Figure 2.

sCD74/MIF‐induced death of myofibroblasts is triggered by RIP1/RIP3‐dependent necroptosis. Cardiac wild‐type myofibroblasts were treated solitarily or simultaneously with MIF and sCD74, and lysates were taken at 10 hours. A and B, Cleaved caspase‐3 levels (Cl Casp 3) as well as (C and D) relative phosphorylation levels of receptor‐interacting serine/threonine‐protein kinase 3 (RIP3) were assessed by Western blotting and immunostaining. Instead of showing the whole blot, only relevant bands were cut out and arranged in the right order. Uncut blots are shown in Figures S3 and S4. Data represent mean±SEM of at least 3 independent experiments. Data were analyzed with a 2‐tailed, unpaired t test. ^$ P<0.05 vs control. E, Cardiac wild‐type fibroblasts were pretreated with dimethyl sulfoxide (DMSO) or a potent necroptosis inhibitor (Nec1s) for 1 hour followed by solitary or co‐treatment with sCD74 and rMIF. After 20 to 24 hours of incubation, cell numbers were quantified by trypan blue staining and automated counting. Data were analyzed with a 2‐tailed, unpaired t test and corrected for multiple comparison (n=9) using Bonferroni's posttest. Data represent mean±SEM of at least 8 independent experiments for inhibition studies. **P<0.01 DMSO vs Nec1s; ^§§§ P<0.001 vs DMSO control; ^$ P<0.05, ^$$$ P<0.001 vs Nec1s control respectively. GAPDH indicates glyceraldehyde 3‐phosphate dehydrogenase; MIF, macrophage migration inhibitory factor; pRIP3, phosphorylated RIP3; rMIF, recombinant MIF; sCD74, soluble CD74.