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: Autophagy

Article Title: Adaptor SH3BGRL drives autophagy-mediated chemoresistance through promoting PIK3C3 translation and ATG12 stability in breast cancers

doi: 10.1080/15548627.2021.2002108

Figure Lengend Snippet: SH3BGRL promotes autophagy flux in breast cancer cells.(A-C) Immunoblots of SQSTM1, LC3B-I and LC3B-II in indicated cells with or without Dox treatment (A). ACTB was used as an internal loading control. Statistical analyses of SQSTM1 expression (B) and LC3B-II:LC3B-I ratio(C) in indicated cells. *** P < 0.001, **** P < 0.0001, n.s., no significance. (D) Immunoblots of SQSTM1, LC3B-I and LC3B-II in indicated cells. Cells were pretreated with 50 nM Baf A 1 for 4 h and cultured in either normal culture medium (upper panel, Normal) or EBSS medium (lower panel, EBSS) for another 2 h, respectively. ACTB was used as an internal loading control. (E) Immunoblots of autophagy-related proteins and the cleaved c-CASP3 in MCF-7 SH3BGRL overexpressing cells or MDA-MB-453 SH3BGRL knockdown cells. Cells were treated with Dox along with or without 50 nM BafA 1 for 4 h. (F,G) Representative immunofluorescence staining of LC3B puncta in indicated cells with Serum-free medium (F) for 12 h or EBSS culture for 6 h (G). Cells were counterstained with DAPI in blue. Bars: 25 μm. (H) Quantifications of LC3B puncta intensity in the assayed cells (F,G) were presented as histograms. *** P < 0.001, **** P < 0.0001.

Article Snippet: Autophagy flux was examined in terms of LC3B-II turnover with immunoblotting LC3B-II (CST, 3868) protein level or the endogenous LC3B puncta number as described [ ].

Techniques: Western Blot, Control, Expressing, Cell Culture, Knockdown, Immunofluorescence, Staining

Journal: Autophagy

Article Title: Adaptor SH3BGRL drives autophagy-mediated chemoresistance through promoting PIK3C3 translation and ATG12 stability in breast cancers

doi: 10.1080/15548627.2021.2002108

Figure Lengend Snippet: SH3BGRL renders autophagy of breast cancers. (A) Immunoblots of SQSTM1 protein in the MCF-7 cells with SH3BGRL overexpression or MDA-MB-453 cells with SH3BGRL knockdown, respectively. Cells were treated with or without 0.5 µg/ml actinomycin D (Act D), 50 μg/ml cycloheximide (CHX), 20 μM MG132, or 50 μM CQ for 12 h, respectively. (B) Immunoblots of MTOR and its phosphorylated form in the indicated cells. (C,D). Representative IHC staining of total LC3B protein expression in 25 paired human breast cancer tissues compared with adjacent normal ones (C) Scale bars: 100 mm (insets, 50 mm). Statistical LC3B expression is shown (D), P = 0.0109. (E) Correlation analysis of SH3BGRL expression level with that of LC3B in breast cancer tissues. (n = 25; r = −0.56; P = 0.0034).

Article Snippet: Autophagy flux was examined in terms of LC3B-II turnover with immunoblotting LC3B-II (CST, 3868) protein level or the endogenous LC3B puncta number as described [ ].

Techniques: Western Blot, Over Expression, Knockdown, Immunohistochemistry, Expressing

Journal: Autophagy

Article Title: Adaptor SH3BGRL drives autophagy-mediated chemoresistance through promoting PIK3C3 translation and ATG12 stability in breast cancers

doi: 10.1080/15548627.2021.2002108

Figure Lengend Snippet: PIK3C3 and ATG12 contribute to tumorigenicity and Dox resistance. (A,B) Xenograft model of chemotherapy in nude mice. Six nude mice in each group were subcutaneously injected with indicated cells for 1 week, mice were treated with Dox combined with or without CQ for two weeks. Mean tumor weights were quantified around another three weeks later (B). **** P < 0.0001. (C) Immunoblots of SH3BGRL, PIK3C3, ATG12, SQSTM1, LC3B, PARP, cleaved (c)-PARP, CASP3 and c-CASP3 in the indicated mice tumor tissues. ACTB was used as a loading control. (D) Statistical analysis of SQSTM1, c-CASP3 and c-PARP expressions in the indicated six group mice tumors. Error bars represent mean ± s.d.* P < 0.05, ** P < 0.01, *** P < 0.001. (E) Transcriptional expression analysis of classical genes involved in Dox resistance from RNA-Seq of MCF-7 and MDA-MB-453 cells with SH3BGRL expression alteration. (F) Translational expression analysis of general genes involved in Dox resistance based on polyribosome profiling results of MDA-MB-453 cells.

Article Snippet: Autophagy flux was examined in terms of LC3B-II turnover with immunoblotting LC3B-II (CST, 3868) protein level or the endogenous LC3B puncta number as described [ ].

Techniques: Injection, Western Blot, Control, Expressing, RNA Sequencing

Journal: Autophagy

Article Title: Adaptor SH3BGRL drives autophagy-mediated chemoresistance through promoting PIK3C3 translation and ATG12 stability in breast cancers

doi: 10.1080/15548627.2021.2002108

Figure Lengend Snippet: Activation of SH3BGRL-PIK3C3/ATG12 autophagy axis in breast cancer patients. (A). Representative IHC staining of SH3BGRL, ATG12, PIK3C3 and LC3B expressions in 25 primary breast cancer specimens along with the matched adjacent normal tissues. Scale bar: 50 µm. (B,C) Statistical analysis of ATG12 (B) and PIK3C3 (C) in tissues of (A). (D) Correlation analysis of SH3BGRL protein level to that of ATG12 (r = 0.42; P = 0.037) or PIK3C3 (r = 0.49; P = 0.01) in breast cancer tissues in (A). (E) Immunoblots of the indicated proteins in 14 fresh breast cancer specimens. ACTB was used as a loading control. (F) Heatmap of protein expression of SH3BGRL, PIK3C3, ATG12, SQSTM1, and LC3B protein band intensity in (E), which was quantified and analyzed with densitometry and Image J software. (G) Correlation analysis of SH3BGRL protein level with that of PIK3C3, ATG12, SQSTM1 or LC3B in (F). (H) Marginal elevation of PIK3C3 mRNA expression in breast cancer tissues, compared with normal breast tissues (T = 1092, N = 111;T:N = 0.83, P < 0.0001; TCGA) and ATG12 mRNA (T = 1092, N = 111;T:N = 1.02, P < 0.0001; TCGA). (I) Kaplan–Meier relapse-free survival curves of breast cancer patients based on mRNA of ATG12 (n = 3951; P = 1.1e-11) or PIK3C3 (n = 3951; P = 1.5e-4). (J) Schematic mechanism of SH3BGRL-PIK3C3/ATG12 autophagy on doxorubicin resistance in breast cancer. All statistical analyses are shown as **** P < 0.0001.

Article Snippet: Autophagy flux was examined in terms of LC3B-II turnover with immunoblotting LC3B-II (CST, 3868) protein level or the endogenous LC3B puncta number as described [ ].

Techniques: Activation Assay, Immunohistochemistry, Western Blot, Control, Expressing, Software