Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, MEFs (p19Arf−/−) were used since these cells are naturally immortalized due to disruption of p19Arf, a component mediating cellular senescence (52), and were easily infected by the recombinant adenoviruses. The MEFs were infected with either control adenoviruses (Ad-GFP) or adenoviruses expressing menin (Ad-menin) at an MOI of 20. Bright-field images of cells are shown (Top panel) 41 h after addition of adenoviruses to the medium. The relative levels of GFP expression are shown using fluorescence microscopy (Bottom panel). B, Ad-menin, but not Ad-GFP, dramatically increases the percentage of Annexin V-positive cells in a dose-dependent manner. Results are representative of three independent experiments.

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Disruption, Infection, Recombinant, Control, Expressing, Fluorescence, Microscopy

Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, The cells were infected at an MOI of 20 and analyzed for the release of free nucleosomal DNA. The assay was performed in duplicate, with standard deviation (SD) presented. This is representative of three independent experiments. B, Detection of expression of menin from Ad-menin. The MEFs infected with either Ad-GFP or Ad-menin were analyzed by Western blotting analysis.

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Infection, Standard Deviation, Expressing, Western Blot

Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, Wild type or Bax−/−/Bak−/− double knock out (DKO) MEFs immortalized using an NIH 3T3 protocol (53) were infected with increasing MOIs (0, 200, 340, 460) of either control Ad-GFP-12 or Ad-menin-22 as indicated. The MOIs used for these cells were higher since they are more refractory to infection. The infected cells were harvested, stained with Annexin V-Cy 5, and analyzed by FACS as described in Fig. 1B. The results are presented as the average of three independent experiments, with SD noted. B, The wild type or DKO cells were infected at an MOI of 200 and processed to detect release of free nucleosomal DNA. The assay was performed in duplicate, with SD presented. This is one of two independent experiments. C, The level of menin expression in the infected (MOI of 200) wild type or DKO cells. Cell lysates (55 μg/lane) from Ad-Menin or Ad-GFP-infected cells were immunoblotted with an anti-menin antibody.

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Knock-Out, Infection, Control, Staining, Expressing

Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, Vector and menin-complemented cell lines were prepared as described in Experimental Procedures. Expression of menin in the cells was detected using Western blotting analysis. B, Complementation of menin-null MEFs with menin increases their sensitivity to UV-induced apoptosis. The vector-complemented cells or menin-complemented cells were treated with UV and stained with trypan blue 24 h later. The assay was performed in duplicate, with SD presented. Results are representative of three independent experiments. C, Vector-complemented or menin-complemented cells were treated with UV and stained with Annexin V 16 hours later. Results are presented as the average of two independent experiments, with SD noted. Complementation of menin-null cells with menin enhances cellular sensitivity to TNF-α, as assessed by trypan blue (D) and Annexin V staining (E).

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Plasmid Preparation, Expressing, Western Blot, Staining

Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, Two independent pairs of menin-expressing and menin-null cell lines, from two distinct groups of littermates (53 and 55 in lanes 1 and 2; 23 and 21 in lanes 3 and 4), were established as described in Experimental Procedures. The expression of menin in the cells was confirmed by Western blotting analysis using an anti-menin antibody (#80). B, RNA (20 μg) was isolated from the above cell lines and was hybridized to a cDNA probe encoding the full-length procaspase 8 (genbank gi 10976309). The bottom panel shows the GAPDH control, demonstrating even loading.

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Expressing, Western Blot, Isolation, Control

Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, Vector and menin-complemented cell lines (27 and 26 cells) were prepared as described in Experimental Procedures. Expression of menin in the cells was detected using an anti-menin antibody (#80). B, RNA (20 μg) was isolated from the above cell lines and was hybridized to the caspase 8 probe (Top panel) or the control GAPDH probe for the loading control (Bottom panel).

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Plasmid Preparation, Expressing, Isolation, Control

Journal:

Article Title: Menin Induces Apoptosis in Murine Embryonic Fibroblasts

doi: 10.1074/jbc.M308073200

Figure Lengend Snippet: A, Three independent pairs of vector and menin-complemented cell lines (26 and 27, AS24 and AS23, and 75 and 78, respectively, lanes 1–6) were prepared as described in Experimental Procedures. The whole cell lysates from these cells (100 μg) were separated by SDS-PAGE and blotted with an anti-caspase 8 antibody (100 μg/mL) (Top panel), or with an anti-actin antibody for loading controls (Bottom panel). B, The vector-complemented and menin-complemented MEFs (27 and 26 cells) were treated with TNF-α, (10 ng/mL)/cycloheximide (5 μg/mL) for 0, 3, 6, 12, or 24 hours, harvested, and processed for the caspase 8 enzymatic activity assay. The assay was performed in triplicate. This is representative of two independent experiments.

Article Snippet: Preparation of Immortalized Murine Embryonic Fibroblasts (MEFs) Men1 ΔN3–8/+ mice ( Men1 +/ − ) heterozygous for the Men1 locus were maintained on a 129s6/SvEvTac background (Taconic, Germantown, New York) ( 15 ).

Techniques: Plasmid Preparation, SDS Page, Enzyme Activity Assay

Journal: Cell

Article Title: ApoE2, ApoE3 and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion

doi: 10.1016/j.cell.2016.12.044

Figure Lengend Snippet: (A) Experimental design. Human neurons (iN cells) were generated from H1 ES cells by forced expression of neurogenin-2 (Ngn2), and cultured either on mouse glia (green) which secrete copious amounts of ApoE, on murine embryonic fibroblasts (MEFs) which secrete no ApoE (blue), or on matrigel (black lines).

Article Snippet: Murine embryonic fibroblasts (MEFs) were isolated from mouse embryos of CF-1 strain (Harlan Laboratories, Inc.) harvested at 12.5–13.5 postcoitum (p.c.).

Techniques: Generated, Expressing, Cell Culture

Journal: Cell

Article Title: ApoE2, ApoE3 and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion

doi: 10.1016/j.cell.2016.12.044

Figure Lengend Snippet: (A) ApoE (10 µg/ml, applied at D10 for 2 h) activates ERK1/2 phosphorylation in human neurons cultured on MEFs with an ApoE4>ApoE3>ApoE2 potency rank order (left, representative immunoblots; right, summary graphs).

Article Snippet: Murine embryonic fibroblasts (MEFs) were isolated from mouse embryos of CF-1 strain (Harlan Laboratories, Inc.) harvested at 12.5–13.5 postcoitum (p.c.).

Techniques: Phospho-proteomics, Cell Culture, Western Blot

Journal: Cell

Article Title: ApoE2, ApoE3 and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion

doi: 10.1016/j.cell.2016.12.044

Figure Lengend Snippet: (A) Human neurons synthesize ~5-fold less APP when cultured on MEFs instead of glia; addition of ApoE2, ApoE3, or ApoE4 (each 10 µg/ml applied from D10-12) stimulates APP synthesis in human neurons on MEFs with a ApoE4>ApoE3>ApoE2 potency rank order, which is blocked by the ApoE-receptor antagonist RAP (left, representative immunoblots; right, summary graphs; see also Fig. S5).

Article Snippet: Murine embryonic fibroblasts (MEFs) were isolated from mouse embryos of CF-1 strain (Harlan Laboratories, Inc.) harvested at 12.5–13.5 postcoitum (p.c.).

Techniques: Cell Culture, Western Blot