Journal: bioRxiv

Article Title: Liquid droplet germ granules require assembly and localized regulators for mRNA repression

doi: 10.1101/382838

Figure Lengend Snippet: GFP reporter transcripts localize to P-granules when tethered (A) Single molecule fluorescent in situ hybridization (smFISH) in nematode germ cells to visualize RNA expression and localization. (B-M) Gonads were extruded from animals harboring the gfp reporter and (B-E) PGL-1::SNAP (n=30); (F-I) PGL-1::SNAP::λN22 (n=27); (J-M) PGL-1 (K126E K129E)::SNAP::λN22 (n=10). Gonads were fixed and imaged for gfp RNA using (B,F,J) smFISH; (C,G,K) GFP protein fluorescence; (D,H,L) DNA (DAPI) and SNAP. The smFISH, DNA and SNAP images are merged in E,I,M. White arrows mark examples of intranuclear puncta; black arrows mark examples of cytoplasmic puncta. Scale bar, 5 µm, for all images, except for 2.5-fold enlarged images in inset. For germline location, see . (N) Quantification of GFP signal in confocal images for the GFP reporter expressed with PGL-1::SNAP (n=23), PGL-1::SNAP::λN22 (n=24), PGL-1 (K126E K129E)::SNAP::λN22 (n=10). A silenced GFP reporter (n=25) served as a negative control. Mean GFP fluorescent signal and standard deviation reported in Arbitrary Units. (O) smFISH and PGL-1::SNAP signal colocalization in germlines expressing the GFP reporter and either PGL-1::SNAP or PGL-1::SNAP::λN22. Reported as the (Sum of smFISH GFP Intensity in SNAP:smFISH colocalized binned signal)/(Total smFISH GFP binned signal). Box plots represent the first and third quartiles, black line is median, whiskers to min and max values. Co-localization greater in germlines expressing PGL-1::SNAP::λN22 ( p <0.01). See Methods for more details.

Article Snippet: The Surface-Surface Colocalization XTension was downloaded and installed from the Bitplane XTension File Exchange at http://open.bitplane.com/tabid/235/Default.aspx?id=111 .

Techniques: In Situ Hybridization, RNA Expression, Fluorescence, Negative Control, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: Liquid droplet germ granules require assembly and localized regulators for mRNA repression

doi: 10.1101/382838

Figure Lengend Snippet: PGL-1-tethered GFP reporter transcripts localize to granules in the presence or absence of WAGO-1 Gonads were extruded from gfp reporter, PGL-1::SNAP::λN22 animals harboring (A-D) wago-1 wild-type (n=22); (E-H) wago-1 null (n=21). Gonads were fixed and imaged for (A,E) gfp RNA using smFISH, (B,F) GFP protein fluorescence, (C,G) DNA (DAPI) and SNAP. gfp RNA smFISH, DNA and SNAP merged in D, H. White arrows mark examples of intranuclear puncta; black arrows mark examples of cytoplasmic puncta. Scale bar, 5 µm, for all images, except for 2.5-fold enlarged images in inset. For germline location, see . (I) smFISH and PGL-1::SNAP signal colocalization in germlines expressing the GFP reporter, PGL-1::SNAP::λN22 in the presence or absence of WAGO-1. Reported as the (Sum of smFISH GFP Intensity in SNAP:smFISH colocalized binned signal)/(Total smFISH GFP binned signal). Box plots represent the first and third quartiles, black line is median, whiskers to min and max values. Co-localization similar in germlines expressing PGL-1::SNAP::λN22 with or without WAGO-1 (p=0.039). See Methods for more details.

Article Snippet: The Surface-Surface Colocalization XTension was downloaded and installed from the Bitplane XTension File Exchange at http://open.bitplane.com/tabid/235/Default.aspx?id=111 .

Techniques: Fluorescence, Expressing

Journal: eNeuro

Article Title: Antioxidant Treatment in Male Mice Prevents Mitochondrial and Synaptic Changes in an NMDA Receptor Dysfunction Model of Schizophrenia

doi: 10.1523/ENEURO.0081-17.2017

Figure Lengend Snippet: NAC protected mitochondrial function against ketamine-induced oxidative stress. A , Acute brain slices of the PFC from adult mice in the four treatment groups were stained with TMRM (red) to measure ketamine-induced changes in mitochondrial membrane potential. PVIs were identified by their GFP expression (green). B , PVIs from ketamine-treated animals exhibited significantly reduced TMRM intensity, which was mitigated by NAC (** p ≤ 0.01, *** p ≤ 0.001; N = 4-5 mice/group). C , Ketamine-induced changes in mitochondrial superoxide levels as indicated by MitoSox Red staining. Acute brain slices from adult mice were stained for MitoSox Red (bottom row); after fixation, the MitoSox Red signal was colocalized with immunostaining for PV (top row, green) and CamKII-α (top row, blue), to identify PVIs and pyramidal cells, respectively. Scale bar, 20 µm. D , Analysis of cell type-specific changes in mitochondrial superoxide levels in PVIs and pyramidal cells (black bars). Ketamine treatment increased mitochondrial ROS levels in both PVIs and pyramidal cells of the PFC; however, changes in PVIs were significantly larger, suggesting relatively greater vulnerability of PVIs to mitochondrial oxidative stress in response to ketamine. The application of NAC significantly ameliorated ketamine-induced mitochondrial ROS accumulation in both PVIs and pyramidal cells. NAC by itself also resulted in small elevations in ROS (* p ≤ 0.01, ** p ≤ 0.01, *** p ≤ 0.001. N = 4–7 mice/group).

Article Snippet: For a cell type-specific analysis of mitochondrial superoxide production z -stacked confocal images were obtained and 3D reconstructions of cells located 10–15 µm from the slice surface were used to colocalize MitoSox Red with PV and CamKII-α staining (NIS Advanced Research, Nikon).

Techniques: Staining, Expressing, Immunostaining