Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: ( a ) 10R UV crosslinked to U87 nuclear extract (NE) and digested with RNAse A (0.5 ug) (lane 3), RNAse T1 (10U) (lane 4), both (0.5ug RNAse A/10U RNAse T1) (lane 5), or nothing (lane 2), and separated by 10% SDS-PAGE. ( b ) 0.1 pmoles of 10R in water (lanes 1, 2 and 4) or 200 mM KCl and 500 μM pyridostatin (PDS) (lanes 3 and 5), IPed with BG4 antibody (lanes 4 and 5) or beads alone (lanes 2 and 3). Products were separated by 10% SDS-PAGE (top) and by 8M Urea/10% PAGE (bottom). ( c ) Representative G-Q folding conformations of 10 repeats of GGGGCC RNA. Numbers indicate G-tracts that participate in GQs or linear fragments. The right most conformation represents 8 consecutive G-tracts forming an octamer. Beneath, a depiction of one quartet, with the N7 (replaced with C in 7-deaza GTP) position surrounded by red dashed oval ( d ) 10R (lanes 1 and 3) and 10R-7dG (lanes 2 and 4) heated to 95°C (lanes 3 and 4) or left on ice (lanes 1 and 2), crosslinked to NE, digested with RNAse A/T1 (.5 ug/10U) and separated by 10% SDS-PAGE. ( e ) 10R-G (lanes 1, 3, 5 and 7) and 10R-G7dG (lanes 2, 4, 6 and 8) heated to 95°C in water (lanes 5 and 6) or 100 mM KCl (lanes 7 and 8) or left on ice (lanes 3 and 4), crosslinked to NE, digested with RNAse A/T1 (0.5 μg/10 U) and separated by 10% SDS-PAGE. This gel was cropped to remove two intervening lanes between lanes 6 and 7. DOI: http://dx.doi.org/10.7554/eLife.17820.002

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: SDS Page

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: Dark exposure of (top) depicting recovery of 10R in water (lane 4) using the BG4 antibody. DOI: http://dx.doi.org/10.7554/eLife.17820.003

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques:

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: Representative nonALS and C9ALS fibroblasts ( a ) and astrocytes ( b ) showing the projection created by 'BG4 Count' that represents all stained area above the determined threshold (0.1) in red, with areas of particularly dense staining in white. Inset depicts source image showing only the red (BG4-FLAG) channel. ( c ) Number of foci and total stained area ( d ) as fold change over the average number of foci, or area, respectively, per control cell for C9 fibroblasts (n replicates = 16), control fibroblasts (n replicates = 12), C9 astrocytes (n replicates = 3), and control astrocytes (n replicates = 3) (*p<0.05, **p<0.01, ***p<0.001). Error bars are plotted to SEM. DOI: http://dx.doi.org/10.7554/eLife.17820.006

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: Staining

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: nonALS and C9ALS astrocytes lacking BG4 antibody incubation. DAP1 = blue; hnRNP H = green; FLAG-BG4 = red. DOI: http://dx.doi.org/10.7554/eLife.17820.007

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: Incubation

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: (Left) Immunofluorescence and BG4 count depiction of nonALS control and C9ALS astrocytes. (Right) Quantification of BG4 foci with and without RNAse A treatment, normalized to the average control, untreated cell. DAP1 = blue; hnRNP H = green; FLAG-BG4 = red. Scale bar = 10 microns. DOI: http://dx.doi.org/10.7554/eLife.17820.008

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: Immunofluorescence

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: C9 patient astrocytes with nuclear hnRNP H/BG4 foci ( a , c ) nuclear redistribution of hnRNP H towards high BG4 staining ( b ) and large cytoplasmic foci ( d ). ( e ) Percentage of control and C9 patient astrocytes containing nuclear and cytoplasmic hnRNP H colocalization events. ( f ) The single, small nuclear hnRNP H/BG4 nuclear foci in control astrocyte. ( g ) Nuclear hnRNP H/BG4 inclusion in C9 patient fibroblasts. ( h ) C9 patient fibroblasts with cytoplasmic hnRNP H/BG4 foci in DAPI-positive cytoplasm. ( i ) Summary of all fibroblasts counted and the number of colocalization event. All objects are indicated with arrow or dashed box ( c ), and all scale bars = 3 microns with the exception of ( h ), 10 microns. DOI: http://dx.doi.org/10.7554/eLife.17820.009

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: Staining

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: Percentage of cells with hnRNP H/BG4 colocalization without treatment and after RNAse A treatment. DOI: http://dx.doi.org/10.7554/eLife.17820.010

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques:

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: ( a ) Western blot analysis of hnRNP H in each of three fractions (soluble, sarkosyl soluble, sarkosyl insoluble). ( b ) Graph of the percentage of insoluble hnRNP H in each of the 14 patients ( c ) IP with BG4 (IP-BG4) from the resuspended sarkosyl insoluble fraction of motor cortex (BA4). (Left) Western blots of input unfractionated tissue probed with anti-GAPDH, hnRNP H and SR protein antibodies. (Right) Western blot of IP-BG4 probed with anti-hnRNP H and SR protein antibodies. HC and LC refer to IgG heavy chain and light chain, respectively. ( d ) Quantification of hnRNP H co-IPed with BG4 (n = 3 replicates) in each of ten non-ALS and C9ALS samples. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars are plotted to SEM. DOI: http://dx.doi.org/10.7554/eLife.17820.017

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: Western Blot

Journal: eLife

Article Title: The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains

doi: 10.7554/eLife.17820

Figure Lengend Snippet: Western blot of BG4 pulldown (IP-BG4) and FLAG-beads alone control IP, probed with anti-hnRNP H antibody. DOI: http://dx.doi.org/10.7554/eLife.17820.018

Article Snippet: BG4 Count is a script written in Matlab (MATLAB and Statistics Toolbox Release 2014b, The MathWorks, Inc., Natick, Massachusetts) that works by reading a multichannel image (given as. tif).

Techniques: Western Blot