Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) In vivo evidence for Hsc70-Cx43 interaction. SDS-PAGE and Coomassie staining of proteins obtained in co-immunoprecipitation assay with anti-Cx43 antibodies using lysates prepared from HuH-7 cells. Arrows indicate the positions of Hsc70 and β-tubulin. Both proteins were identified by MALDI/Q-TOF mass spectrometry analysis. (b) Binding of Hsc70 to GST-Cx43CT. Proteins extracted from HuH-7 cells were mixed either with GST or with GST-Cx43CT residues 228-382 resin. After washing, the bound proteins were released and analyzed on 12% SDS-PAGE followed by western blotting (WB) with anti-Hsc70 antibody. Full-length blots are presented in . (c and d) Co-immunoprecipitation of Hsc70 with Cx43. Lysates were prepared from HuH-7 cells co-transfected with wild-type Cx43 and T7-tagged Hsc70 expression plasmids. Lysates were immunoprecipitated (IP) either by anti-Cx43 antibodies and control IgG (c) or by anti-T7 antibody and the IgG (d). The immunocomplexes were analyzed by western blotting (WB) using anti-T7 antibody (c) or anti-Cx43 antibodies (d). Full-length blots are presented in . (e) Co-localization of endogenous Hsc70 and Cx43. HuH-7 cells were fixed and stained with anti-Cx43 antibodies and anti-Hsc70 antibody. The nuclei were stained with TO-PRO3. Co-localization of both proteins was observed in the cytoplasm.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: In Vivo, SDS Page, Staining, Co-Immunoprecipitation Assay, Mass Spectrometry, Binding Assay, Western Blot, Immunoprecipitation, Transfection, Expressing

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) A schematic representation of GST-Cx43CT fusion proteins used as a ligand. Consensus protein domains and regions with putative signaling functions are indicated. Abbreviations in Cx43: TM1-4, transmembrane domains 1–4; JM, juxtamembrane domain that binds microtubules; Y265, Src phosphorylation site; PP, proline-rich sequence that binds the SH3 domain of v-Src; DLEI, carboxy-terminal sequence involved in ZO-1 binding. (b) Identification of Hsc70-binding domain in Cx43. Whole cell lysates of HuH-7 cells and various forms of the GST-Cx43CT fusion proteins were used for GST pulldown assays. Binding experiments and western blotting (WB) were carried out as described in . (c) A schematic representation of Hsc70 and its deletion mutants used for overexpression. (d) Identification of Cx43 binding domain in Hsc70. Expression vectors encoding various Hsc70s were transfected into HuH-7 cells, and binding experiments were carried out as described in . The immunocomplexes were analyzed by western blotting (WB) using anti-T7 antibody.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Sequencing, Binding Assay, Western Blot, Over Expression, Expressing, Transfection

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) Hsc70 interacts with cyclin D1 or Cx43. Co-immunoprecipitation of Hsc70 with cyclin D1 or Cx43. Lysates were prepared from HuH-7 cells co-transfected with T7-tagged Hsc70 and cMyc-tagged cyclin D1 or wild-type Cx43 expression plasmids. Lysates were immunoprecipitated (IP) either by anti-cMyc antibody and control IgG (left) or by anti-Cx43 antibodies and the IgG (right). The immunocomplexes were analyzed by western blotting (WB) using anti-T7 antibody. (b) Absence of interaction between cyclin D1 and Cx43. Co-immunoprecipitation of Cx43 with cyclin D1. Lysates were prepared from HuH-7 cells co-transfected with wild-type Cx43 and cMyc-tagged cyclin D1 expression plasmids. Lysates were immunoprecipitated (IP) either by anti-cMyc antibody and control IgG (left) or by anti-Cx43 antibodies and the IgG (right). The immunocomplexes were analyzed by western blotting (WB) using anti-Cx43 antibodies (left) or anti-cMyc antibody (right). (c) Competition assay of Cx43 and cyclin D1 for binding with Hsc70. HuH-7 cells were transfected with equivalent plasmids of both T7-tagged Hsc70 and cMyc-tagged cyclin D1, and with increasing amounts of Cx43. Lysates were immunoprecipitated (IP) by anti-T7 antibody. The immunocomplexes were analyzed by western blotting (WB) using anti-T7-tag, anti-Cx43, and anti-cMyc-tag antibodies.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Competitive Binding Assay, Binding Assay

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) BrdU incorporation assays were examined in HuH-7 cells. HuH-7 cells were transiently transfected with vectors encoding empty vector EGFP, Hsc70-EGFP, Cx43-EGFP alone, or both Hsc70- and Cx43-EGFP, respectively. Forty-eight hours after transfection, the cells were labeled with bromodeoxyuridine (BrdU) for 2 hr and subsequently fixed and stained with anti-BrdU antibody. Green indicates transfectants, and red indicates BrdU-incorporated cells. (b) The fraction of BrdU-positive cells over total number of transfected cells was determined. The data shown in (b) are means and SDs (error bars) of a representative experiment performed in triplicate at least. * p < 0.01. N.S.; not significant.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: BrdU Incorporation Assay, Transfection, Plasmid Preparation, Labeling, Staining

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) A schematic representation of Hsc70 and its splice variant, Hsc54, used for overexpression. (b) Pulldown assays with GST-Cx43CT fusion protein were performed using lysates prepared from Hsc70- or Hsc54-transfected HuH-7 cells. Expression vectors encoding Hsc70-EGFP and Hsc54-EGFP were transfected into HuH-7 cells, and binding experiments were carried out as described in . The bound proteins were analyzed by western blotting (WB) using anti-GFP antibody. (c) BrdU incorporation assays in HuH-7 cells. HuH-7 cells were transiently transfected with vectors encoding empty vector EGFP, Hsc54-EGFP, Cx43-EGFP alone, or both Hsc54- and Cx43-EGFP, respectively. BrdU incorporation and immunofluorescence staining were carried out as described in . (d) The fraction of BrdU-positive cells over total number of transfected cells. (e) Inhibition assay of protein-protein interaction. To inhibit the binding of proteins to Hsc70, HuH-7 cells were cultured with 10 μg/ml of 15-DSG for 24 hr. The fraction of BrdU-positive cells over total cells was determined. The data shown in (d) and (e) are means and SDs (error bars) of a representative experiment performed in triplicate at least. * p < 0.01. N.S.; not significant.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Variant Assay, Over Expression, Transfection, Expressing, Binding Assay, Western Blot, BrdU Incorporation Assay, Plasmid Preparation, Immunofluorescence, Staining, Inhibition, Cell Culture

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) Hsc70 translocated into the nucleus under heat shock. HuH-7 cells were heat shocked at 42°C for 2 hr and stained with anti-Hsc70 antibodies. (b) Overexpression of Cx43 prevented the nuclear translocation of Hsc70 under heat shock. HuH-7 cells were transfected with control vector EGFP or Cx43-EGFP. Forty-eight hours after transfection followed by heat shocked at 42°C for 2 hr, cells were stained with anti-Hsc70 antibodies. (c) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in Hsc70. After heat shock, at least 30 transfected HuH-7 cells of control EGFP or Cx43-EGFP were examined and quantified by confocal scanning microscopy. The data shown in (b) were plotted. The horizontal lines represent mean values. * p < 0.01.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Staining, Over Expression, Translocation Assay, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) Effect of Cx43 on nuclear accumulation of cyclin D1. HuH-7 cells were transfected with control plasmid mRFP or Cx43-mRFP, or both Cx43-mRFP and Hsc70-DsRed2. Immunofluorescence staining with anti-cyclin D1 antibodies was carried out. Nuclear accumulation of cyclin D1 was examined and quantified by confocal laser scanning microscopy. (b) Ratio of nuclear/cytoplasmic fluorescence intensities in cyclin D1. At least 30 transfected HuH-7 cells of control mRFP or Cx43-mRFP, or both Cx43-mRFP and Hsc70-DsRed2 were examined and quantified. The data shown in (a) were plotted. The horizontal lines represent mean values. * p < 0.01.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Fluorescence

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RNAi screening identifies RABGAP1L as an IAV restriction factor (A) Schematic representation of recombinant IAV WSN/33 in which the coding region for the hemagglutinin (HA) glycoprotein has been replaced by Renilla luciferase (WSN/33- Renilla ). (B) RNAi-screening experimental workflow. (C) MRC-5-HA cells were transfected for 30 h with individual siRNAs targeting MX1 or IFITM3 or with a non-targeting (NT) control siRNA. Following stimulation with IFNα2 (1,000 U/mL or mock) for 16 h, cells were infected with WSN/33- Renilla (MOI 5 PFU/cell) in the presence of the live-cell substrate EnduRen. Luciferase activity was monitored up to 12 h post-infection (p.i.), and the area under the curve (AUC) was calculated as indicated. Mean values from 50 technical replicates across two independent biological experiments are plotted, with error bars representing SDs. (D) Hit criteria for RNAi screening. In a primary screen following the workflow in (B), 100 putative ISGs were silenced with four individual siRNAs each. Twenty-two genes met the threshold, and 20 were re-tested in a confirmation screen. Applying the same hit criteria, a total of 8 putative ISGs were confirmed in both screening rounds. (E) Heatmap showing Z scores of positive controls ( MX1 and IFITM3 ) and the top 8 hits from the two RNAi-screening rounds. Columns represent individual siRNAs targeting genes listed in rows. See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Luciferase, Transfection, Infection, Activity Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: IFN-mediated restriction of IAV by RABGAP1L (A) A549 cells were transfected with the indicated siRNAs for 32 or 60 h prior to lysis and assessment of cell viability using CellTiter-Glo. An NT siRNA and an siRNA targeting IRF9 were used as negative controls. siRPS is an siRNA targeting the essential gene RPS27A and thus acted as a positive control for cell toxicity. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (B and C) A549 cells were transfected with the indicated siRNAs 30 h prior to IFNα2 treatment (1,000 U/mL or mock). Sixteen hours post-IFN stimulation, cells were infected with WSN/33- Renilla (MOI 1 PFU/cell), and luciferase activity was monitored every 2 h for a total of 12 h. The NT siRNA and siRNA targeting IRF9 were used as controls. (C) The AUC was calculated from measured relative light units (RLUs) over time. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (D) In parallel to (B) and (C), cells were harvested for western blot analysis 16 h post-IFN stimulation. Proteins of interest were detected as indicated. RABGAP1L (RG1L) isoforms corresponding to detected bands are highlighted. (E) Schematic representation of RABGAP1L isoforms A, G, H, and I, showing the phosphotyrosine-binding (PTB) domain, the kinesin-like (kin) domain, and the Tre-2/Bub2/Cdc16 (TBC) domain. Isoform G further contains a domain of unknown function (DUF3084). (F) Immunofluorescence analysis of A549 cells stably expressing either empty vector (EV) or RABGAP1L isoforms A, G, H, and I. Cells were fixed and stained for RABGAP1L (red); nuclei were stained with DAPI (blue). Scale bar represents 25 μm. Representative confocal-microscopy images from at least two biologically independent experiments are shown. (G) Cells described in (F) were harvested for western-blot analysis. Proteins of interest were detected with the indicated antibodies. Images are representative of three biologically independent experiments. (H) Cells described in (F) and (G) were treated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected 48 h p.i. and titrated on Madin-Darby canine kidney (MDCK) cells to determine viral titers. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance in (C) and (H) was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001; ns, non-significant). See also and .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Transfection, Lysis, Positive Control, Infection, Luciferase, Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L overexpression restricts selected positive- and negative-sense RNA viruses (A) A549 cells stably expressing GFP or RABGAP1L (RG1L) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with different Renilla luciferase-encoding IAVs: H1N1 (WSN/33, MOI 1 PFU/cell), pdmH1N1 (Neth/09, MOI 5 PFU/cell), or H5N1 (Viet/04, MOI 0.5 PFU/cell). EnduRen live-cell substrate was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs up to 11 h p.i. (B) Huh-7 cells stably expressing GFP or RG1L were treated as described in (A) and infected with WSN/33- Renilla (MOI 1 PFU/cell) or HCoV-229E- Renilla (MOI 5 PFU/cell). EnduRen was supplemented, and the luciferase activity was measured every 2 h for a total of 11 h. RLUs were used to calculate the AUC. (C–E) A549 cells expressing EV or RG1L were stimulated with IFNα2 (10, 100 or 1,000 U/mL or mock) for 4 h prior to infection with VSV-GFP (MOI 1 PFU/cell) (C) or for 16 h prior to infection with SeV-GFP (MOI ∼1 PFU/cell) (D) and NDV-GFP (MOI 1 PFU/cell) (E). GFP intensity was measured every 2 h for up to 72 h. The AUC was calculated from total green integrated intensity. (F and H) Calu-3 (F) or Vero-CCL81 (H) cells stably expressing GFP or RG1L were treated with IFNα2 (10, 100, or 1,000 U/mL or mock) for 16 h, followed by infection with WSN/33- Renilla (MOI 1 PFU/cell). EnduRen was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs. (G and I) Calu-3 (G) or Vero-CCL81 (I) cells stably expressing GFP or RG1L were treated as described in (F) prior to infection with SARS-CoV-2 (MOI 0.1 PFU/cell). Supernatants were collected 24 h p.i., and viral titers were determined by plaque assay in Vero-E6 cells. (A–I) Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined comparing GFP-overexpressing with RG1L-overexpressing cells in equal treatment conditions in all panels using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.0002, ∗∗∗∗ p < 0.0001; ns, non-significant).

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Over Expression, Stable Transfection, Expressing, Infection, Luciferase, Activity Assay, Plaque Assay, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: The antiviral function of RABGAP1L relies on its catalytically active TBC domain and residues implicated in endosomal trafficking (A) Schematic representation of RG1L WT and the 421 mutant (RG1L 421) which lacks the C-terminal region downstream of the kin domain. (B) Immunofluorescence analysis of A549 cells stably expressing EV, RG1L WT, or RG1L 421. Cells were fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (C) A549 cells stably expressing GFP, RG1L WT, or RG1L 421 were stimulated with IFNα2 (1,000 U/mL or mock) for 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected after 48 h and titrated on MDCK cells. (D) Schematic representation of the TBC domain of RABGAP1L and the localization of mutants R584A (R mut ), Q621A (Q mut ), R584A-Q621A (RQ mut ), and KK784EE (KK mut ). KK mut has previously been shown to prevent interaction with the AnkB death domain (DD). (E) Western blot validation of RABGAP1L expression in A549 cells stably expressing RG1L WT or the indicated mutants. (F) Immunofluorescence analysis of cells described in (E) (here, EV was used as a control), fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (G) Cells described in (E) were infected with WSN/33- Renilla (MOI 1 PFU/cell) following treatment with IFNα2 (1,000 U/mL or mock) for 16 h. The AUC was calculated from RLU values taken up to 11 h p.i. For (B), (E), and (F), representative data from three biologically independent experiments are shown. For (C) and (G), mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001). See also Figure S3 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Mutagenesis, Immunofluorescence, Stable Transfection, Expressing, Staining, Infection, Western Blot, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: Proximity-labeling-based proteomics identifies the RABGAP1L host interactome (A) Schematic representation of TurboID-V5-tagged (T-V5) GFP (negative control) carrying a nuclear-export sequence (NES) or T-V5-tagged RABGAP1L (T-V5-RG1L). (B) Constructs described in (A) were stably expressed in A549 cells, and their expression was validated by immunofluorescence using an α-V5 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (C) Western blot analysis of cells described in (B) compared with A549 cells stably expressing untagged GFP or RABGAP1L (RG1L). Proteins of interest were detected with the indicated antibodies. (D) Cells described in (C) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33- Renilla (MOI 1 PFU/cell). The AUC was calculated from RLU values taken up to 11 h p.i. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (E) Workflow of the TurboID proximity-labeling approach. Cells described in (B) were treated with IFNα2 (1,000 U/mL or mock) for 16 h, followed by treatment with biotin (500 μM) for 15 min. Following streptavidin-based affinity purification, peptides were generated and subjected to mass-spectrometry analyses. (F) Interactors specific to RABGAP1L (as compared to GFP-NES) identified using the protocol described in (E). Hits are listed with their gene names and sorted according to previously described functions. Most hits were identified in non-IFNα2-treated samples. Hits marked with an asterisk ( ∗ ) were identified in the presence and absence of IFNα2, and hits marked in bold were only identified in IFNα2-treated samples. (G) A549 cells stably expressing constructs introduced in (A) or T-V5-tagged RABGAP1L KK mut and RQ mut were subjected to the proximity labeling approach outlined in (E). Following streptavidin-based affinity purification (samples termed “eluates”), total lysates and eluates were analyzed by western blot. Proteins were detected with the indicated antibodies. Data obtained in (B), (C), and (G) are representative of three biologically independent experiments. For (D), statistical significance was determined using one-way ANOVA following log transformation (ns, non-significant). See also and Figure S4 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Labeling, Negative Control, Sequencing, Construct, Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot, Infection, Affinity Purification, Generated, Mass Spectrometry, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L expression impacts host endosomal function and IAV uptake (A–C) A549 cells stably expressing RABGAP1L WT, the 421-truncation mutant, or EV were infected with WSN/33 (MOI 5 PFU/cell) for 1 h on ice. Three hours after incubation at 37°C, cells were fixed and stained with antibodies against RABGAP1L (red) and NP (green) (A). Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (B and C) Green mean fluorescent intensities (MFIs) of nuclear NP signals were quantified from fluorescent-microscopy images from (A) using ImageJ software. Individual cells are represented by single dots (B). Mean values of data from three biologically independent experiments in (B), normalized to EV, are shown in (C). (D) MDCK cells, expressing the constructs described in (A), were infected for 4 h at 37°C with WSN/33-pseudotyped β-lactamase-matrix protein (BlaM1) fusion protein VLPs prior to quantification of entry-positive cells via flow cytometry. Data represent means, with error bars showing SDs, from three biologically independent experiments. Individual data points are shown. (E) Experimental setup for immunofluorescence-based confocal microscopy to track early stages during IAV entry. Following infection with WSN/33 (MOI 25 PFU/cell or mock) for 1 h at 4°C, cells were fixed at the indicated timepoints. (F) A549 cells stably expressing RABGAP1L (RG1L) or EV were subjected to the experimental setup described in (E). The MFI of HA signals (green) at 0 min p.i. were quantified from confocal-microscopy images shown in Figure S6 A using ImageJ. Individual cells are represented by single dots. (G) Quantification of co-localizations between EEA1 and HA from confocal images shown in (H) and Figure S6 A using Imaris. Individual cells are represented by single dots. (H) Immunofluorescence analysis of RABGAP1L or EV-expressing A549 cells treated as described in (E). Cells were stained for early endosomes (EEA1, magenta), viral proteins (HA, green), and nuclei (DAPI, blue). Scale bar represents 25 μm. White arrows indicate co-localizations between EEA1 and HA. Representative images of at least nine analyzed cells per time point from at least two biologically independent experiments. (I) Quantification of co-localizations between EEA1 and HA from confocal images shown in Figure S6 B. Individual cells are represented by single dots. (J and K) Cells described in (A) were serum starved for 2 h prior to treatment with Dynasore (Dyn.; 100 μm) or DMSO for 1 h at 37°C. Cells were then incubated with Alexa-Fluor-488-conjugated transferrin (Tf-488) for 1 h at 4°C followed by a 10-min incubation at 37°C prior to fixation. (J) MFI quantification of Tf-488 signals from confocal-microscopy images shown in (K) using ImageJ software. Individual cells are represented by single dots. (K) Cells were stained with anti-transferrin receptor (TfR) antibody (magenta) and DAPI (blue) prior to analysis by confocal microscopy. Scale bar represents 25 μm. Representative images of at least 25 analyzed cells from two biologically independent experiments. Statistical significance was determined using unpaired nonparametric t test (B, F, G, and J), unpaired one-way ANOVA (C and D), or ordinary two-way ANOVA (I) ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, non-significant). See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Expressing, Stable Transfection, Mutagenesis, Infection, Incubation, Staining, Microscopy, Software, Construct, Flow Cytometry, Immunofluorescence, Confocal Microscopy

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Cell Viability Assay, Mutagenesis, Clone Assay, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 2 High SERPINE1 expression in GC cells promotes macrophage M2 polarization. tSNE visualization of nine single-cell clusters partitioned by unsu pervised cluster analysis, SERPINE1 expression of each single-cell, and SERPINE1 expression abundance of different single-cell clusters in the GSE134520 (A–C) and GSE167297 (D–F) datasets. (G) Flow cytometry analysis of the proportion of CD68+CD206+ macrophages in a Transwell co-culture system, with MKN45 and AGS cells overexpressing (oe_SERPINE1) or silencing SERPINE1 (shRNA#3 or sh_SERPINE1#3) in the upper chamber, and THP1 cells treated with PMA in the lower chamber. (H) Immunofluorescence staining of xenograft tumor tissues. Comparison of the proportion of M1 or M2 macrophage infiltra tion. Green indicates F4/80. Red indicates iNOS or Arg1 expression

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, shRNA, Immunofluorescence, Staining, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 5 SERPINE1-mediated gastric cancer-derived exosomes facilitate the polarization of THP1 cells into M2 macrophages. (A) Schematic representation of the extraction and identification of exosomes and the induction of macrophage polarization. Transmission electron microscopy (B), nanoparticle tracking analysis (C), and western blotting (D) were used to identify the morphology, particle size, and markers of exosomes. (E) Confocal laser scanning microscopy detected Dil-labeled exosomes (red) internalized by DAPI-labeled macrophages (blue). (F–G) Immunofluorescence analysis of the proportion of CD206+ cells in THP1 cells treated with exosomes. (H–I) Flow cytometry analysis of the proportion of CD68+CD206+ cells in THP1 cells treated with exosomes. (J–K) qRT-PCR analysis of M1 markers (iNOS and TNF-α) and M2 markers (TGF-β, IL-10, and Arg-1) in THP1 cells treated with exosomes. (L–N) Transwell migration and invasion assays of GC cells (upper chamber) co-cultured with macrophages (lower chamber) ingesting exosomes

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Derivative Assay, Extraction, Transmission Assay, Electron Microscopy, Western Blot, Confocal Laser Scanning Microscopy, Labeling, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Migration, Cell Culture

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Hepatic MTH1+CD3+ T cells correlated with disease activity in AIH. (A) Representative confocal staining of CD3 (red), MTH1 (green), and DAPI (for nuclei in blue) (magnification ×400) in the liver of patients with AIH. Scale bar, 20 μm. (B) Number of MTH1+CD3+ T cells in portal areas was positively correlated with degree of hepatic inflammation but showed no clear link with fibrosis stages in AIH. (C) Number of MTH1+CD3+ T cells in portal areas had a significant positive correlation with levels of serum ALT and AST in patients with AIH. (D) Number of MTH1+CD3+ T cells in portal areas was positively correlated with levels of serum ALP and GGT. Bars reflect the mean ± SEM. Abbreviations: DAPI, 4 0, 6‐diamidino‐2‐phenylindole; hpf, high‐power field.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: Activity Assay, Staining

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib significantly inhibited T‐cell proliferation in human T cells in vitro . Isolated human CD3+ T cells were cultured with/without anti‐CD3/CD28 beads for 72 hours. Representative western blot analyses of MTH1 72 hours after anti‐CD3/CD28 beads stimulation. (B,C) Isolated T cells were activated with anti‐CD3/CD28 beads with or without 2 μM karonudib for 72 hours. The percentage of CD25+ and CD69+ T cells was determined on day 3 by flow cytometry. (D) Analysis of T‐cell number treated with karonudib for 72 hours after anti‐CD3/CD28 beads stimulation. (E) Statistical analysis of the T‐cell proliferation assay treated with/without 2 μM karonudib for 72 hours from HCs and patients with AIH who were treatment naive. (F) Representative western blot analyses of P53, P21, P27, CDK2, and cyclin E 72 hours after anti‐CD3/CD28 beads stimulation. The GAPDH blot was used as a loading control. Data are from one experimental representative of at least three independent experiments and represent triplicate wells. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Cell Culture, Western Blot, Flow Cytometry, Proliferation Assay, Control

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib rendered activated T cells more susceptible to DNA damage in vitro . (A) Representative fields corresponding to each treatment were photographed. Isolated human T cells were activated with/without anti‐CD3/CD28 beads in the presence of DMSO or 2 μM karonudib for 72 hours. The alkaline comet assay was conducted and nucleoids were visualized by epifluorescence microscopy using a fluorescein isothiocyanate filter. (B) Quantification of comet tail moment. Values represent mean ± SEM from three independent experiments (100 comets per experiment). (C) Levels of cleaved‐PARP and phosphorylated histone H2AX (γ‐H2AX) were determined by immunoblot analysis. The GAPDH blot was used as a loading control. (D,E) Intracellular flow cytometry assessment of annexin V and propidium iodide expression in anti‐CD3/CD28 beads‐stimulated CD4+ and CD8+ T lymphocytes treated with 2 μM karonudib or DMSO after 72 hours. Data are from one experimental representative of at least three independent experiments. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Alkaline Single Cell Gel Electrophoresis, Epifluorescence Microscopy, Western Blot, Control, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A) Structures of human Bcl-rambo and its mutants. (B) S2 cells were transfected with pMT-V5-His A, pMT-V5-His A/ Bcl-rambo , or pMT-V5-His A/ Bcl-rambo (ΔTM) and then incubated in the presence of CuSO 4 (500 μM) and Z-VAD-fmk (20 μM) for 24 h. S2 cells were stained for Bcl-rambo (green) and with DAPI (blue) and MitoTracker ® Red (red). The stained cells in at least five different fields were observed by confocal laser scanning microscopy. Optical sections containing single transfected cells are shown. Data were representative of two independent experiments. Scale bars indicate 10 μm. (C) S2 cells were transfected with pMT-V5-His A, pMT-V5-His A/ Bcl-rambo , or pMT-V5-His A/ Bcl-rambo (ΔTM) and incubated in the presence of CuSO 4 (500 μM) for 24 h. S2 cells were homogenized and separated into PNL, P10, P100, and S100 fractions. The fractions were analyzed by Western blotting using anti-Bcl-rambo, anti-HSP60, and anti-α-tubulin antibodies. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Transfection, Incubation, Staining, Confocal Laser Scanning Microscopy, Western Blot

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A) S2 cells were transfected with pAct5C- GAL4 together with pUAST, pUAST- Bcl-rambo , pUAST- Bcl-rambo (ΔTM) , pUAST- Bcl-rambo (TM) , or pUAST- DsRed-monomer for 24 h. Cells were stained with Hoechst 33342. Nuclear morphology was observed by fluorescent microscopy. Apoptotic cells (%) are shown as the mean ± S.E. of three independent experiments. ** P <0.01, significantly different from the control. n.s., not significant. Transfection efficiency was measured by counting DsRed-monomer-expressing cells, and calculated to be 17.0 ± 0.7% (the mean ± S.E of three independent experiments). (B) S2 cells were transfected with (+) or without (–) pMT-V5-His A/ Bcl-rambo (no tag), or pMT-V5-His A/ Drob-1 (FLAG tag) for 20 h and then incubated in the presence of CuSO 4 (500 μM) for the indicated times. The expression of Bcl-rambo and Drob-1 was analyzed by Western blotting using anti-Bcl-rambo and anti-FLAG antibodies, respectively. Data were representative of three independent experiments. (C) S2 cells were transfected with pMT-V5-His A (open circles), pMT-V5-His A/ Bcl-rambo (filled circles), or pMT-V5-His A/ Drob-1 (filled squares) for 20 h and then incubated in the presence of CuSO 4 (500 μM) for the indicated times. Cells were stained with Hoechst 33342. Apoptotic cells (%) are shown as the mean ± S.E. of three independent experiments. ** P <0.01, significantly different from the control. (D) S2 cells were transfected with (+) or without (–) pMT-V5-His A/ Bcl-rambo or pMT-V5-His A/ Drob-1 for 20 h and then incubated in the presence of CuSO 4 (500 μM) for 8 h. The cytosolic fraction and organelle fraction containing mitochondria were both analyzed by Western blotting using anti-cytochrome c (Cyt. c ), α-tubulin, and anti-HSP60 antibodies. Data are representative of two independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Transfection, Staining, Microscopy, Control, Expressing, FLAG-tag, Incubation, Western Blot

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using dpp - GAL4 driver fly lines. (a) w ; +; dpp-GAL4 /+, (b) w ; UAS-Bcl-rambo /+; dpp-GAL4 /+, (c) w ; UAS-Bcl-rambo(ΔTM)/+ ; dpp-GAL4 /+. The morphology of the wings was observed under light microscopy. The white arrow indicates morphological aberrations in the wing vein. The L3-L4 area was measured by Image J software (B). Data are shown as the mean ± S.E. of three independent experiments (n = 4–5 for each experiment). ** P <0.05, significantly different from control. n.s., not significant. (C) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using pnr - GAL4 driver lines. (a) w ; +; pnr-GAL4 /+, (b) w ; UAS-Bcl-rambo /+; pnr-GAL4 /+, (c) w ; UAS-Bcl-rambo (ΔTM)/+ ; pnr-GAL4/+ . The morphology of adult thoraxes was observed by SEM. Scale bars indicate 100 μm. Data were representative of three independent experiments. (D and E) Cell lysates of third instar larval salivary glands were prepared from sg-GAL4/w ; +; +, sg-GAL4/w ; UAS-Bcl-rambo/+ ; +, and sg-GAL4/w ; UAS-Bcl-rambo (ΔTM)/ +; + flies, and analyzed by Western blotting. The amount of Bcl-rambo and Bcl-rambo (ΔTM) was normalized to that of α-tubulin. The protein expression of Bcl-rambo (%) is shown as the mean ± S.E. of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy, Software, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR - GAL4 driver fly lines. (a, e, i) GMR-GAL4 / w ; +/ CyO ; +, (b, f, j) GMR-GAL4 / w ; UAS-GFP /+; +, (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO ; +, (d, h, l) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) / CyO ; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Light microscopy and SEM photographs were taken from different individuals. Data were representative of three independent experiments. (B) Eye imaginal discs were stained for Bcl-rambo (green) and with Hoechst 33342 (blue). (a and d) GMR-GAL4 / w ; +/ CyO or Sp ; +, (b and e) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO or Sp ; +, (c and f) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM) / CyO or Sp ; +. White arrows show the morphogenetic furrow. Scale bars indicate 50 μm. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy, Staining

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) (a, d) GMR-GAL4 / w ; +/ CyO or Sp ; +, (b, e) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO or Sp ; +, (c, f) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) / CyO or Sp ; +. The eye imaginal discs were labeled for activated caspase-3/7 (green) and with Hoechst 33342 (blue). Scale bars indicate 50 μm. White arrows indicate the morphogenetic furrow. The number of fluorescent cells harboring activated caspases from the morphogenetic furrow to the posterior region of imaginal eye discs was measured (B). Data are shown as the mean ± S.E. of three independent experiments (n = 6 for each experiment). ** P <0.01, significantly different from control. n.s., not significant.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Labeling, Control

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR - GAL4 driver fly lines. (A) (a, e, i) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, f, j) GMR-GAL4 / w ; UAS-Bcl-rambo /+; UAS-p35 /+, (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo /+; UAS-Diap1 /+, (d, h, l) GMR-GAL4/w ; UAS-Bcl-rambo/+ ; GMR-Diap2/+ . (B) (a, e, i) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; +, (b, f, j) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; UAS-p35/+ , (c, g, k) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; UAS-Diap1/+ , (d, h, l) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; GMR-Diap2/+ . The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Light microscopy and SEM photographs were taken from different individuals. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Pupal retinae 42 h after pupal formation were stained with an anti-discs large antibody (green). (a) w/+ ; +; +, (b) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (c) GMR-GAL4 / w ; UAS-Bcl-rambo /+; UAS-p35 /+, (d) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (e) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; UAS-p35 /+. Scale bars indicate 10 μm. The numbers of normal and aberrant ommatidia were counted. Aberrant morphology (%) is shown as the mean ± S.E. of three independent experiments (n = 5 for each experiment). ** P <0.01. n.s., not significant.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Eye imaginal discs were stained for DAPI (blue) and Elav (green), together with Prospero (red) (A) or Cut (red) (B). (a–d) GMR-GAL4 / w ; +/ CyO or Sp ; +, (e–h) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO or Sp ; +, (i–l) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM) / CyO or Sp ; +. Scale bars indicate 10 μm. Data were representative of at least two independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR - GAL4 driver fly lines. (A) (a, f, k) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, g, l) GMR-GAL4 / w ; UAS-Bcl-rambo / Drob-1 E26 ; +, (c, h, m) GMR-GAL4 / w ; UAS-Bcl-rambo / Drob-1 W105 ; +, (d, i, n) GMR-GAL4/w ; UAS-Bcl-rambo/Drob-1 E26 Buffy H37 ; +, (e, j, o) GMR-GAL4/w ; UAS-Bcl-rambo/Drob-1 W105 Buffy H37 ; +. (B) (a, f, k) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; +, (b, g, l) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 E26 ; +, (c, h, m) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 W105 ; +, (d, i, n) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 E26 Buffy H37 ; +, (e, j, o) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 W105 Buffy H37 ; +. The morphology of the adult eyes was observed by light microscopy (a–e) and SEM (f–o). Scale bars in f–j and k–o indicate 50 μm and 14.2 μm, respectively. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: HEK293T cells were transfected with (+) or without (–) expression vectors for FLAG-tagged Bcl-rambo or FLAG-tagged Buffy together with VSV-tagged Buffy or VSV-tagged Drob-1/Debcl for in the presence of zVAD-fmk (20 μM) for 16 h. Cell lysates were immunoprecipitated with anti-FLAG antibody-conjugated beads. Immunoprecipitates (IP) were analyzed by Western blotting using an anti-VSV antibody. Cell lysates were analyzed by Western blotting using anti-FLAG, anti-VSV, and β-actin antibodies. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A to C) (A) (a, d, g) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, e, h) sesB org / GMR-GAL4 ; UAS-Bcl-rambo /+; +, (c, f, i) w 67c23 P{lacW}Ant2 G0247 sesB G0247 / GMR-GAL4 ; UAS-Bcl-rambo /+; +. (B) (a, d, g) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (b, e, h) sesB org / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +, (c, f, i) w 67c23 P{lacW}Ant2 G0247 sesB G0247 / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +. The morphology of adult eyes was observed by light microscopy (a–c) and SEM (d–i). (C) (a, e, i) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, f, j) w 67c23 P{lacW}schlank G0061 schlank G0061 / GMR-GAL4 ; UAS-Bcl-rambo /+; + (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (d, h, l) w 67c23 P{lacW}schlank G0061 schlank G0061 / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Data were representative of two independent experiments. (D) (a, e, i) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, f, j) w 1118 P{EP}Atg8a EP362 / GMR-GAL4 ; UAS-Bcl-rambo /+; + (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (d, h, l) w 1118 P{EP}Atg8a EP362 / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , Schematic depiction of the 30 SARS-CoV-2 encoded proteins in the order they appear in the genome. The polyprotein ORF1a(b) is (auto)proteolytically cleaved into 16 non-structural proteins (Nsp, turquoise). The structural proteins (yellow) are Spike (S), Membrane (M), envelope (E) and nucleoprotein (N). The set is complemented by the accessory proteins (red) ORF 3a, 3b, 3c, 6, 7a, 7b, 8, 9b, 9c and 10. b-e , Schematic depiction of the assay setup (top panel) and heatmap (red = inhibition, blue = induction) depicting modulation of innate immune pathways by overexpression of indicated SARS-CoV-2 proteins. Stimuli of the immune pathways are indicated. (a, b) Readout by Luciferase reporter gene assay (color represents the mean of n=3) using indicated promotor constructs in HEK293T cells, (c) autophagosome measurement by quantification of membrane-associated GFP-LC3B in HEK293T-GFP-LC3B cells (color represents the mean of n=4) or (d) Quantification of ASC specks in THP-1 cell lines by flow cytometry; cell lines doxycycline-inducible expressing the indicated transgenes were treated with Bacillus anthracis PA and LFn-MxiH to stimulate NLRC4 inflammasomes (color represents the mean of n=2). The vector/control is set to 1 (white). SeV, Sendai Virus. Rapa, Rapamycin. BafA, Bafilomycin A1, PA, protective antigen of B. anthracis , MxiH, Needle protein of S. flexneri.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Inhibition, Over Expression, Luciferase, Reporter Gene Assay, Construct, Flow Cytometry, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , Schematic depiction of the type-I IFN signaling pathway. b , Exemplary immunoblot analysis showing activation of type-I IFN signaling markers using whole cell lysates (WCLs) of HEK293T cells expressing indicated proteins and stimulated with IFN-β (1000 U/mL, 45 min). Blots were stained with anti-pSTAT1, anti-STAT1, anti-pSTAT2, anti-STAT2, anti-IFNAR, anti-strep II and anti-actin. c , Quantification of the band intensities in (b) for IFNAR normalized to the band intensities of actin. Bars represent mean of n=3±SEM. d , Quantification of the band intensities in (b) for phospho-STAT1 (pSTAT1) normalized to the band intensities of actin. Bars represent mean of n=3±SEM. e , Schematic depiction of autophagy. f , Exemplary immunoblot analysis showing autophagy activity markers using WCLs of HEK293T cells expressing indicated proteins. Blots were stained with anti-SQSTM1/p62, anti-LC3B-II, anti-Beclin-1, anti-ULK1, anti-strep II and anti-actin. g , Quantification of the band intensities in (f) for LC3B-II normalized to the band intensities of actin. Bars represent mean of n=3±SEM. h , Quantification of the band intensities in (f) for p62 normalized to the band intensities of actin. Bars represent mean of n=3±SEM. i , Exemplary confocal laser scanning microscopy images of autophagy activation via GFP-LC3B (green) puncta formation. Indicated strep II-tagged SARS-CoV-2 proteins (red) were overexpressed in HeLa GFP-LC3B cells (green). CQ, Chloroquine (4 h 10 μM) was used as a positive control. Nuclei, DAPI (blue). Scale bar, 25 μM. j , Quantification by area of GFP-LC3B puncta divided by cell number from the images in (i). Bars represent the mean of n=38-100 cells±SEM.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Western Blot, Activation Assay, Expressing, Staining, Activity Assay, Confocal Laser Scanning Microscopy, Positive Control

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , Heatmap (red = downregulation, blue = upregulation) depicting the fold changes of cellular and viral proteins during overexpression of indicated single SARS-CoV-2 proteins in HEK293T cells or b , SARS-CoV-2 infection (MOI 1) of Caco-2 cells 24 or 48 h post infection as assessed by mass spectrometry. c , Scatter plots of log2 fold enrichment and p-value of the GO-Term ‘late endosome’ in protein sets regulated more than 4-fold upon expression of indicated viral protein (a) or SARS-CoV-2 infection (b). d , Quantification of co-localization by Pearson Correlation of Rab9 and indicated viral proteins in HeLa cells transiently transfected with the indicated viral protein and GFP-Rab9. Bars represent the mean of n=7-15 cells±SEM. e , Exemplary confocal microscopy images of HeLa cells transiently expressing indicated viral proteins (red) and a marker of late endosomes GFP-Rab9 (green). Cells were stained with anti-strep II (red). Nuclei, DAPI (blue). Scale bar, 10 μm. f , Exemplary confocal microscopy images of the quantification in (g) stained with anti-TGN46 (green) and anti-strep II (red). Nuclei, DAPI (blue). Scale bar, 10 μm. g , Pearson’s correlation indicating colocalization between TGN46 and the indicated viral proteins from the image in (f). Bars represent the mean of n=6 cells±SEM. h, Quantification of non-Golgi associated vesicles per cell as puncta/cell of (f). Bars represent the mean of n=15-25 cells ±SEM.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Over Expression, Infection, Mass Spectrometry, Expressing, Transfection, Confocal Microscopy, Marker, Staining

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a-c , Immune activation of type-I IFN induction (a), type-I IFN signaling (b) or autophagy (c) in the presence of indicated proteins (Nsp1, Nsp3, Nsp7, Ndsp15, M, N, ORF3a, ORF6, ORF7a) of SARS-CoV-2 (blue), RaTG13-CoV (purple) or SARS-CoV-1 (red) assessed by IFN-β-promotor luciferase reporter gene assays stimulated with Sendai Virus (SeV, a). ISRE-promotor luciferase reporter gene assays stimulated with IFN-β (1000 U/ml, b). Membrane-associated GFP-LC3B (c) (n=4±SEM). Vector induction set to 100% (black). Controls, RABV P, MeV V or TRIM32 (grey). Bars represent the mean of n=3±SEM (a,b) or n=4±SEM (c). d , Dose dependent effect of SARS-CoV-2, RaTG13-CoV or SARS-CoV-1 Nsp15 expression on IFN-β induction stimulated with SeV (24 h). Quantification by IFN-β promotor dependent luciferase reporter activity. Lines represent one individual replicate. e , Dose dependent effect of Nsp15 expression on IFN-β signaling in HEK293T cells, stimulated with IFN-β (1000 U/ml, 8 h). Quantification by ISRE promotor dependent luciferase reporter activity. Lines represent one individual replicate.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-γ and -λ

doi: 10.1101/2020.10.15.340612

Figure Lengend Snippet: a , SARS-CoV-2 N RNA in the supernatant of SARS-CoV-2 (MOI 0.05, 48h p.i.) infected Calu-3 cells that were left untreated and/or were treated with the indicated amounts of indicated IFNs or pro-inflammatory cytokines as assessed by qPCR. Lines represent the mean of n=2±SD. b , Correlation between average inhibition of the indicated innate immune signaling pathway and impact on replication of SARS-CoV-2 after treatment with the respective cytokine. r, Pearson’s correlation. c , SARS-CoV-2 N RNA in the supernatant of SARS-CoV-2 (MOI 0.05, 48h p.i.) infected Calu-3 cells that were left untreated and/or were treated with the indicated combinations of indicated IFNs (5 U/ml) or Rapamycin (125 nM) either 24 h before the infection (Pre-treatment) or 6 h post infection (Post-treatment). Dots represent individual experiments, line the mean. Fold reduction compared to control is indicated. d , Immunoblot analysis of the SARS-CoV-2 infection using the WCLs of Calu-3 cells in (c). Blots were stained with anti-SARS-CoV-2 S, anti-SARS-CoV-2 N, and anti-GAPDH.

Article Snippet: Proteins were stained using primary antibodies against β-actin (1:10,000, AC-15, Sigma), strep II-tag (1:1,000, NBP2-43735, Novus), strep II-tag (1:2,000, ab76949, abcam), GAPDH (1:1,000, 607902, Biolegend), pSTAT1 (1:1,000, Y701, Cell Signaling Technology), STAT1 (1:1,000, 9172S, Cell Signaling Technology), pSTAT2 (1:1,000, Y690, Cell Signaling Technology), STAT2 (1:1,000, 4594S, Cell Signaling Technology), IFNAR1 (1:1,000, ab45172, abcam), p62 (1:1,000, GP62-N, ProGen), LC3a/β (1:200, G-4, Santa Cruz Biotechnology), Beclin-1 (1:1,000, 3738S, Cell Signaling Technology), ULK1 (1:1,000, D8H5, Cell Signaling Technology), Rab5 (1:1,000, C8B1, Cell Signaling Technology), SARS-CoV-2 Nsp3 (1:1,000, GTX135614, GeneTex), Flag-tag (1:5,000, M2, Sigma), V5-tag (1:1,000, D3H8Q, Cell Signaling Technology), SARS-CoV-2 (COVID-19) spike antibody (1:1000, 1A9, Biozol), SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (1:1000, MM05, SinoBiological), and Infrared Dye labelled secondary antibodies (1:20,000, LI-COR IRDye), diluted in 0.05% Casein in PBS.

Techniques: Infection, Inhibition, Western Blot, Staining

Journal: PLoS ONE

Article Title: Agonist-Directed Desensitization of the β 2 -Adrenergic Receptor

doi: 10.1371/journal.pone.0019282

Figure Lengend Snippet: (a)–(d) Confocal images of cells after immunostained with anti-β 2 AR. The cells were first stimulated with 2 nM epinephrine for 1 min (a), 2 min (b), 5 min (c) and 30 min (d). After epinephrine removal, the cells were further incubated in medium at 37°C for 29 min (a), 28 min (b), 25 min (c) and 0 min (d), respectively, such that equal time post stimulation was achieved for all. The staining was performed using anti-β 2 AR antibody and Alexa Fluor 488 goat anti-rabbit antibody. The nuclei were stained with DAPI. Confocal microscopy images were acquired on Zeiss confocal laser scanning microscope (oil-immersion, 63× objective). The green color is from the β 2 AR, the blue from the nuclei. (e) The agonist exposure time and Ro31-8220 sensitivity of pERK stimulated by epinephrine in A431 cells. The cells were incubated with the vehicle (0 min) or 2 nM epinephrine for a specific time (1 min, 2 min, 30 min), followed by further incubation at 37°C for another time to ensure all reach 30 min post simulation. Ro31-8220 when used was presented throughout the incubation. Equal amounts of cell lysate were separated by SDS-PAGE and analyzed for pERK by Western blotting. Equivalent gel loading was confirmed by probing with antibodies against GAPDH.

Article Snippet: Rabbit polyclonal anti-β 2 AR (#sc-569), mouse monoclonal anti-pERK1/2 (#sc-81492) and rabbit polyclonal anti-GAPDH (#sc-25778) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Incubation, Staining, Confocal Microscopy, Laser-Scanning Microscopy, SDS Page, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Beclin1-mediated autophagy was upregulated with cardiac hypertrophy in vivo and in vitro . (A) The expression of LC3, beclin1 and p62 in hypertrophic heart were determined by western blotting at indicated time point after TAC (Student t test, n =5-6, * P < 0.05 or ** P < 0.01 vs. sham. The expression of proteins in TAC mice was normalized to that of the sham mice in each time point, and β-actin served as a loading control). (B) Cardiomyocytes were challenged by ET-1 and the expression of LC3, beclin1 and p62 were examined by western blotting at indicated time point (one-way ANOVA, n =5-6, * P < 0.05 vs. 0h). (C) After infected with mRFP-GFP-LC3 adenoviral particles for 24h, the cells were challenged by ET-1. Fluorescent signals were captured with the confocal laser scanning microscopy at indicated time point and the number of autolysosomes and autophagosomes was determined by counting of red puncta or yellow puncta, respectively (one-way ANOVA, * P < 0.05 or ** P < 0.01 vs. 0h in yellow puncta. # P < 0.05, ## P < 0.01 vs. 0h in red puncta. Thirty randomly selected cells per experimental group were analyzed).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Control, Infection, Confocal Laser Scanning Microscopy

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Inhibition of Beclin1-mediated autophagy mitigated adverse cardiac remodeling and cardiac hypertrophy. (A) Ultrastructure analysis showing the autophagosomes (multimembrane vacuoles, arrow heads) and autolysosomes (electron dense structures, arrows) in the myocardium 8 weeks after TAC. (B) Representative cross-sectional images with H&E staining. (C) Representative cross-sectional images showing interstitial fibrosis with Masson staining. (D) Analysis results for cardiomyocyte cross-sectional area (one-way ANOVA, n =5, * P < 0.05 vs . sham, # P < 0.05 vs. TAC). (E) Analysis results for fibrosis (one-way ANOVA, n =5, * P < 0.05 vs. sham, # P < 0.05 vs. TAC). (F) mRNA level of β-MHC and BNP in hypertrophic heart were determined by real time RCR from mice in each experimental group (one-way ANOVA, n =5, * P < 0.05 vs. sham and # P < 0.05 vs. TAC, β-actin served as a loading control). (G) Neonatal cardiomyocytes pretreated with Beclin1 shRNA were subjected to ET-1 for 24h. Autolysosomes and autophagosomes were determined by use of a tandem mRFP-GFP-LC3 adenovirus in cardiomyocytes. The red puncta indicated autolysosomes and the yellow puncta indicated autophagosomes, respectively (one-way ANOVA, * P < 0.05 or # P < 0.05 vs. Control in yellow puncta or red puncta, respectively. $ P < 0.05 vs. ET in yellow puncta. △ P < 0.05 vs. ET in red puncta. Thirty randomly selected cells per experimental group were analyzed). (H) The cell size was assessed by Image J (one-way ANOVA, n =30, * P < 0.05 vs. control, # P < 0.05 vs. ET). (I) mRNA levels of β-MHC and BNP in cardiomyocytes were determined by real time RCR (one-way ANOVA, n =5, * P < 0.05 vs. control, # P < 0.05 vs. ET-1, β-actin served as a loading control).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Inhibition, Staining, Control, shRNA

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: DGKζ was decreased in response to the prohypertrophic stimuli in vivo and in vitro, and DGKζ deficiency linked to Beclin1-mediated autophagy during myocardial hypertrophy (A) Cardiac hypertrophy was induced by TAC in mice and the expression of DGKζ in the heart was determined by western blotting at indicated time point after surgery (Student t test, n =5, * P < 0.05 or ** P < 0.01 vs. sham. The expression of proteins in TAC mice was normalized to that of the sham mice in each time point, and β-actin served as a loading control). (B) The expression of DGKζ was determined by western blotting in ET-1-treated cardiomyocytes for indicated time point (one-way ANOVA, n =5, * P <0.05 or ** P <0.01 vs. 0h). (C) The level of DGKζ was successfully downregulated in cardiomyocytes by infecting lentiviral DGKζ shRNA. Lentiviral plasmid containing no targeted sequences or scramble sequences served as vector control and non-silencing control (one-way ANOVA, n =5, * P <0.05 vs. control). (D) The level of DAG was measured by ELISA kits in heart tissue 12 weeks after TAC and in cardiomyocytes after ET-1 treatment for 24h (one-way ANOVA, n =5). (E) Cardiomyocytes were challenged by ET-1 and the expression of LC3, beclin1 and p62 were examined by western blotting (one-way ANOVA, n =5-6, * P < 0.05 vs. control, # P < 0.05 vs. ET). (F) Autolysosomes and autophagosomes were determined by use of a tandem mRFP-GFP-LC3 adenovirus in cardiomyocytes. The red puncta indicated autolysosomes and the yellow puncta indicated autophagosomes, respectively (one-way ANOVA, ** P < 0.01 vs. control in yellow puncta. # P < 0.05 or ## P < 0.01 vs. control in red puncta. $ P < 0.05 vs. DGKζ shRNA+ET in yellow puncta. △ P < 0.05 vs. DGKζ shRNA+ET in red puncta. Thirty randomly selected cells per experimental group were analyzed). (G) The cell size was assessed by Image J (one-way ANOVA, n =30, ** P <0.01 vs. control, ## P <0.01 vs. ET). (H) mRNA level of β-MHC and BNP in cardiomyocytes were determined by real time RCR (one-way ANOVA, n =3, * P < 0.05 vs. control, # P < 0.05 vs. ET, $ P < 0.05 vs. DGKζ shRNA+ET, β-actin served as a loading control).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Control, shRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Deficiency of DGKζ promoted cardiac dysfunction by augmenting Beclin1-mediated autophagy. (A) Representative M-mode echocardiograms, transmitral flow and tissue doppler echocardiograms. (B) The analyzed results of LVAW and LVPW obtained from mice in each experimental group ((one-way ANOVA, n =6, * P <0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (C) The analyzed results of EF and FS obtained from mice in each experimental group ((one-way ANOVA, n =6, * P <0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (D) The analyzed results of E/A and e'/a' obtained from mice in each experimental group ((one-way ANOVA, n =6, * P <0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (E) Representative cross-sectional images with H&E staining. (F) Representative cross-sectional images with masson staining. (G) Analysis results for cardiomyocyte cross-sectional area (one-way ANOVA, n =5, * P < 0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (G) Analysis results for fibrosis (one-way ANOVA, n =5, * P < 0.05 vs. TAC, # P < 0.05 vs. DGKζ shRNA+TAC).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: shRNA, Staining

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: AKT/mTOR dependent signaling is involved in the enhancement of cardiac autophagy by DGKζ deficiency Cardiomyocytes infected with lentivirus expressing DGKζ shRNA were subjected to ET-1 for 24h. The total protein expression and phosphorylated level of AKT, mTOR, AMPK, ULK1 and Beclin1 were examined by western blotting, respectively. (A) Representative blots depicting total and phosphorylated proteins. (B) The analyzed results of p-AKT/AKT level, p-mTOR/mTOR level, p-AMPK/AMPK level, p-ULK1/ULK1 level and p-Beclin1 level (one-way ANOVA, n =5, *P< 0.05 vs. control. # P< 0.05 or ## P< 0.01 vs. ET).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, shRNA, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Deficiency of DGKζ impaired the interaction of mTOR with TFEB and favored the nuclear translocation of TFEB. (A) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in heart tissues after TAC (Student t test, n =7-8, * P < 0.05 or ** P < 0.01 vs. sham. The expression of proteins in TAC mice was normalized to that of the sham mice in each time point, and β-actin served as a loading control). (B) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in ET-1-treated cardiomyocytes (one-way ANOVA, n =6, * P <0.05 vs. 0h). (C) Immunofluorescent staining showing the translocation of TFEB from cytoplasm to nucleus induced by ET-1. (D) The representative image of immunoprecipitation assay showing that mTOR interacted with TFEB in cardiomyocytes. Input was used as a positive control and IgG was used as a negative control. (E) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in cardiomyocytes after DGKζ knockdown (one-way ANOVA, n =5-6, * P <0.05 vs. control, # P< 0.05 vs. ET). (F) Immunofluorescent staining showing the translocation of TFEB from cytoplasm to nucleus induced by ET-1 after DGKζ knockdown.

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Expressing, Control, Staining, Immunoprecipitation, Positive Control, Negative Control, Knockdown

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Genetic knockdown of TFEB abrogates Beclin1-mediated autophagy and ameliorate ET-1-induced myocardial hypertrophy. (A) Representative blot and the analyzed results of the expression of TFEB and Beclin1 in ET-1-treated cardiomyocytes (one-way ANOVA, n =5 , *P< 0.05 vs. scramble+ET, # P <0.05 vs. (scramble+DGKζ) shRNA+ET). (B) mRNA levels of Beclin1 in cardiomyocytes were determined by real time RCR (one-way ANOVA, n =3, * P < 0.05 vs. control, # P < 0.05 vs. ET, & P < 0.05 vs. DGKζ shRNA+ET, β-actin served as a loading control). (C) Schematic representation of conserved binding sequences identified in the promoter regions of Becn1 using JASPAR. (D) Occupancy of Becn1 promoter regions -1420 to -1290, 540 to -340, and -880 to -670 by TFEB, as determined by ChIP assay (one-way ANOVA, n =3, * P < 0.05 vs. control, # P <0.05 vs. ET, β-actin served as a loading control). (E) Luciferase activity in cardiomyocytes transfected with the Becn1 luciferase reporter constructs (one-way ANOVA, n =3, * P <0.05 vs. control, # P <0.05 vs. ET, & P <0.05 vs. DGKζ shRNA+ET). (F) Autolysosomes and autophagosomes were determined by use of a tandem mRFP-GFP-LC3 adenovirus in cardiomyocytes. The red puncta indicated autolysosomes and the yellow puncta indicated autophagosomes, respectively (Student t test, * P < 0.05 vs. ET in yellow puncta. ## P < 0.01 vs. ET in red puncta. Thirty randomly selected cells per experimental group were analyzed). (G) The cell size was assessed by Image J (one-way ANOVA, n =30, ** P < 0.01 vs. control, ## P < 0.01 vs. ET). (H) mRNA level of β-MHC and BNP in cardiomyocytes were determined by real time RCR (one-way ANOVA, n =3, * P < 0.05 vs. control, # P <0.05 vs. ET, & P <0.05 vs. DGKζ shRNA+ET, β-actin served as a loading control).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, shRNA, Control, Binding Assay, Luciferase, Activity Assay, Transfection, Construct