Journal: Cell Host & Microbe

Article Title: Complement C4 Prevents Viral Infection through Capsid Inactivation

doi: 10.1016/j.chom.2019.02.016

Figure Lengend Snippet: C1 and C4 Prevent Adenoviral Protein VI Exposure and Endosomal Escape (A) IP of Ad5 with 9C12 after Ad5 was incubated at the indicated temperatures for 30 min. WB: anti-adenovirus. (B) IP after Ad5 and 9C12 were complexed with C1 or C1/C4 and then incubated at 37°C or 49°C for 30 min. WB: anti-adenovirus. (C and D) HeLa TRIM21 KO cells were infected for 30 min in the presence of 9C12 or 9C12+ complement. Error bars depict the mean ± SEM of the indicated number of cells (n) acquired in three independent experiments. Scale bar, 5 μm. (C) Left: Ad5 staining is displayed in green; protein VI staining is depicted in red. Right: quantification of protein VI puncta per cell in the indicated conditions. (D) Left: Ad5 staining is displayed in green; Galectin-3 staining is depicted in red. Right: quantification of Galectin-3 puncta per cell in the indicated conditions. Original western blots are included in Figure S6 .

Article Snippet: Z-stacks with 1 μm thick optical slices were acquired and protein VI and Galectin 3 puncta were quantified using NIS Elements 4.30 (Nikon).

Techniques: Incubation, Infection, Staining, Western Blot

Journal: Nature Communications

Article Title: VAV-1 acts in a single interneuron to inhibit motor circuit activity in Caenorhabditis elegans

doi: 10.1038/ncomms6579

Figure Lengend Snippet: ( a – c ) Expression of mCherry, tagged with a 2 × -nuclear localization signal, under control of the vav-1 promoter, is shown. ( a ) vav-1 reporter expression co-localizes with a GFP marker for the ALA interneuron. Anterior is to the left. ( b ) vav-1 reporter expression is found in cholinergic motor neurons of the ventral nerve cord. Shown are two posterior cholinergic motor neurons (VA11 and VA12) and their co-localization with cytoplasmic cholinergic GFP reporter. Arrows mark these two cell bodies. Anterior is to the left. ( c ) Only two GABAergic neurons (RME dorsal and ventral, head motor neurons) express the vav-1 reporter. Asterisks mark vav-1 expression in the pharynx, a dashed arrow marks the ALA neuron and solid arrows mark the two RME neurons that express the vav-1 reporter and co-localize with a GABA neuron-specific GFP marker. Anterior is up. Scale bars, 5 μm.

Article Snippet: Metamorph software was used to analyse GFP cholinergic synaptic puncta ( nuIs152 [Punc-129::GFP::snb-1] ).

Techniques: Expressing, Control, Marker

Journal: Nature Communications

Article Title: VAV-1 acts in a single interneuron to inhibit motor circuit activity in Caenorhabditis elegans

doi: 10.1038/ncomms6579

Figure Lengend Snippet: ( a ) Representative confocal micrographs of WT (top panel) and vav-1 mutant (bottom panel) animals expressing GFP in all GABAergic neurons ( oxIs12 [Punc-47::GFP] ). In WT (top panel), arrows and lines point to GABA neuron representative cell bodies and circumferential axons (commissures), respectively. In vav-1 mutants (bottom panel), these features are also present. Dashed arrows indicate fluorescent coelomocytes, a marker of the vav-1 mutant strain that should be ignored. Asterisk marks non-specific posterior intestinal GFP. Anterior is to the left, and scale bars, 100 μm. ( b ) Quantification of the number of GABA neuron cell bodies and commissures indicates that vav-1 mutants show grossly normal GABA neuron development. n =13 (WT) and 19 ( vav-1 ). ( c ) Representative images of GABAergic neuron synapses and ( f ) cholinergic synapses in the dorsal nerve cord of WT (left) and vav-1 mutant (right); fluorescent puncta are synaptobrevin tagged with GFP at the neuromuscular junction (NMJ). GABAergic animals are juIs1 [Punc-25::GFP::snb-1] , and cholinergic are nuIs152 [Punc-129::GFP::snb-1] . ( d , g ) Quantification of density of fluorescent puncta (left y axis) and the mean area of puncta (right y axis). For d , n =10 (WT) and 11 ( vav-1 ). For g , n =11 (WT) and 16 ( vav-1 ). ( e , h ) Quantification of fluorescence intensity of puncta (left y axis), and of nerve cord between puncta (right y axis). For e , n =10 (WT) and 11 ( vav-1 ). For h , n =28 (WT) and 36 ( vav-1 ). ( i ) The structure of the ALA neuron in WT and vav-1 mutants is shown using a fluorescent ALA reporter analysed by confocal microscopy ( Pida-1::ida-1::GFP ). In the left panels, solid arrows point to the ALA neuron cell body and a dashed arrow points to one ALA axonal projection. The right panels show the same axons projecting to the tails of these animals. Asterisks mark GFP associated with coelomocytes and rectal epithelial cells, and can be ignored. n =3(WT) and 11 ( vav-1 ). Quantified data are displayed as mean ±s.e.m. and were analysed by two-tailed Student’s T -tests (no significant differences were found). NS, not significant.

Article Snippet: Metamorph software was used to analyse GFP cholinergic synaptic puncta ( nuIs152 [Punc-129::GFP::snb-1] ).

Techniques: Mutagenesis, Expressing, Marker, Fluorescence, Confocal Microscopy, Two Tailed Test

Journal: Aging Cell

Article Title: Disrupted‐in‐schizophrenia‐1 protects synaptic plasticity in a transgenic mouse model of Alzheimer’s disease as a mitophagy receptor

doi: 10.1111/acel.12860

Figure Lengend Snippet: Disrupted‐in‐schizophrenia‐1 (DISC1) promotes mitophagy in a LIR‐dependent manner. (a) HeLa cells transfected with LC3‐GFP, mito‐dsRed, and DISC1‐Flag or DISC1 FSFI mutant‐Flag were stained for Flag and imaged. (b) The densities of GFP‐LC3 + puncta were counted and expressed as numbers of GFP‐LC3 + puncta per cell. (c) Percentages of cells with fragmented mitochondria. (d) Colocalization ratio between LC3‐GFP + and mito‐dsRed + puncta. (e) Mitophagosomes in HeLa cells transfected with DISC1 or DISC1 FSFI mutant were imaged by EM. (f) Numbers of mitophagosomes per cell. (g) Western blot analysis of levels of TOMM20 in HeLa cells transfected with DISC1‐Flag (WT) or DISC1 FSFI mutant‐Flag. (h) Quantification of relative levels of TOMM20. Data are presented as mean ± SEM . n = 3 or 4 independent experiments. * p < 0.05; *** p < 0.001. One‐way ANOVA. Scale bars: 10 μm (a), 500 nm (e)

Article Snippet: In comparison with empty vector‐transfected cells, WT DISC1‐transfected HeLa cells harbored more fragmented mitochondria as described previously (Millar, James, Christie, & Porteous, ) (Figure a,c), more LC3‐GFP + puncta (Figure a, b) and increased numbers of mitophagosomes as indicated by enhanced colocalization between LC3‐GFP + and Mito‐dsRed + puncta (Figure a,d).

Techniques: Transfection, Mutagenesis, Staining, Western Blot

Journal: bioRxiv

Article Title: Deciphering the Genome Protection Roles of Autophagy in Primary Human Dermal Fibroblasts (HDFs) against Ultraviolet-(B) –Induced Skin Photodamage

doi: 10.1101/2020.09.28.316273

Figure Lengend Snippet: (3A-3E) Western blotting analysis of key autophagy marker proteins showing time and intensity dependent response of autophagy in UV-B 10, 20 and 30mJ/cm 2 exposed HDFs. (3F & 3G) GFP-RFP-LC3B PUNCTA assay for quantification of puncta (+) cells in UV-B 30mJ/cm 2 exposed HDFs and effect of Rapamycin (100nM) and Chloroquine (50µM) on autophagy response in 6h UV-B post-irradiation. (3H & 3I) Immunoflorescence analysis of autophagy proteins P62 and LC3B and DNA damage marker proteins pχH2AX and pP53 in UV-B 30mJ/cm 2 exposed HDFs in 1h UV-B post-irradiation in florescent microscopy. (3J & 3K) Reactive Oxygen Species (ROS) estimation in UV-B 30mJ/cm 2 exposed HDFs treated with Salubrinal (25µM) and Everolimus (200nM) in 1h UV-B post-irradiation. (**p≤ 0.01, #p≤ 0.001, were considered statistically significant).

Article Snippet: Fura 3 AM, DAPI, ER tracker, Acridine Orange, Ethidium Bromide, Salubrinal, Rapamycin, Chloroquine, Bafilomycin A1, Everolimus, GFP RFP LC3B Puncta kit from (Thermo Scientific).

Techniques: Western Blot, Marker, Irradiation, Microscopy