Journal: Biology

Article Title: Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

doi: 10.3390/biology10070571

Figure Lengend Snippet: Component list.

Article Snippet: #30 , IX73 Microscope Frame (1 deck) , Single deck , IX73P1F , Olympus , 1.

Techniques: Inverted Microscopy, Microscopy, Solvent, Adhesive

Journal: Oncotarget

Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis.

doi: 10.18632/oncotarget.9959

Figure Lengend Snippet: 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 a-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.

Article Snippet: To find out whether overexpression of wild-type LACE1-FLAG (NM_145315) leads to correct mitochondrial targeting of the fusion protein, we have transiently transfected wild-type HEK293 cells with a C-FLAG-fusion human LACE1 ORF construct (OriGene Technologies).

Techniques: Membrane, Knockdown, shRNA, Activity Assay, Transfection, Construct, Isolation, Disruption, Extraction, FLAG-tag, Control, Plasmid Preparation, Stable Transfection, Cell Culture, Titration, Blue Native PAGE, Western Blot

Journal: Oncotarget

Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis.

doi: 10.18632/oncotarget.9959

Figure Lengend Snippet: Figure 2: Loss of LACE1 leads to increased apoptotic resistance whereas its overexpression results in increased apoptotic sensitivity. A. Overexpression of LACE1-FLAG in both wild-type HEK293 cells and LACE1 KD cells results in increased PARP cleavage. Wild-type HEK293 cells and LACE1 KD cells were transfected with LACE1-FLAG expression vector or with the empty vector. Whole-cell lysates were analyzed with western blotting using antibody to cleaved PARP. Antibody to α -tubulin was used to monitor equal protein loading. B. LACE1 KD cells exhibit increased resistance to staurosporine-induced apoptosis but respond normally to TNFalpha-treatment. Control and LACE1 KD cells were treated with staurosporine (STS; 2 μM) or TNFalpha (10 ng/ml) and IFN- gamma (80 ng/ml) for 0, 3 and 6 hours. Whole cell lysates were prepared from treated cells and used for immunoblotting with antibody to cleaved PARP. Antibody to α-tubulin was used to control for equal protein loading. C. Cells were treated with staurosporine (STS; 2 μM) for 0, 3 and 6 hours and then harvested and used for subcellular fractionation using dounce homogenization and differential centrifugation. The resulting cytoplasmic fractions were subjected to SDS-PAGE western blotting with antibody to cytochrome c. Western blotting of alpha-tubulin was used as loading control. D. Cells grown on coverslips were treated with staurosporine (STS; 2 μM) for 0, 3 and 6 hours and then fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100. Cells were blocked by 10% Fetal Bovine Serum and primary detection was performed with anti-cytochrome c antibody. After secondary fluorescent detection, cells were analyzed at 24°C using a Nikon Diaphot 200 inverted microscope equipped with a Plan-Apochromat 60×, numerical aperture 0.95, oil objective. The images were acquired with an Olympus DP50 CCD camera and Viewfinder Lite 1.0 software. Bar, 10 μM. E. Whereas knockdown of LACE1 leads to moderate upregulation of PUMA and BAX mRNAs, overexpression of LACE1-FLAG results in their significant downregulation. Relative mRNA quantification was performed using TaqMan Gene Expression Assays according to the manufacturer’s instructions (Applied Biosystems). Data were collected in duplicate in two separate runs using a 7300 Real-Time PCR System (Applied Biosystems). HPRT1 (hypoxanthine phosphoribosyltransferase 1), TUBA1A (tubulin, alpha 1a) and TBP (TATA box– binding protein) were used as reference genes.

Article Snippet: To find out whether overexpression of wild-type LACE1-FLAG (NM_145315) leads to correct mitochondrial targeting of the fusion protein, we have transiently transfected wild-type HEK293 cells with a C-FLAG-fusion human LACE1 ORF construct (OriGene Technologies).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Fractionation, Homogenization, Centrifugation, SDS Page, Inverted Microscopy, Software, Knockdown, Gene Expression, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Oncotarget

Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis.

doi: 10.18632/oncotarget.9959

Figure Lengend Snippet: Figure 3: LACE1 expressed in LACE1 KD background physically interacts with p53 and promotes its increased mitochondrial accumulation and nuclear reduction. A. Overexpression of LACE1 leads to increased mitochondrial accumulation of p53 and its nuclear reduction. Whole-cell lysates (WCL), mitochondrial protein lysates (MITO) and nuclear extracts (NUCLEAR) were prepared from control cell line (Control) and from LACE1 KD cell line transfected with either the empty expression vector or LACE1-FLAG construct and subsequently immunoblotted with antibodies to p53, Bax and PUMA. Equal protein loading and fractionation efficiency was monitored using antibodies to TIMM50, histone H3 and a-tubulin. B. Loss of LACE1 leads to upregulation of PARL and downregulation of HTRA2. Mitochondrial protein lysates (MITO) were prepared from control cell line (Control) and from LACE1 KD cell line transfected with either the empty expression vector or LACE1-FLAG construct and subsequently immunoblotted with antibodies to OMA1, SCO2, HTRA2, FLAG, PARL and mtHSP70. C. P53 co-immunoprecipitates with LACE1-FLAG expressed in LACE1 KD background. LACE1 KD cells were transiently transfected with the empty expression vector, LACE1-FLAG construct or CLPP-FLAG construct. Mitochondrial fractions were solubilized with 1% Triton X-100 and subjected to anti-FLAG affinity purification. The bound antigens were eluted with 3x FLAG peptide solution under native conditions and processed for western blotting. Five percent (vol) of the purification input was loaded on the gels along with purified samples and immunoblotted with antibodies to FLAG, p53 and CLPP.

Article Snippet: To find out whether overexpression of wild-type LACE1-FLAG (NM_145315) leads to correct mitochondrial targeting of the fusion protein, we have transiently transfected wild-type HEK293 cells with a C-FLAG-fusion human LACE1 ORF construct (OriGene Technologies).

Techniques: Over Expression, Control, Transfection, Expressing, Plasmid Preparation, Construct, Fractionation, Affinity Purification, Western Blot, Purification

Journal: Oncotarget

Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis.

doi: 10.18632/oncotarget.9959

Figure Lengend Snippet: Figure 4: Loss of LACE1 abrogates mitomycin c-induced translocation of p53 into mitochondria whereas its overexpression promotes mitochondrial translocation of p53. A-B. Overexpression of LACE1-FLAG in wild-type HEK293 cells promotes increased mitochondrial accumulation of p53. Cells grown on coverslips were transfected with the LACE1-FLAG construct or the empty vector and 48 hours post-transfection were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were then blocked by 10% Fetal Bovine Serum and primary detection was performed with antibodies to FLAG, p53 and COX1. After fluorescent secondary detection, cells were analyzed at 24°C using a Nikon Diaphot 200 inverted microscope equipped with a Plan- Apochromat 60×, numerical aperture 0.95, oil objective. The images were acquired with an Olympus DP50 CCD camera and Viewfinder Lite 1.0 software. Bar, 10 μM. C. Loss of LACE1 abrogates mitomycin c-induced mitochondrial translocation of p53. Cells grown on coverslips were treated with mitomycin c (MMC; 5g/mL) for 0, 3, 6 and 12h and then processed for immunofluorescence microscopy essentially as describe in (A-B) except that anti-p53 antibody and DAPI staining were used. Bar, 10 μM.

Article Snippet: To find out whether overexpression of wild-type LACE1-FLAG (NM_145315) leads to correct mitochondrial targeting of the fusion protein, we have transiently transfected wild-type HEK293 cells with a C-FLAG-fusion human LACE1 ORF construct (OriGene Technologies).

Techniques: Translocation Assay, Over Expression, Transfection, Construct, Plasmid Preparation, Inverted Microscopy, Software, Immunofluorescence, Microscopy, Staining

Journal: Oncotarget

Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis.

doi: 10.18632/oncotarget.9959

Figure Lengend Snippet: Figure 5: The LACE1-mediated apoptosis is dependent on p53. A. Transfection of human dermal fibroblasts with LACE1 stealth siRNA leads to efficient knockdown of LACE1 protein. Normal human dermal fibroblasts (NHDFs) were transfected with control and LACE1 siRNA, harvested 48 hours post-transfection and processed for western blotting. The blots were developed with antibodies to LACE1 and mtHSP70. B. Knockdown of LACE1 in NHDFs by siRNA renders cells resistant towards STS-induced apoptosis. Both control siRNA- and LACE1 siRNA-transfected NHDFs were treated with 0-h, 3-h and 6-h exposures to staurosporine (2 μM). Whole-cell lysates were subsequently western blotted with antibody to cleaved PARP. Equal protein loading was controlled with antibody to α-tubulin. C. The LACE1-mediated apoptosis is dependent on p53. NHDFs in which p53 was downregulated using siRNA as well as appropriate siRNA control NHDFs were transfected with either LACE1-FLAG or empty vector constructs. The resulting whole-cell extracts were western blotted with antibodies to p53, cleaved PARP, FLAG. Equal protein loading was monitored with α-tubulin detection.

Article Snippet: To find out whether overexpression of wild-type LACE1-FLAG (NM_145315) leads to correct mitochondrial targeting of the fusion protein, we have transiently transfected wild-type HEK293 cells with a C-FLAG-fusion human LACE1 ORF construct (OriGene Technologies).

Techniques: Transfection, Knockdown, Control, Western Blot, Plasmid Preparation, Construct