSS in sterile drinking H2O. (B) Macroscopic scores on day 5 post-DSS. (C) Representative photomicrographs of H&E stained colon cross sections on day 5 post-DSS visualized using Nikon Eclipse 80i microscope. Original magnification, ×10. (D) Histologic damage scores. (E) MPO activity on day 5 post-DSS. (F) Western blot analysis performed on protein extracts obtained from the colon homogenates of the recipient mice administered with DSS. Representative Western blot with β-actin is presented as loading controls. Pro-Casp8, pro-caspase-8; Casp-8, caspase-8. Each lane represents an individual mouse. Results are the representative of 3 independent experiments performed on at least 3 mice per group. (G) Cytokine levels in the colon of Tph1-/- and Tph1+/- littermates on day 5 post-DSS after transfer of gut microbiota from Tph1+/- mice to Tph1-/- mice and vice versa. Data are from 1 representative experiment of 2 independent experiments performed. Data are represented as mean ± standard deviation from 8 to 10 mice per group; *P < .05 by Student t test. (→) denotes microbiota transfer.

Fig 4: Role of death receptor signaling in ER stress-induced cell death. (A and B) casp9−/− MEFs were pretreated with 50 ng/mL of TNF blocking antibody, 100 ng/mL of FAS:Fc or 500 ng/mL of TNFS10 blocking antibody for 1 h. For the next 24 h cells were treated with 0.5 μM of Tg (C) or 0.5 μg/ml Tm (D) and cell death estimated by propidium iodide staining. Protein samples from the same treatments were also harvested and expression of cleaved CASP8 was evaluated by protein gel blot (E). Actin was used as a loading control. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD.

Fig 5: Immunoblotting of full length caspase-8 proenzyme in unstimulated Casp8 −/− and control cells (A) and cleaved caspase-8 in doxorubicin stimulated Casp8 −/− and control cells (B). Cell proliferation of Casp8 −/− cells compared to control cells evaluated by MTT test (C).