Journal: Stem Cells Translational Medicine

Article Title: Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation

doi: 10.1093/stcltm/szae013

Figure Lengend Snippet: Silencing of agrin expression in neurospheres significantly improves cell migration following transplantation in vivo. Two neurospheres were implanted subserosally into the distal colon of wild-type mice ( A ), with tdT fluorescence confirming proper placement ( B ). LMMP of wild-type mouse colon was examined 7 days after in vivo transplantation ( C ), where Wnt1;tdT marks the transplanted cells, TUJ1 stains intrinsic and transplanted nerve fibers, and GFP represents the reporter in the lentivurus. Adult Wnt1;tdT control neurospheres ( D ) migrating significantly less than neurospheres expressing agrin-shRNA lentivirus ( E, F ).

Article Snippet: Immediately after cell sorting, tdT + cells were transduced with an agrin shRNA lentivirus containing 4 unique 29mer agrin targeting shRNA and a GFP reporter (purchased from Origene; CAT#: TL512313V, and expanded and packaged at the MGH Vector Core Facility, Massachusetts General Hospital Neuroscience Center, Charlestown, MA to have a final concentration of 10 12 TU/ml) or a control scramble shRNA with a GFP reporter (purchased from Origene; CAT# TR30021V, and expanded and packaged at the MGH Vector Core Facility to have a final concentration of 10 12 TU/mL).

Techniques: Expressing, Migration, Transplantation Assay, In Vivo, Fluorescence, Control, shRNA

Journal: iScience

Article Title: The prion protein is required for normal responses to light stimuli by photoreceptors and bipolar cells.

doi: 10.1016/j.isci.2024.110954

Figure Lengend Snippet: Figure 7. Morphometric analysis of ribbon synapses and cone pedicle anatomy (A and B) Confocal maximum intensity projections show representative retinal sections from 129/Ola WT and PrPKO mice. PKCa (green) marks cell bodies and dendrites of rod bipolar cells, CtBP2 (yellow) marks synaptic ribbons and Pikachurin (magenta) is a protein present in the cleft of ribbon synapses. (C and D) 3D surfaces were rendered using IMARIS software 9.9.1. to enable quantitation of ribbons and synaptic puncta, assessment of 3D morphology, and relative associations of synaptic ribbons, Pikachurin and rod-bipolar cell dendrites. (E and F) Quantitation of ribbons (CtBP2), synaptic cleft puncta (Pikachurin) and ratio of ribbons to synaptic puncta are shown in graphs for both 129/Ola and C57BL/6J retinas. (G and H) Cone pedicle (green) and ribbon synapse (yellow) 3D surfaces were rendered using IMARIS software 9.9.1. For 129/Ola -WT and PrPKO, top panels show pre-rendered confocal image and bottom panels show 3D rendered images. Example cone pedicles with volume calculation are shown at right. Each cone pedicle was rotated in 3D to study associations with ribbon synapses (yellow). (I and J) Quantitative data from analysis of confocal datasets in E, are shown for cone pedicle volumes, and the number of ribbon synapses per cone pedicle. Scale bars: A, B = 4 mm (left), 2 mm (right) C, D = 1 mm G, H = 5 mm (left), 1 mm (right). Statistical analysis by Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, bars represent mean values, +/ SD.

Article Snippet: Antibodies D13-anti prion protein (Human) 1:100 Matsuanaga66 N/A Anti-Cone Arrestin - Arrestin 3/Cone photoreceptors – Rabbit - 1:100 Millipore AB15282; RRID:AB_1163387 Anti-CtBP2 - C-terminal binding protein 2/ribeye protein of ribbon synapses - Mouse 1:100 Santa Cruz sc-17759; RRID:AB_627310 Anti-PKCa - Protein kinase C/Rod bipolar cells - Goat 1:100 RD Systems AF5340; RRID:AB_2168552 Anti -Pikachurin (ribbon synaptic cleft) - Rabbit 1:100 Proteintech 14578-1-AP; RRID:AB_2277653 Anti- EAAT1 - Excitatory amino acid transporter - Rabbit 1 1:100 Abcam ab416; RRID:AB_304334 Anti-EAAT5 - Excitatory amino acid transporter 5 - Rabbit 1:100 Abcam ab230217 Anti -PSD-95 - Post synaptic density-95 - Mouse 1:100 Neuromics MO50000 Anti-Calbindin – horizontal cells – Rabbit 1:100 Abcam Ab108404; RRID:AB_10861236 Critical commercial assays Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 10X Genomics v3.1

Techniques: Software, Quantitation Assay, MANN-WHITNEY