Journal: Cell Death & Disease
Article Title: A novel neurodegenerative spectrum disorder in patients with MLKL deficiency
Figure Lengend Snippet: MLKL variant expression and effect on necroptosis. a MLKL protein expression in PBMCs of the sister II-1, unrelated controls C1–C5 and the brothers, II-3 and II-2. The molecular weight, in kilodaltons (kDa), of full-length MLKL and of the predicted, truncated MLKL variant (which was not detectable), relative to the marker, are denoted by the arrows. GAPDH was used as the loading control. b Relative expression of MLKL messenger RNA (mRNA) in PBMCs from controls (sister II-1, and unrelated controls C1–C5; shown in blue) and the patients (brothers II-3 and II-2; shown in red), assessed in triplicate. c MLKL protein expression in unstimulated fibroblasts (−) and fibroblasts stimulated with 1000 ng/ml of IFNγ for 24 h (+). Lysate of HEK-293 cells transiently transfected with the truncated MLKL variant protein was used as a control. d Relative expression and upregulation of MLKL mRNA upon stimulation of fibroblasts from controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) with varying concentrations of IFNγ for 24 h. Three independent experiments were performed. e Proportion of dying fibroblasts from the controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) over an 87-h period. Necroptosis sensitization was achieved by RIPK3 transduction in the presence of 5 ng/ml IFNγ and 50 μM zVAD, and cell death was measured based on the uptake of IncuCyte® Cytotox Red Reagent. Four independent experiments were performed. Blue and dark blue asterisks (*) denote a statistically significant difference ( P
Article Snippet: Real-time quantitative PCR was performed using TaqMan® Gene Expression Assays MLKL_Hs00930421_m1 and GAPDH_Hs02758991_g1.
Techniques: Variant Assay, Expressing, Molecular Weight, Marker, Transfection, Transduction