Fig 1: Impact of LONP1, PMPCA and PARL on parthanatos. A WT L929Ts cells or MEF were nucleofected with a non-targeting siRNA that does not elicit an RNA interference response (siNT) or with siRNAs specific for murine LONP1 (si LONP1) or PMPCA (siPMPCA). 48 h after nucleofection, the cells were left untreated or stimulated with 0.5 mM MNNG for 15 min and further incubated for 16 h with fresh medium without MNNG before loss of membrane integrity as a marker for cell death was measured by PI staining and flow cytometry (upper panels). Each measurement represents the mean of nine determinations, error bars indicate the corresponding SD. ns not significant (two-tailed unpaired Student’s t test). Expression of the respective target proteins was analyzed in Western blots for LONP1 and PMPCA. Detection of actin served as control for equal loading (lower panels). B PARL-deficient MEF (PARL KO) or PARL-deficient MEF reconstituted with WT FLAG-tagged PARL (PARL KO * WT) were left untreated or stimulated with 0.5 mM MNNG for 15 min and further incubated for 16 h with fresh medium without MNNG before loss of membrane integrity as a marker for cell death was measured by PI staining and flow cytometry (upper panel). Each measurement represents the mean of nine determinations, error bars indicate the corresponding SD. ns, not significant (two-tailed unpaired Student’s t test). Lower panel: immunofluorescence analysis to validate the presence of PARL in PARL KO * WT MEF. PARL is indicated by green fluorescence (top left), mitochondria (red) were stained with MitoTracker Orange (top right), cell nuclei (blue) were stained with Hoechst 33342 (bottom left), and an overlay of all stainings is shown at the bottom right. Scale bars, 10 µm