Journal: Neurobiology of disease

Article Title: A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-β toxicity

doi: 10.1016/j.nbd.2019.104668

Figure Lengend Snippet: Tau-PxxP 5/6 inhibits the Tau-Fyn interaction in cells. A: Timeline of Tau-Fyn PLA experiments in HEK-293 cells. B: Representative immunofluorescent images of HEK-293 cells treated with Tau-PxxP 5/6 (rrrqrrkkrgRSRTPSLPTPPT-REPKK) or Tau-CTD (rrrqrrkkrgENAKAKTDHGAEIVYKS) after Tau-Fyn proximity ligation assay (PLA) was performed (full description in the section). Green puncta represent sites of Tau-Fyn interaction, red is Tau-mKate2, and magenta is immunofluorescently labeled Fyn. Scale bar = 10 μm. Quantification of Tau-Fyn PLA density demonstrates that Tau-SH3 inhibitor Tau-PxxP 5/6 reduced Tau-Fyn interaction by 40% while negative control peptide Tau-CTD did not (ANOVA, F(2, 33) = 10.14, p = .0004, average of 5–7 images per coverslip from n =11–13 coverslips from N = 6 distinct passages, see for details of quantification and analysis). Differences indicated: ** p < .01, and *** p < .001 by Dunnetťs post hoc test with correction for multiple comparisons relative to control. C: Western blot of lysate from HEK-293 cells demonstrates that Tau-PxxP 5/6 did not change Tau, Fyn, or GAPDH levels (Studenťs t -test, t(10)=1.86, p = .092; t(9)=0.28, p = .79; t(10) =1.78, p = .10, respectively, n =5–6 wells per group).

Article Snippet: Trypsin was inactivated by diluting it 1:10 in DMEM (Fisher MT10013CV) supplemented with 10% premium select fetal bovine serum and 1% Pen Strep (ThermoFisher 15140122) and the cells were plated to 10% confluency in 500 μL supplemented DMEM onto 12 mm No 1 glass coverslips (Carolina Biological 633029) coated overnight at 4 °C with 0.1 mg/mL Poly-D-Lysine and 0.02 mg/mL laminin in a 24-well plate (Fisher 08–772–1).

Techniques: Proximity Ligation Assay, Labeling, Negative Control, Control, Western Blot

Journal: Neurobiology of disease

Article Title: A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-β toxicity

doi: 10.1016/j.nbd.2019.104668

Figure Lengend Snippet: Tau-PxxP 5/6 inhibits endogenous Tau-Fyn interaction in neurons. A: Representative immunofluorescent image of DIV21 primary neurons after Tau-Fyn PLA. Cyan puncta represent sites of Tau-Fyn interaction and red is immunofluorescently labeled Tau. Scale bar = 50 μm. Inset: magnification of a neurite showing Tau-Fyn PLA (cyan) adjacent to Tau-positive neurites (red). Scale bar = 5 μm. B: Timeline of endogenous Tau-Fyn PLA inhibition experiments. C: Representative images of endogenous Tau-Fyn PLA after Tau-PxxP 5/6 treatment demonstrating reduction of endogenous Tau-Fyn PLA in neurons by Tau-PxxP 5/6 . Tau-PxxP 5/6 reduced endogenous Tau-Fyn interaction in neurons by 62% (Studenťs t -test, t(13) = 6.124, p < .0001, average of 5–7 images per coverslip from n =7–8 coverslips). Scale bar = 25 μm.

Article Snippet: Trypsin was inactivated by diluting it 1:10 in DMEM (Fisher MT10013CV) supplemented with 10% premium select fetal bovine serum and 1% Pen Strep (ThermoFisher 15140122) and the cells were plated to 10% confluency in 500 μL supplemented DMEM onto 12 mm No 1 glass coverslips (Carolina Biological 633029) coated overnight at 4 °C with 0.1 mg/mL Poly-D-Lysine and 0.02 mg/mL laminin in a 24-well plate (Fisher 08–772–1).

Techniques: Labeling, Inhibition

Journal: Bio-protocol

Article Title: Invadopodia Detection and Gelatin Degradation Assay

doi: 10.21769/BioProtoc.997

Figure Lengend Snippet: 1-Add pre-warmed gelatin solution to the tip of sterilized coverslips; 2-Hold coverslip with forceps and spread gelatin with the help of a pipet tip. 3-Remove the excess gelatin solution over the next coverslip to prepare. 4-Carefully aspirate the excess gelatin from the lower side of the coverslip. Refer to text for further details.

Article Snippet: Round 18 mm diameter glass coverslips (0.13-0.17 mm thickness) (Carolina Biological Supply Company, catalog number: 633033) Microscope glass slides (75 × 25 mm) (Thermo Fisher Scientific, catalog number: 1255015) 12-well Microplates (Corning, catalog number: 3513) Thermomixer or water bath set at 60 °C Tissue culture incubator Hemocytometer (LabSource, catalog number: 0267154) http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/counting-cells-in-a-hemacytometer.html Fluorescence Microscope Vacuum source

Techniques:

Journal: MicrobiologyOpen

Article Title: Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae

doi: 10.1002/mbo3.197

Figure Lengend Snippet: Appressoria of Magnaporthe oryzae strain 70-15, ΔMohik5 , ΔMohik8 , and the complemented strains ΔMohik5/HIK5 and ΔMohik8/HIK8 on hydrophobic plastic cover slips. The photographs were taken after an incubation period of 16 h at RT in H 2 O on a hydrophobic surface. HIK, histidine kinases.

Article Snippet: Appressorium development was assayed by monitoring germination of conidia on hydrophobic plastic slides (thickness 1, 76 × 51 mm, no. 653081; Greiner Bio-One, Kremsmünster, Austria).

Techniques: Incubation

Journal: MicrobiologyOpen

Article Title: Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae

doi: 10.1002/mbo3.197

Figure Lengend Snippet: Appressorium formation of Magnaporthe oryzae strain 70-15 and the HIK mutants. The diagram shows the number of appressoria after an incubation period of 16 h at RT in H 2 O on a hydrophobic surface or induced by 1,16-HDD. The error bars represent the standard deviation of three experiments with five replicates each. HIK, histidine kinases.

Article Snippet: Appressorium development was assayed by monitoring germination of conidia on hydrophobic plastic slides (thickness 1, 76 × 51 mm, no. 653081; Greiner Bio-One, Kremsmünster, Austria).

Techniques: Incubation, Standard Deviation