Journal: BMC Cell Biology

Article Title: Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage

doi: 10.1186/s12860-014-0043-3

Figure Lengend Snippet: Silencing of LPCAT1 and LPCAT2 by siRNA leads to enlarged lipid droplets in A431 cells. A) A431 cells were either left untreated (untreated, wt), mock transfected (mock), transfected with control siRNA (eg5 as transfection control, leads to cell death, or scrambled#5 + #6 as non-targeting siRNAs) or the four possible combinations of two sequences each against LPCAT1 or LPCAT2 as indicated. After 48 h cells were lysed and subjected to SDS-PAGE/Western blotting for LPCAT1, LPCAT2 and glycerol-3-phosphate dehydrogenase (GAPDH, as a load control). B) Confocal images of controls and LPCAT1/LPCAT2 double-knock-downs as described in panel A . Nuclei (blue), LDs (green), scalebar = 10 μm. C) Confocal images as described in panel B were quantified with Image J for LD size distribution as described in . Data are mean LD size ± StdDev, calculated from >50 individual cells in 3 independent experiments. Significances relative to non-targeting siRNAs were calculated by unpaired two-sided T -test analysis (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). D) Confocal image as described in panel B were quantified with ImageJ. Data show mean lipid droplet number per frame, corrected for variations in cell density, calculated from >50 individual cells in 3 independent experiments. Control: scrambled#5 + #6, LPCAT siRNA: average of all siRNA treatments. Significance was calculated by unpaired two-sided T -test analysis (*** p ≤ 0.001).

Article Snippet: Polyclonal rabbit antisera against the C-terminal peptide of human LPCAT1 CNSDAGRKPVRKKLD, conjugated to keyhole limpet hemocyanin, and against purified recombinant 6His-hLPCAT2 310-545 were raised by Eurogentec (Seraing, Belgium) and were affinity purified against the respective antigen.

Techniques: Transfection, SDS Page, Western Blot

Journal: BMC Cell Biology

Article Title: Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage

doi: 10.1186/s12860-014-0043-3

Figure Lengend Snippet: Silencing of LPCAT1 by siRNA leads to enlarged lipid droplets and reduced lipoprotein secretion in HuH7 cells. A) HuH7 cells were either left untreated (untreated, wt), mock transfected (mock), transfected with control siRNA (scrambled#5 or scrambled#6 as non-targeting siRNAs) or the two different siRNA sequences against LPCAT1 as indicated. After 72 h cells were subjected to a LPCAT activity assay or Western blotting (WB) for LPCAT1 using GAPDH, as load control. B) Confocal images of controls and LPCAT1 knock-downs as described in panel A . Nuclei (blue), LDs (green), scale bar = 10 μm C + D) Confocal images as described in panel B were quantified for LD size distribution as described in . Data represented mean LD size ± StdDev, calculated from > 50 individual cells in 3 independent experiments. Significances were calculated by unpaired two-sided T -test analysis relative to non-targeting siRNA (scrambled#5) (*** p ≤ 0.001). For analysis of LD size distribution (panel D ), LDs were grouped into size classes, and the distribution displayed as percentage of total LDs per size class. Controls (black, light and dark grey and white), siRNAs against LPCAT1 (red, blue). E) Confocal images as described in panel B were quantified with Image J. Data show mean lipid droplet number per frame, corrected for variations in cell density, calculated from >50 individual cells in 3 independent experiments. Control: average of scrambled#5 and scrambled#6, LPCAT siRNA: average of both siRNA treatments. Significance was calculated by unpaired two-sided T -test analysis (*** p ≤ 0.001). F) HuH7 cells were transfected with non-targeting siRNA (control) or different siRNA sequences targeting LPCAT1 as indicated. ApoB secretion was measured by ELISA (dark grey, data represent mean ± StdDev, n = 4) or by Western blotting (light grey, data represent mean ± StdDev, n = 5). [3H]lipid secretion after labeling with 1 μCi [3H]oleate was calculated as % of total radioactivity recovered (supernatant + cells) and normalized to control. Data represent mean ± StdDev, n = 4. p-Values were obtained by unpaired T -test relative to the respective control (** p ≤ 0.01, * p ≤ 0.05).

Article Snippet: Polyclonal rabbit antisera against the C-terminal peptide of human LPCAT1 CNSDAGRKPVRKKLD, conjugated to keyhole limpet hemocyanin, and against purified recombinant 6His-hLPCAT2 310-545 were raised by Eurogentec (Seraing, Belgium) and were affinity purified against the respective antigen.

Techniques: Transfection, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Radioactivity

Journal: BMC Cell Biology

Article Title: Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage

doi: 10.1186/s12860-014-0043-3

Figure Lengend Snippet: Increase in LD size upon LPCAT1/2 knock-down is independent of neutral lipid synthesis and accumulation. A) A431 cells were transfected with siRNA as described in Figure A. Seventy hours after siRNA transfection growth medium was exchanged to medium containing 10% delipidated FCS and 50 μM alkyne-oleate. After two hours cells were washed and lipids extracted. Extracts were subjected to quantitative click-analysis for the ratio of incorporation of alkyne fatty acid into TAG and PC. Data are mean ± StdDev, n = 3. Significances were calculated by unpaired two-sided T -test analysis relative to non-targeting siRNA (scrambled#5 + #6) and were found to be insignificant. B) A431 cells were transfected as described in Figure A. After 48 h, total lipid extracts were analyzed by mass spectrometry in duplicate samples, and species abundances were normalized to the corresponding internal standard. The molar contents of each species of the same class were summed up and normalized to the total content of all detectable lipids. C) HuH7 cells were transfected with siRNA as described in Figure A. Seventy hours after siRNA transfection as indicated, growth medium was exchanged to medium containing 10% delipidated FCS and 50 μM alkyne-oleate. After two hours, cells were washed, lipids extracted and extracts were subjected to quantitative click-analysis as in panel A . Data are mean ± StdDev, n = 3. Significances were calculated compared to non-targeting siRNAs (scrambled#5) and were found to be insignificant. D) HuH7 cells were transfected as described in Figure A. After 72 h, total lipid extracts were analyzed by mass spectrometry in duplicate samples. Species abundances were normalized as described for panel B .

Article Snippet: Polyclonal rabbit antisera against the C-terminal peptide of human LPCAT1 CNSDAGRKPVRKKLD, conjugated to keyhole limpet hemocyanin, and against purified recombinant 6His-hLPCAT2 310-545 were raised by Eurogentec (Seraing, Belgium) and were affinity purified against the respective antigen.

Techniques: Transfection, Mass Spectrometry

Journal: BMC Cell Biology

Article Title: Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage

doi: 10.1186/s12860-014-0043-3

Figure Lengend Snippet: The LPCAT1/2 fly ortholog CG32699 resembles human LPCAT1 and 2. A) Drosophila melanogaster S2 Schneider cells were transfected with GFP-CG32699, which is the fly ortholog of mammalian LPCAT1 and LPCAT2. The growth medium was supplemented with 100 μM oleate. In the merged image, LDs are shown in red and CG32699 in green. Lower panels show a higher magnification of the central region of the pictures in the upper panels. Scalebar = 10 μm. Note the colocalization of GFP and LD540 signals. Resolution is limited due to the small size and round shape of S2 cells. B) Female virgin flies of a tubulin-Gal4 containing fly strain were crossed with male flies of three different CG32699-specific RNAi containing fly strains. L3 larvae were selected, fat bodies were isolated and representative DIC images of those fat bodies are shown for wildtype (wt), white mutant (white) and the three RNAi strains 32382, 32924 and 104570. The amount of CG32699 mRNA was measured by quantitative PCR. Scale bar = 10 μm. C + D) Bright field images as shown in panel B were quantified with ImageJ. Mean lipid droplet number per frame and mean lipid droplet size is presented for 3 frames per condition of 4 different experiments. significances were calculated by unpaired T -test analysis (*** p ≤ 0.001, ** p ≤0.01) compared to control. E) Female virgin flies of a tubulin-Gal4 containing fly strain were crossed with male flies of 3 different CG32699-specific RNAi containing fly strains or white mutant (white). From each cross three L3 larvae were selected and subjected to a LPCAT activity assay. Data were analyzed with Gel Pro Analyzer. Standard deviations and significances were calculated from three experiments by unpaired T -test analysis (** p ≤ 0.01, * p ≤0.05) compared to control (white).

Article Snippet: Polyclonal rabbit antisera against the C-terminal peptide of human LPCAT1 CNSDAGRKPVRKKLD, conjugated to keyhole limpet hemocyanin, and against purified recombinant 6His-hLPCAT2 310-545 were raised by Eurogentec (Seraing, Belgium) and were affinity purified against the respective antigen.

Techniques: Transfection, Isolation, Mutagenesis, Real-time Polymerase Chain Reaction, Activity Assay

Journal:

Article Title: Temporal dissection of Bax-induced events leading to fission of the single mitochondrion in Trypanosoma brucei

doi: 10.1038/sj.embor.7400095

Figure Lengend Snippet: Bax-induced changes of cytochrome c localization and of mitochondrial energy metabolism. (A) A transgenic T. brucei cell line allowing tetracycline-inducible expression of human Bax was analysed for the release of cytochrome c. Using immunoblots, 50 μg each of cytosolic extract (upper panel) and of isotonically isolated gradient-purified mitochondria (middle panel; Hauser et al, 1996) from uninduced cells (−Tet) and from cells expressing Bax for 3 or 5 h (+Tet (h) 3 or 5), respectively, were analysed for cytochrome c content. All mitochondrial fractions were also analysed for the presence of Hsp60 (lowest panel). (B) Mitotracker staining of an uninduced cell with an intact mitochondrial membrane potential (−Tet) and a cell expressing Bax for 6 h that lacks a membrane potential (+Tet, 6 h). (C) Time course of Bax-induced changes in mitochondrial energy metabolism. Four parameters were measured at the indicated time points after induction of Bax expression: (i) in organello ATP production in response to succinate and glycerol-3-phosphate to monitor oxidative phosphorylation; (ii) in organello ATP production in response to pyruvate to measure mitochondrial substrate level phosphorylation (van Hellemond et al, 1998; Bochud-Allemann & Schneider, 2002); (iii) intracellular ATP levels (ATP production and ATP content are indicated relative to uninduced cells); and (iv) the presence or absence of a mitochondrial membrane potential in individual cells (n=150–300) as determined by Mitotracker staining (expressed as the percentage of the total population that retains a membrane potential).

Article Snippet: Mitochondrial and cytosolic protein extracts from uninduced cells and cells induced for Bax expression for 3 and 5 h, respectively, were resolved on 16% SDS–PAGE, blotted to nitrocellulose and probed with polyclonal rabbit antisera directed against trypanosomal cytochrome c . The rabbit antiserum was produced by Eurogentec using the peptides PPKERAALPPGDAVR and QERADL IAYLETLKD as antigens.

Techniques: Transgenic Assay, Expressing, Western Blot, Isolation, Purification, Staining

Journal: PLoS ONE

Article Title: The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway

doi: 10.1371/journal.pone.0010054

Figure Lengend Snippet: (A) Protein extracts from wt flies and flies expressing either the UAS-dmfn-RNAi Vienna or UAS-dmfn-RNAi Guo transgenes targeting the dmfn gene were subjected to western blot analysis with an anti-dMfn antiserum. The hsp70-GAL4 driver was used to induce expression of the dmfn-RNAi transgenes, and flies were collected for analysis 24 to 48 hrs after a 3-hr heat shock protocol. The arrow indicates the location of the dMfn band at 94 kDa. The asterisks (*) indicate two nonspecific bands detected at 80 kDa and 110 kDa. (B) Protein extracts from wt flies, flies bearing the UAS-Opa1-FLAG transgene and the mesoderm-specific 24B-GAL4 driver, and flies bearing the UAS-opa1RNAi transgene and the muscle-specific dmef2-GAL4 driver were subjected to western blot analysis with an anti-Opa1 antiserum. (C) Protein extracts from wt flies, flies bearing the UAS-Drp1-HA transgene and the muscle-specific dmef2-GAL4 driver, and flies heterozygous for the Df(2L)Excel6008 deletion chromosome (which removes the drp1 gene) were subjected to western blot analysis with an affinity-purified anti-Drp1 antiserum.

Article Snippet: Rabbit polyclonal antisera recognizing the Drosophila Drp1 and dMfn were generated by a commercial source (Pocono Rabbit Farm, Canadensis, PA) using synthetic peptides corresponding to sequences in these proteins (Drp1: PPAPTRPDSIENST; dMfn: FTGKVRERSKKGQP).

Techniques: Expressing, Western Blot, Affinity Purification

Journal: PLoS ONE

Article Title: The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway

doi: 10.1371/journal.pone.0010054

Figure Lengend Snippet: Protein extracts from wt males (m), PINK1 B9 hemizygous males, wt males and females (m and f) and park 25 males and females were subjected to western blot analysis with an affinity-purified anti-dMfn antiserum (A), an anti-Opa1 antiserum (C), an affinity-purified anti-Drp1 antiserum (D), an anti-complex V β antiserum (A, C, D), an anti-VDAC antiserum (A, C, D), and an anti-actin antiserum (A, C, D). Arrow in (A) indicates location of the dMfn band at 94 kDa and the asterisks (*) indicate nonspecific bands. (B) Quantification of dMfn abundance in PINK B9 and park 25 null mutants relative to wt controls. Because PINK1 B9 males are sterile and the PINK1 gene resides on the X chromosome, we are only able to generate male PINK1 B9 mutants and thus used wt males as the control population for these mutants. The ratio of dMfn to actin abundance was obtained from three independent blots for each sample analyzed; the ratios for wt controls were set at a value of 1 and mutant ratios were normalized to the wt ratios. *p<0.05 by Student’s t-test. (E) Protein extracts from mitochondrial and cytosolic fractions (see section for details on fractionation) were subjected to western blot analysis with an affinity-purified anti-Drp1 antiserum, an anti-complex V β (CompV) antiserum, an anti-VDAC antiserum, and an anti-actin antiserum. (F) Protein extracts from wt flies, flies overexpressing Parkin ( hsp70-GAL4/UAS-Parkin ) and flies overexpressing PINK1 ( hsp70-GAL4/UAS-PINK1 ) were subjected to western blot analysis with an affinity-purified anti-dMfn antiserum, an anti-complex V β antiserum, an anti-VDAC antiserum, and an anti-actin antiserum. Flies were subjected to a 1-hr heat shock and collected for analysis 24 hrs following the heat shock. The arrow indicates the location of the dMfn band at 94 kDa and the asterisks (*) indicate the two nonspecific bands.

Article Snippet: Rabbit polyclonal antisera recognizing the Drosophila Drp1 and dMfn were generated by a commercial source (Pocono Rabbit Farm, Canadensis, PA) using synthetic peptides corresponding to sequences in these proteins (Drp1: PPAPTRPDSIENST; dMfn: FTGKVRERSKKGQP).

Techniques: Western Blot, Affinity Purification, Sterility, Control, Mutagenesis, Fractionation