Journal: Neurology: Genetics

Article Title: Adult-Onset Alexander Disease: New Causal Sequence Variant in the GFAP Gene

doi: 10.1212/NXG.0000000000000681

Figure Lengend Snippet: Patient's pedigree carrying the missense mutation in GFAP : c.173T>C; p.L58P. The pedigree has been modified for confidentiality (diamonds). Wild type (+) and mutated (M) allelic forms of GFAP . AAO = age at onset.

Article Snippet: To test whether this new mutation c.173T>C; p.L58P in GFAP affects intermediated filament network formation, we introduced the point mutation in the plasmid encoding human GFAP (OriGene Technologies, SC118873 by mutagenesis (QuikChange XL Site-Directed Mutagenesis Kit, Agilent)).

Techniques: Mutagenesis, Modification

Journal: Neurology: Genetics

Article Title: Adult-Onset Alexander Disease: New Causal Sequence Variant in the GFAP Gene

doi: 10.1212/NXG.0000000000000681

Figure Lengend Snippet: (A) Representative Western blot performed in lysates from HeLa cells transfected with plasmids encoding human GFAP-WT or mutant GFAP-L58P (GFAP: mAb #3670 cell signaling 1:1000, GAPDH: sc-25778 Santa Cruz, 1:1000). (B) Representative confocal images showing HeLa cells transiently transfected with plasmids encoding human wild-type GFAP or mutant GFAP-L58P and labeled with GFAP antibody (mAb #3670 cell signaling 1:300, green fluorescence). The image shows that wild-type GFAP assembled in filament networks, whereas mutant L58P formed dot-like aggregates. Cells were costained with αβ-crystallin antibody (sc-137129 Santa Cruz 1:100, red fluorescence) in cells transfected with mutant GFAP -L58P, and αβ-crystallin formed dot-like aggregates that colocalized with the GFAP signal. Images are representative of 50 analyzed cells from 3 independent experiments. DAPI is indicated in blue in the merge images on the right.

Article Snippet: To test whether this new mutation c.173T>C; p.L58P in GFAP affects intermediated filament network formation, we introduced the point mutation in the plasmid encoding human GFAP (OriGene Technologies, SC118873 by mutagenesis (QuikChange XL Site-Directed Mutagenesis Kit, Agilent)).

Techniques: Western Blot, Transfection, Mutagenesis, Labeling, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model

doi: 10.1073/pnas.1311660110

Figure Lengend Snippet: Characterization of MELAS iPSCs. (A) m.3243A>G MELAS mutant mtDNA amount in parental fibroblast cultures (Fb), the reprogrammed iPSC lines (iPSC), and in fibroblast clones from the parental lines (Fb_cl). (B) RT-PCR analysis of ES cell-specific transcripts (OCT4, SOX2, NANOG, REX, and DNMT3B). All patient-derived iPSC clones expressed ES-specific genes similarly to the healthy control iPSCs and ES cells (HDF, human dermal fibroblasts; HES, human embryonic stem cells). (C) Immunofluorescence staining for ES cell-marker proteins TRA-1–60 (green), SSEA4 (green), and NANOG (red). Blue staining for nucleus is DAPI. Colony morphology and ES cell-marker protein expression were similar in all of the clones. (Scale bar, 50 μm.) (D) Expression of OCT4, SOX2, KLF4, and c-MYC compared with that in human ES cells and normalized against cyclophilin, expression. Expression of viral transgenes was down-regulated in all clones. (E) MELAS iPSC lines generated teratomas that differentiated toward all three germ layers independent of the mutation load. (Left) Mesoderm, cartilage; (Center) ectoderm, pigmented epithelia; and (Right) endoderm, intestinal epithelia. (Scale bar, 200 μm.) (F) iPSCs differentiated in vitro into neural cultures consisting of MAP2 and βIII-tubulin positive neurons (green) and GFAP-positive glia (red). DAPI staining for nuclei (blue). (Scale bar, 50 μm.) (G) m.3243A>G mutant mtDNA load in the iPSC lines during culture and in the differentiated cells. The heteroplasmy levels did not change significantly during culture or upon differentiation. MH, MELAS-high; ML, MELAS-low; p., passage number. See also Fig. S1.

Article Snippet: To reveal antigenic sites, the rehydrated sections were treated in a microwave in 1 mM EDTA, pH 8, for 3 min. Primary antibodies were against Tra1-60 (1:40, ab90232, Millipore), SSEA4 (1:1,000, ab90231, Millipore), Nanog (1:500, D73G4, Cell Signaling Technology), β-III-tubulin [1:100 (immunofluorescence, IF), 1:500 (immunohistochemistry, IHC), T2200, Sigma], MAP2 (1:2,000, ab5543, Millipore), GFAP (1:100, mab2594, R&D Systems), DNA (1:20, AC-30-10, Progen Biotechnik), GM130 (1:250, sc16268, Santa Cruz Biotechnology), COX1 [1:200 (IF), 1:500 (western blotting, WB), M404, Mitosciences], C1-NDUFS3 (1:142, ab110246, abcam), CI-NDUFB4 (1:2,000, ab110243, abcam), CI-NDUFA9 (1:2,000, ab14713, abcam), CII-70kDa [1:2,000 (IF), 1:10000 (WB), 1:142 (IHC), MS204, Mitosciences], CIII-UQCRC2 [1:2,500 (WB), 1:1,000 (IF), 1:142 (IHC), ab14745, abcam], CV-α (1:1,000, MS507, Mitosciences), LC3B (1:200, NB600-1384, Novus Biologicals), PINK1 [1:250 (IF), 1:500 (WB), ab75487, abcam], PRK8 1:250 (IF), 1:1,000 [(WB) ab15954, abcam], β-tubulin (1:5,000, 2146, Cell Signaling Technology), β-Actin (1:2,000, sc1616, Santa Cruz Biotechnology), and TOM20 [1:250 (IF), 1:500 (WB), sc11415, Santa Cruz Biotechnology].

Techniques: Mutagenesis, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Control, Immunofluorescence, Staining, Marker, Expressing, Generated, In Vitro