Journal: Aging Cell

Article Title: Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes

doi: 10.1111/acel.12835

Figure Lengend Snippet: Phospholipase A2 receptor (PLA2R1) promotes progerin‐induced misshapen nuclei. (a, b) MRC5 cells were infected with retroviral vectors encoding lamin A‐GFP or progerin‐GFP together with control or PLA2R1 shRNA sequences. Ten days after selection, cells were analyzed by confocal microscope. Representative pictures are shown in panel a, and counting of normal, mildly misshapen, and severely misshapen nuclei was performed and is displayed in panel b. (c) Ten days after the end of the selection, immunofluorescence using a lamin A/C antibody was performed in HGPS‐derived fibroblasts encoding either control or shRNA directed against PLA2R1. The same classification as in b was used. Around 200 nuclei were counted for each condition (mean ± SEM s). The experiments shown are representative of at least three biological repeats. Statistical analysis was performed with Student's t test (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: The following vectors were supplied by Addgene: pBABE‐puro‐GFP‐wt‐lamin A (#17662) and pBABE‐puro‐GFP‐progerin (#17663).

Techniques: Infection, shRNA, Selection, Microscopy, Immunofluorescence, Derivative Assay

Journal: Aging Cell

Article Title: Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes

doi: 10.1111/acel.12835

Figure Lengend Snippet: A progerin/PLA2R1 pathway increases FDPS expression and thereby induces senescence and misshapen nuclei. (a, b) Fifteen days after selection, RNAs were extracted from MRC5 or HGPS‐derived cells, and mRNA levels of FDPS were quantified by RT–qPCR for each indicated condition. Results were normalized against ACTB levels. (c) RNA was extracted from the bone of mice of the indicated genotypes before RT–qPCR experiments to measure FDPS levels. FDPS levels were normalized against ACTB levels. The number of mice per group ranged from 3 to 5. (d–k) MRC5 cells were infected with retroviral vectors encoding lamin A‐GFP or progerin‐GFP. After selection and seeding the same quantity of cells, cells were treated every 3 days with 5 µM pamidronate (a bisphosphonate FDPS inhibitor). (d) After 15 days of treatment, cells were stained for SA‐β‐Gal activity, and the percentage of SA‐β‐Gal‐positive cells was calculated. (e) Eight days after selection, the same amounts of cells were seeded. Ten days later, cells were fixed and stained with crystal violet. (f) At each passage, cells were counted and the number of population doublings was calculated, and the same number of cells was reseeded. (g–i) Fifteen days after selection, RNA was prepared, reverse‐transcribed, and indicated transcript levels were quantified by qPCR and normalized against ACTB levels. (j, k) Fourteen days after selection, cells were analyzed by confocal microscopy. Representative pictures are shown, and counting of normal, mildly misshapen, and severely misshapen nuclei was performed. The experiments shown are representative of at least three biological repeats. Statistical analysis was performed with Student's t test (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: The following vectors were supplied by Addgene: pBABE‐puro‐GFP‐wt‐lamin A (#17662) and pBABE‐puro‐GFP‐progerin (#17663).

Techniques: Expressing, Selection, Derivative Assay, Quantitative RT-PCR, Infection, Staining, Activity Assay, Confocal Microscopy

Journal: Aging Cell

Article Title: Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes

doi: 10.1111/acel.12835

Figure Lengend Snippet: p53 promotes FDPS expression and misshapen nuclei in progerin‐expressing cells. (a) MRC5 cells infected with retroviral vectors encoding lamin A or progerin together with or without a retroviral vector encoding shRNA directed against PLA2R1. Fifteen days after selection, protein extracts were prepared and analyzed using antibodies targeting the indicated proteins. (b–e) MRC5 cells were infected with retroviral vectors encoding lamin A or progerin together with or without a retroviral vector encoding a dominant‐negative form of p53 (p53DN). (b) Twelve days after selection, photographs of nuclei and (c) quantification of normal, mildly misshapen, and severely misshapen nuclei were performed. (d) Ten days after infection and selection, RNAs were prepared, reverse‐transcribed, and FDPS transcripts were quantified by qPCR. Results were normalized against ACTB mRNA levels. (e) Twelve days after selection, protein samples were prepared. Samples were analyzed by immunoblot using antibodies targeting the indicated proteins. (f) MRC5 cells were treated with nutlin‐3, and 5 days later, RT–qPCR against FDPS and p21 was performed. Results were normalized against ACTB mRNA levels. (g) DNA pulldown assay showing p53 binding to a DNA motif in FDPS promoter. A lysate of MRC5 primary human fibroblasts treated with nutlin‐3 was incubated with FDPS probe or CDKN1A probe, harboring either a wild‐type (WT) p53‐binding motif or a mutant one with two base substitutions (mut). p53 binding was assessed by western blot. The experiments shown are representative of at least two biological repeats. Statistical analysis was performed with Student's t test (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: The following vectors were supplied by Addgene: pBABE‐puro‐GFP‐wt‐lamin A (#17662) and pBABE‐puro‐GFP‐progerin (#17663).

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Selection, Dominant Negative Mutation, Western Blot, Quantitative RT-PCR, Binding Assay, Incubation, Mutagenesis