Figure 1. LACE1 is a mitochondrial integral membrane protein whose knockdown by shRNA leads to reduced PARP cleavage and diminished activity of respiratory chain complexes II-IV.

A. LACE1 is a mitochondrial protein. Whole cell lysate (WCL), mitochondrial (MITO) and cytosolic (CYTO) fractions were prepared from HEK293 cells and immunoblotted with antibody to LACE1 (Abcam, UK), to mitochondrial matrix heat shock protein mtHsp70 (Lonza, Switzerland) and to β-actin. B. LACE1-FLAG expressed in HEK293 cells is a mitochondrial integral membrane protein. Wild-type HEK293 cells were transiently transfected with a C-FLAG-fusion human LACE1 (NM_145315) ORF construct (OriGene). Submitochondrial fractions were prepared from isolated mitochondria using either sonic disruption (SONIC) or extraction with 100 mM sodium carbonate (pH 11.5). The resulting supernatant (S) and pellet (P) fractions and untreated mitochondria (MITO) were immunoblotted with M2 monoclonal antibody to FLAG epitope (Sigma Aldrich, Germany), and with antibodies to intermembrane space protein PNPase and matrix localized ClpP (Abcam, UK). C. Stable knockdown (KD) of LACE1 by shRNA leads to markedly diminished PARP cleavage. Whole-cell lysates (~20 μg of protein) from two different stable shRNA knockdown cell lines (denoted LACE1sh4 and sh5) were immunoblotted with antibodies to LACE1, Cl-PARP, mtHSP70 and α-tubulin. The control cell line was prepared by transfecting HEK293 cells with the non-silencing (scrambled) shRNAmir vector. D. LACE1 KD cells exhibit moderate growth impairment. Stably transfected HEK293 cells were seeded in six-well plates at 5 × 10⁴ cells per well and cultured in DMEM containing 1 μg/ml puromycin. Viable cells were counted every 24 h for a total of 7 d using a Scepter Handheld Automated Cell Counter. E. Loss of LACE1 leads to diminished activity of respiratory chain complexes II-IV. The oxygen consumption of digitonin-permeabilized cells was measured at 37°C using an OROBOROS Oxygraph-2k (OROBOROS INSTRUMENTS Corp, Innsbruck, Austria) in 2ml chamber by substrate-inhibitor titration. * p<0.05; ** p<0.01 F. LACE1 KD cells have moderately increased content of complex I and slightly reduced content of complex III. Blue-native PAGE immunoblotting analysis of mitochondria isolated from LACE1 KD cells were performed. Isolated mitochondria were solubilized with 1% (w/v) dodecyl maltoside, resolved on 5-15% blue-native PAGE gels and immunoblotted with antibodies to Ndufb6 (CI), Core 2 (CIII), Cox1 (CIV) and SDHA (CII). CI-CIV denotes four mitochondrial respiratory chain complexes.