Journal: Molecular Psychiatry

Article Title: Novel animal models for studying complex brain disorders: BAC-driven mi RNA-mediated in vivo silencing of gene expression

doi: 10.1038/mp.2010.1

Figure Lengend Snippet: Generation and validation of the Tg ( Npy-eGFP/miRNA:Gad1 ) 1KM mice. ( a ) Schematic linear representation of the construct. Flanking regions of the construct are derived from the driver NPY BAC that provides interneuron subtype-specific expression of the construct. The 5′ region of the driver BAC (NPY promoter region) ensures specific expression of eGFP, and the SV40-pA ensures proper polyadenylation. The construct, containing a non-functional (not translated) part of β-globin exons 1 and 2, and intron 1 in its entirety, is spliced by the cellular machinery, liberating the intron that contains the 70–100 nucleotide long double-stranded miRNA directed against GAD1. The spliced intron- miRNA:Gad1 is processed through DROSHA and exported from the nucleus. In the cytoplasm the miRNA binds to and degrades the endogenous GAD1 mRNA through an RNA-induced silencing complex (RISC). , The eGFP mRNA is translated, thus fluorescently labeling cells of interest. The presence of LoxP sites facilitates generation of animals lacking the silencing part of the miRNA:Gad1 construct, but still expresses eGFP, to serve as controls. ( b ) miRNA-mediated downregulation of GAD1 in vitro . CHO cells co-transfected with GFP reporter vector alone (−), two different intermediate constructs (GFP reporter vectors containing two different synthetic miRNAs directed against GAD1—nos. 1 and 2), and GAD1 expression vector. The cells were harvested at 24 and 48 h after transfection. Western blot analysis confirmed that the two different miRNAs against GAD1 were correctly processed from the β-globin intron and that both miRNAs strongly downregulated GAD1 expression. Similar results have been obtained in HEK293 cells with stable expression of GAD1 that were engineered in our laboratory (data not shown). The same cells transfected with CMV-eGFP,miRNA:Gad1 show high levels of eGFP expression, confirming that the construct performs as expected. In addition, the processing of the miRNA from the β-globin intron in the construct does not interfere with eGFP protein translation. ( c ) Identification of founder animals by PCR-based genotyping of founder animals using eGFP primers with genomic DNA as template. The first lane represents size marker, and the last lane corresponds to positive control, with the remaining lanes containing eGFP amplification products from the genomic DNA of individual animals. The 550-nt product on a 1% agarose gel indicates construct incorporation into the genome of four founder animals (NPY 1, 3, 4 and 5). Similar results were obtained by Southern hybridization (data not sown). ( d ) Quantitative PCR (qPCR) amplification plot from frontal cortex of founder animals using eGFP construct-specific primers. The y axis denotes PCR product accumulation, and the x axis denotes amplification cycle number. Note that two (NPY1 and NPY4) of the four founder lines that incorporated the NPY-BAC/GAD1-miRNA construct reported functional eGFP expression. These lines were used for further characterization. ( e ) The NPY-BAC/GAD1-miRNA construct showed the expected tissue distribution in the brain. Micrographs depict the fidelity of co-localized eGFP and NPY in adult transgenic animals from line Tg ( Npy-eGFP/miRNA:Gad1 ) 1KM . The left column micrographs denote eGFP immunostaining, middle column micrographs represent sections labeled with anti-NPY and the right column micrographs illustrate pseudocolored composite of eGFP-NPY co-localization in the same tissue sections. In the cortex, roman numerals denote cortical laminae. Hippocampus abbreviations: gcl, granule cell layer; h, hilus; ml, molecular layer. Note that all GFP+ neurons are also NPY+, and all NPY+ cells are GFP+, suggesting that the construct is specifically and exclusively expressed in the phenotypically appropriate target cell population. Calibration bar=100 μm. ( f , g ) Tg ( Npy-eGFP/ miRNA:Gad1 ) 1KM animals show undetectable GAD1 levels in the frontal cortex and hippocampus of NPY+ cells compared with NPY+ neurons in control animals. eGFP-GAD1 double-immunohistochemistry (eGFP, green; GAD1, red) was performed from a coronal section through the frontal cortex and hippocampus of a transgenic animal. Wild-type control littermate was double stained against NPY and GAD1. Same cells are denoted by arrowheads. In the cortex, roman numerals denote cortical laminae. Hippocampal abbreviations: f, hippocampal fissure; gcl, ganglion cell layer; h, hilus; ml, molecular layer; slm, stratum lacunosum moleculare; sp, stratum pyramidale; sr, stratum radiatum. Note that all NPY+ cells (white arrows) are GAD1+ in control animals, whereas none of the eGFP+ (and thus NPY+) neurons (white arrows) show GAD1 staining in the transgenic mice. Moreover, note the large number of single-labeled GAD1+ neurons that are eGFP– in the transgenic line. These data indicate selective, miRNA-mediated, cell type-specific downregulation of GAD1 in NPY+ interneurons. Calibration bars=60 μm (overview figures) and 100 μm.

Article Snippet: Intermediate construct DNA (CMV-eGFP, miRNA:Gad1) was co-transfected into HEK-293 cells (American Type Culture Collection) along with a GAD1 expression construct (IMAGE clone 5358787) in a 3:1 ratio using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Biomarker Discovery, Construct, Derivative Assay, Expressing, Functional Assay, Labeling, In Vitro, Transfection, Plasmid Preparation, Western Blot, Marker, Positive Control, Amplification, Agarose Gel Electrophoresis, Hybridization, Real-time Polymerase Chain Reaction, Transgenic Assay, Immunostaining, Control, Immunohistochemistry, Staining

Journal:

Article Title: Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y 13 receptor-type

doi: 10.1038/sj.bjp.0705588

Figure Lengend Snippet: P2Y receptor mRNA expression and immunohistochemistry in HEK 293-N26 cells. (A) Subsequent to total RNA extraction and RT–PCR amplification with primers specific for distinct P2Y receptor cDNA fragments (P2Y13, 25 cycles; P2Y1–P2Y12, 35 cycles), cDNA products were analyzed by agarose gel (1.5%) electrophoresis. A representative gel with ethidium bromide-stained cDNA fragments is shown: P2Y1 (528 bp), P2Y4 (431 bp), P2Y6 (380 bp), P2Y11 (410) and P2Y13 (575 bp). P2Y2 and P2Y12 transcripts were not detectable. (B) Representative fluorescence image of HEK 293-N26 cells after immunocytochemical labeling of P2Y1 (a) and P2Y4 receptors (c), with rabbit anti-P2Y1 and P2Y4 receptor antibodies (scale bar, 20 μm). P2Y2 receptor immunoreactivities (b) could not be detected.

Article Snippet: After fixation, washing with Tris-buffered saline (TBS, 0.05 M; pH 7.6) and blocking with 5% fetal calf serum (FCS), the cells were incubated with the rabbit anti-P2Y receptor antibodies (anti-P2Y 1 : 1 : 1500, SmithKline Beecham Pharmaceuticals, Harlow, Essex, U.K.; anti-P2Y 2 : 1 : 1000, Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, MD, U.S.A.; anti-P2Y 4 : 1 : 1000, Alomone Labs, Jerusalem, Israel) with 0.1% Triton X-100, 5% FCS in TBS for 12 h at 4°C.

Techniques: Expressing, Immunohistochemistry, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Fluorescence, Labeling

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Upregulated expression of Sox9 in medial vSMCs of aortic allografts and HMGB1-treated vSMCs (A) Heatmap shows differentially regulated genes (|log2FC(allograft/isograft)| > 1; p adjust <0.01) in the medial cells of non-transplanted aorta from Lewis (n = 4) and BN (n = 4) rats, isografts (Lewis-Lewis. n = 10 rats) and allografts (BN-Lewis. n = 9 rats) transfected with lentiviruses expressing negative control shRNA with a specific tagln promoter (shNC) 2 weeks after transplantation, as detected by preforming RNA sequencing. (B) GO enrichment analysis shows selected GO terms of biological process of differentially regulated genes (DEGs). Size of circles represents the number of DEGs. p adjust values are displayed. (C) Venn diagram shows the overlap between genes in indicated GO terms. (D) qRT-PCR analysis of Sox9 mRNA expression within medial cells of shNC isografts and shNC allografts 2 weeks after transplantation (means ± SEM ∗∗p < 0.01. n = 8 rats per group). (E) Representative cross sections of aortic grafts coimmunostained for Sox9 (red) and Acta2 (green) 2 weeks after transplantation. Cell nuclei were stained with hoechst (blue). White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of Sox9 positive cells in the media of aortic grafts (means ± SEM ∗∗p < 0.01. n = 15 rats per group).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Expressing, Transfection, Negative Control, shRNA, Transplantation Assay, RNA Sequencing Assay, Quantitative RT-PCR, Staining

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Sox9 drives the phenotypic modulation of vSMC following transplantation (A) qRT-PCR analysis of Sox9 , Cnn1 , Acta2 and Tagln mRNA expression within the media of aortic isografts and allograft transfected with sh Sox9 or shNC 2 weeks after transplantation (means ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 8 rats per group). (B) Representative cross sections of non-transplanted aorta, and sh Sox9 -or shNC-transfected aortic grafts immunostained for Cnn1 (green), Acta2 (green) and Tagln (green) 2 weeks after transplantation (Bi). Cell nuclei were stained with hoechst (blue). Red boxes indicate the total sections. White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of Cnn1, Acta2 and Tagln positive area in the media of aortic grafts (Bii) (means ± SEM ∗p < 0.05, ∗∗∗p < 0.001. n = 15 rats per group). (C and D) qRT-PCR and western blotting analyses of Myocd , Srf , Sox9 , Cnn1 , Acta2 and Tagln expression in cultured vSMCs transfected with lentiviruses carring Sox9 -targeting shRNA (sh Sox9 ) with a specific Tagln promoter or negative control shRNA (shNC) followed by HMGB1 (100 ng/mL) stimulation or not (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Transplantation Assay, Quantitative RT-PCR, Expressing, Transfection, Staining, Western Blot, Cell Culture, shRNA, Negative Control

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Sox9 facilitates vSMC proliferation, migration and transplant arteriosclerosis in rat aortic allografts (A) Representative cross sections of shNC- or sh Sox9 -transfected isografts and allografts immunostained for PCNA 2 weeks after transplantation. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of PCNA-positive cells in the media of aortic grafts (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 15 rats per group). (B and C) vSMCs transfected with sh Sox9 or negative control shNC were stimulated with or without HMGB1 (100 ng/mL), cell proliferation was evaluated by CCK-8 and BrdU incorporation assays as the measured absorbance at 450 nm (means ± SEM ∗p < 0.05. n = 5). (D) Representative images of migrated vSMCs on the bottom of transwell membrane as detected by Transwell assays (means ± SEM ∗p < 0.05. n = 5). (E) Representative images to show H&E staining, EVG staining and immunofluorescence staining of vSMC marker Acta2 in cross sections of aortic isografts and allografts transfected with shNC or sh Sox9 8 weeks after aortic transplantation. Green arrows denote the internal elastic lamina. White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. (F–H) Quantification of of intimal area, intima/media ratio and lumen stenosis ratio (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 18 rats per group).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Migration, Transfection, Transplantation Assay, Negative Control, CCK-8 Assay, BrdU Incorporation Assay, Staining, Immunofluorescence, Marker

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Autophagy is associated with increased Sox9 expression in vSMCs (A) Representative cross sections of non-transplanted aorta and shNC-transfected aortic grafts coimmunostained for LC3Ⅰ/Ⅱ (red) and Acta2 (green) 2 weeks after transplantation. Cell nuclei were stained with hoechst (blue). White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of LC3Ⅰ/Ⅱ-positive area in the media of aortic grafts (means ± SEM ∗∗∗p < 0.001. n = 15 rats per group). (B) Transmission electron microscopy images of double membrane autophagosome and single membrane autophagolysosome structures in aortic isografts and allografts transfected with shNC. Red boxes show the enlarged sections of aortic grafts. (C) LC3Ⅰ/Ⅱ-stained fluorescent microscopy images of vSMCs treated with HMGB1 (100 ng/mL) for 48 h, transmission electron microscopy images of autophagosomes or autophagolysosomes in vSMCs stimulated with HMGB1 (100 ng/mL). Red boxes show the enlarged sections. (D) vSMCs were transfected with small interfering RNA targeting Atg5 (si Atg5 ) or its corresponding negative control (siNC) followed by HMGB1 (100 ng/mL) stimulation, Sox9 mRNA expression was measured by qRT-PCR (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (E) Western blotting analysis of Sox9, Atg5, p62 and LC3Ⅰ/Ⅱ expression in vSMCs transfected with si Atg5 or siNC followed by HMGB1 (100 ng/mL) stimulation (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (F and G) vSMCs pretreated with or without Bafilomycin A1 (200 nM) were stimulated with HMGB1. qRT-PCR analysis of Sox9 mRNA expression. n = 4. Western blotting analysis of Sox9, p62 and LC3Ⅰ/Ⅱ protein expression (means ± SEM ∗p < 0.05. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Expressing, Transfection, Transplantation Assay, Staining, Transmission Assay, Electron Microscopy, Microscopy, Small Interfering RNA, Negative Control, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Autophagy promotes vSMC phentopic modulation by upregulating Sox9 (A and B) qRT-PCR and western blotting analyses of Cnn1 , Acta2 and Tagln mRNA and protein expression in vSMCs transfected with si Atg5 or siNC followed by HMGB1 stimulation (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (C and D) vSMCs pretreated with or without bafilomycin A1 were stimulated with HMGB1, mRNA and protein expression of Cnn1 , Acta2 and Tagln were measured by qRT-PCR and western blotting (means ± SEM ∗p < 0.05. n = 4). (E and F) qRT-PCR and western blotting analyses of Sox9 , Cnn1 , Acta2 and Tagln expression in vSMCs transfected with plasmids overexpressing Sox9 ( Sox9 oe) or its control vector (vector) (means ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 4). (G) vSMCs were cotransfected with Atg5 siRNA and Sox9 overexpressing plasmid together with HMGB1 (100 ng/mL) stimulation, protein expression of Sox9, Cnn1, Acta2 and Tagln were determined (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (H and I) BrdU incorporation assay was performed in si Atg5 -transfected and Bafilomycin A1-pretreated vSMCs exposed to HMGB1 (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 3). (J and K) The migration ability of si Atg5 -transfected and Bafilomycin A1-pretreated vSMCs exposed to HMGB1 was evaluated by Transwell assay (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 3).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, BrdU Incorporation Assay, Migration, Transwell Assay

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Autophagy-mediated Sox9 expression relies on p27 degradation (A and B) qRT-PCR and western blotting analyses of p27 Kip1 ( p27 ) expression in vSMCs exposed to HMGB1 (100 ng/mL) for indicated time (means ± SEM ∗p < 0.05. n = 4). (C and D) vSMCs were stimulated with HMGB1 for 48 h, co-IP assay was performed to analyze the ubiquitination of p27 using anti-ubiquitin (n = 4); Binding of p27 and p62 was determined by immunoprecipitating p27 (n = 4). (E) vSMCs pretreated with Bafilomycin A1 (200 nM) or MG132 (5 μM) were stimulated with or without HMGB1 (100 ng/mL) for 48 h, p27 protein level was determined by western blotting analysis, and densitometric analysis of blots was performed (means ± SEM ∗p < 0.05. n = 4). (F) western blotting analysis and quantification of p27 expression in vSMCs transfected with si Atg5 or siNC followed by HMGB1 stimulation (means ± SEM ∗p < 0.05. n = 4). (G and H) qRT-PCR and western blotting analyses of p27 expression in vSMCs transfected with si p27 or siNC (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (I and J) vSMCs were cotransfected with sh Sox9 and si p27 , Cnn1, Acta2 and Tagln expression were detected by western blotting (means ± SEM ∗p < 0.05. n = 4); Cell proliferation was evaluated by BrdU incorporation assay (means ± SEM ∗p < 0.05. n = 3); Cell migration was detected by Transwell assay (means ± SEM ∗p < 0.05. n = 3). (L and M) vSMCs were cotransfected with si Atg5 and si p27 together with or without HMGB1, mRNA expression of p27 and Sox9 were detected by qRT-PCR (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 3); p27 and Sox9 protein expression were measured by western blotting (means ± SEM ∗p < 0.05. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Transfection, BrdU Incorporation Assay, Migration, Transwell Assay

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: p27 associates with p130/E2F4 complex on Sox9 promoters (A) vSMCs were cotransfected with pGL3- Sox9 reporter construct and p27 overexpressing plasmid or empty vector for 48 h, Sox9 promoter activity was measured by dual luciferase assays (means ± SEM ∗p < 0.05. n = 3). (B) Dual luciferase assays were performed to evaluate Sox9 promoter activity in vSMCs cotransfected with pGL3- Sox9 reporter construct and si p27 for 48 h (means ± SEM ∗p < 0.05. n = 3). (C) Confirmation of the binding of p27 to Sox9 promoter in vSMCs by ChIP assay followed by qRT-PCR and agarose gel electrophoresis. An anti-IgG antibody was used as the negative control, an anti-Histone H3 antibody was used as the positive control and no antibody was added as the empty control (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 4). (D) Coimmunoprecipitation (co-IP) of p27 and p130/E2f4 complex in vSMCs (n = 4). (E) ChIP of p130 and E2F4 protein in Sox9 promoter in vSMCs (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 4). (F) vSMCs were cotransfected with pGL3- Sox9 reporter construct and p27 overexpressing plasmid or p130 siRNA or E2F4 siRNA, Sox9 promoter activity was measured by dual luciferase assays (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (G and H) qRT-PCR and western blotting analyses of Sox9 , Cnn1 , Acta2 and Tagln expression in vSMCs transfected with si p130 , si E2F4 or siNC for 48 h (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Construct, Plasmid Preparation, Activity Assay, Luciferase, Binding Assay, Quantitative RT-PCR, Agarose Gel Electrophoresis, Negative Control, Positive Control, Co-Immunoprecipitation Assay, Western Blot, Expressing, Transfection

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Recombinant, CCK-8 Assay, Labeling, Sonication, Chromatin Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Sequencing, Plasmid Preparation, Software, Imaging

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal: Viruses

Article Title: Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

doi: 10.3390/v14010107

Figure Lengend Snippet: SwSCV-1 DAg antiserum cross-reactivity with the DAg of different kolmiovirids. ( A ) I/1Ki cells transfected with 1.2× SwSCV-1, HDV-1, TSRV-1, DabDV-1, and CITV-1 REV constructs were stained for the DAg at 4 days post transfection using rabbit α-SwSCV-1 DAg antiserum (1:100 dilution). ( B ) I/1Ki cells transfected with 1.2× SwSCV-1, HDV-1, TSRV-1, DabDV-1, and CITV-1 FWD constructs and clean cell control were stained for the DAg 4 days post transfection using rabbit α-SwSCV-1 DAg antiserum (1:100 dilution). Hoechst 33342 served for detection of the nuclei ( left panels ), and AlexaFluor 488-labeled donkey anti-rabbit IgG as the secondary antibody for DAg detection ( middle panels ). The ( right panels ) show overlay of the nuclear and DAg staining. The images were captured using Opera Phenix High Content Screening System (PerkinElmer, Waltham, MA, USA) with 20× objective. ( C ) I/1Ki cells transfected with 1.2× SwSCV-1, HDV-1, TSRV-1, DabDV-1, and CITV-1 REV constructs ( left panel ) and FWD constructs ( right panel ) were submitted for western blot at 4 days post transfection. The samples were separated on 4–20% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA), transferred onto nitrocellulose, and the membranes were probed with rabbit α-SwSCV-1 DAg antiserum and affinity purified α-HDAg antibody. We loaded 1/3 volume of the 1.2× SwSCV-1 REV and FWD samples. The bands corresponding to the different DAgs are marked with the black rectangle. The results were recorded using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Article Snippet: The Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was employed to record the results.

Techniques: Transfection, Construct, Staining, Labeling, High Content Screening, Western Blot, Affinity Purification, Imaging

Journal: Viruses

Article Title: Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

doi: 10.3390/v14010107

Figure Lengend Snippet: Western blot of I/1Ki cells after transfection with 2× and 1.2× SwSCV-1 (2×Δ and 1.2×Δ, respectively) FWD and REV constructs. ( A ) Samples of I/1Ki cells transfected with 2×Δ-FWD, 2×Δ-REV, 1.2×Δ-FWD, and 1.2×Δ-REV constructs collected at 1–4 days post transfection were separated on 4–20% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA), transferred onto nitrocellulose, and the membranes were probed with rabbit α-SwSCV-1 DAg antiserum and mouse monoclonal anti-pan actin antibody. The left panel shows 2× and the right panel 1.2× genome constructs. The results were recorded using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). ( B ) Samples of I/1Ki cells transfected with 2×Δ-FWD, 2×Δ-REV, 1.2×Δ-FWD, and 1.2×Δ-REV constructs collected at 5 days, analyzed as described in ( A ). ( C ) RNA isolated from I/1Ki cells transfected with 2×Δ-FWD, 2×Δ-REV, 1.2×Δ-FWD, and 1.2×Δ-REV constructs at 3 and 6 days post transfection were analyzed by qRT-PCR targeting genomic SwSCV-1 RNA. In vitro transcribed RNA target served for obtaining a standard curve to convert cycle threshold values into copy numbers. qRT-PCR targeting GAPDH mRNA served for normalizing the results between samples. The y-axis shows copy numbers/reaction. The error bars represent standard deviation.

Article Snippet: The Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was employed to record the results.

Techniques: Western Blot, Transfection, Construct, Imaging, Isolation, Quantitative RT-PCR, In Vitro, Standard Deviation

Journal: Viruses

Article Title: Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

doi: 10.3390/v14010107

Figure Lengend Snippet: Comparison of persistently SwSCV-1-infected I/1Ki cells generated following transfection with 2× and 1.2× SwSCV-1 FWD constructs by immunofluorescence, and western and northern blot. The 2× SwSCV-1 (I/1Ki-2×Δ) cell line was analyzed at approximately 2.5 years and the 1.2× SwSCV-1 (I/1Ki-1.2×Δ) at approximately 8 months after initial transfection, during which the cell lines were passaged at 1–2 week interval. ( A ) Rabbit α-SwSCV-1 DAg antiserum and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody served for IF staining of the fixed cells, and Hoechst 33342 for staining the nuclei. The top panels show staining of I/1Ki-2×Δ cells, and the bottom panels the staining of I/1Ki-1.2×Δ cells. The left panels show staining of nuclei in blue, the middle panels show DAg staining in green, and the right panels show an overlay. The images were captured using Opera Phenix High Content Screening System (PerkinElmer, Waltham, MA, USA) with 20× objective. ( B ) Samples of naïve I/1Ki cells, I/1Ki-2×Δ cells, I/1Ki-1.2×Δ cells, and the brain homogenates of SwSCV-1-infected boa constrictors (F18-4 and F-18-5, of ) were separated on 4–20% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA), transferred onto nitrocellulose, and the membranes probed with rabbit α-SwSCV-1 DAg antiserum and mouse monoclonal anti-pan actin antibody. The results were recorded using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). ( C ) Indicated amounts of total RNA isolated from I/1Ki-2×Δ, I/1Ki-1.2×Δ, and clean I/1Ki cells and an in vitro-transcribed control RNA (~850 nucleotides long) were prepared using two different loading dyes (2X RNA loading dye [NEB] or “in-house” loading dye prepared according to Mansour and Pestov ), separated on agarose gel and transferred onto nylon membrane. Probes were targeting SwSCV-1 genomic RNA and SwSCV-1 DAg mRNA (left and middle panels) and antigenomic RNA and SwSCV-1 DAg mRNA (right panel); the bands of the marker served for visualizing the RNA targets. The results were recorded using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Article Snippet: The Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was employed to record the results.

Techniques: Infection, Generated, Transfection, Construct, Immunofluorescence, Western Blot, Northern Blot, Labeling, Staining, High Content Screening, Imaging, Isolation, In Vitro, Agarose Gel Electrophoresis, Marker

Journal: Viruses

Article Title: Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

doi: 10.3390/v14010107

Figure Lengend Snippet: SwSCV-1 infection on naïve I/1Ki cells. Supernatants from I/1Ki cells transfected six months ago with 1.2× or 2× SwSCV-1 FWD were collected three days post superinfection with HISV-1 and subsequently used to inoculate naïve I/1Ki cells at 1:5 and 1:100 dilutions. ( A ) Rabbit α-SwSCV-1 DAg antiserum and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody served for IF staining of the fixed cells, and Hoechst 33,342 for staining the nuclei. The left panels show an overlay of DAg (green) and nuclear (blue) staining of I/1Ki-2×Δ cells and the right panels the staining of I/1Ki-1.2×Δ cells fixed at 3, 6, or 9 dpi. The images were captured using Opera Phenix High Content Screening System (PerkinElmer, Waltham, MA, USA) with 20× objective. ( B ) Opera Phenix High Content Screening System (PerkinElmer, Waltham, MA, USA) served for enumerating the number of infected cells at each time point. The dark bars represent cells inoculated with 1:5 dilution of HISV-1 superinfected 2× SwSCV-1 and the light bars cells inoculated with 1:5 dilution of HISV-1 superinfected 1.2× SwSCV-1 cell culture supernatant. ( C ) RT-PCR served to quantify the amount of SwSCV-1 RNA in the cells at each time point. The number of SwSCV-1 RNA copies in the reaction (corresponding to 1/20 of RNA extracted from cells of a single 24-well plate well) normalized against housekeeping gene (GAPDH). ( D ) Samples of cells inoculated with 1:5 or 1:100 diluted supernatant collected from HISV-1 superinfected 2× SwSCV-1 FWD or 1.2× SwSCV-1 FWD transfected cells were separated on 4–20% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA), transferred onto nitrocellulose, and the membranes probed with rabbit α-SwSCV-1 DAg antiserum, rabbit α-HISV NP antiserum, and mouse monoclonal anti-pan actin antibody. The top panels show the results recorded using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and the bottom panels show results for the quantification (using Image Studio Lite Ver 2) of the HISV NP and DAg bands normalized against the actin signal.

Article Snippet: The Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was employed to record the results.

Techniques: Infection, Transfection, Labeling, Staining, High Content Screening, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Imaging

Journal: Circulation Research

Article Title: Activation of Transient Receptor Potential Vanilloid Type-1 Channel Prevents Adipogenesis and Obesity

doi: 10.1161/01.res.0000262653.84850.8b

Figure Lengend Snippet: Figure 2. Reduced TRPV1 expression in visceral adipose tissue from obese db/db mice (upper panel), obese ob/ob mice (middle panel) and obese human male subjects (lower panel). Picture of body shape and abdominal situs showing visceral fat (a and e); expres- sion of TRPV1 in visceral adipose tissue (b and f); representative agarose gel electrophoresis of PCR products from mRNA of TRPV1 and GAPDH genes in visceral adipose tissue (c and g), M denotes bp marker; and summary data (d and h); each n4. **P0.01 between groups. Lower panels show data from age-matched human male control subjects and obese human male subjects. Waist cir- cumference (i); each n8. ***P0.001 between groups; expression of TRPV1 in visceral adipose tissue and subcutaneous fat (j); repre- sentative quantitative real-time RT-PCR from visceral adipose tissue (k) from 1 human male control subject (black line) and from 1 obese human male subject (red line); y-axis denotes fluorescence at 530 nm in arbitrary units, and x-axis denotes number of cycles of the quantitative real-time RT-PCR; The shift of the red line to the right compared with the black line indicates reduced TRPV1 tran- scripts in obese subjects; summary data of quantitative real-time RT-PCR are given in (l); n4. *P0.05 between groups. Quantitative in-cell Western assay of TRPV1 expression in visceral adipose tissue (m), representative capsaicin-induced calcium influx into visceral adipose tissue from 1 human control subject (open circles) and 1 obese human subject (filled circles) (n); and summary data (o); each n4; *P0.05).

Article Snippet: In-cell Western assay of TRPV1 channels human visceral adipose tissue was performed using the Odyssey infrared imaging system (Licor biosciences) as described by our group.9 Primary rabbit anti- human TRPV1-antibodies were obtained from Alomone labs (Jerusalem, Israel) and secondary IRDye800CW-infrared fluorescent dye-conjugated goat anti-rabbit antibodies were obtained from Biomol (Hamburg, Germany).

Techniques: Expressing, Agarose Gel Electrophoresis, Marker, Control, Quantitative RT-PCR, In-Cell ELISA

Journal: Circulation Research

Article Title: Activation of Transient Receptor Potential Vanilloid Type-1 Channel Prevents Adipogenesis and Obesity

doi: 10.1161/01.res.0000262653.84850.8b

Figure Lengend Snippet: Figure 3. The TRPV1 channel agonist capsaicin prevents obesity in mice on high fat diet. a, Time course of body weight from control mice (open circles), mice on high fat diet (filled circles), control mice with capsaicin (open squares), and mice on high fat diet with cap- saicin (filled squares; each n10); Body weight (b), daily food intake per mouse during the first 10 days after the start of capsaicin administration in control mice (open circles), mice on high fat diet (filled circles), control mice with capsaicin (open squares), and mice on high fat diet with capsaicin (filled squares; each n10) (c), picture of body shape of mice (d), waist circumference (e), serum triglyc- erides (f), visceral adipose tissue (g), subcutaneous fat (h), brown fat (i) at the age of 120 days of control mice (n10), mice on high fat diet (n10), control mice plus capsaicin (n10), and mice on high fat diet plus capsaicin (n10). **P0.01 for the comparison high fat vs control; P0.01 for the comparison high fat plus capsaicin vs high fat.

Article Snippet: In-cell Western assay of TRPV1 channels human visceral adipose tissue was performed using the Odyssey infrared imaging system (Licor biosciences) as described by our group.9 Primary rabbit anti- human TRPV1-antibodies were obtained from Alomone labs (Jerusalem, Israel) and secondary IRDye800CW-infrared fluorescent dye-conjugated goat anti-rabbit antibodies were obtained from Biomol (Hamburg, Germany).

Techniques: Control, Comparison

Journal: Circulation Research

Article Title: Activation of Transient Receptor Potential Vanilloid Type-1 Channel Prevents Adipogenesis and Obesity

doi: 10.1161/01.res.0000262653.84850.8b

Figure Lengend Snippet: Figure 4. The TRPV1 channel agonist capsaicin prevents adipocyte hypertro- phy and the reduction of TRPV1 expres- sion in visceral adipose tissue from mice on high fat diet. a, Representative cross sections of visceral adipose tissue (vis fat), subcutaneous fat (sc fat), and brown fat (br fat) from control mice, mice on high fat diet, control mice plus capsaicin, and mice on high fat diet plus capsaicin. Scale bar indicates 10 m; magnifica- tion, 400. Summary data of adipocyte size of visceral adipose tissue (b), subcu- taneous fat (c), and brown fat (d) in mice. ***P0.001 for the comparison high fat vs control; P0.001 for the com- parison high fat plus capsaicin vs high fat. e, Expression of TRPV1 in visceral adipose tissue (vis fat) from mice. ***P0.001 for the comparison high fat vs control; P0.001 for the com- parison high fat plus capsaicin vs high fat. f, Representative agarose gel electro- phoresis of PCR products from mRNA of TRPV1 and GAPDH genes in visceral adipose tissue mice M denotes bp marker. Panels are representative of 3 separate experiments showing similar results.

Article Snippet: In-cell Western assay of TRPV1 channels human visceral adipose tissue was performed using the Odyssey infrared imaging system (Licor biosciences) as described by our group.9 Primary rabbit anti- human TRPV1-antibodies were obtained from Alomone labs (Jerusalem, Israel) and secondary IRDye800CW-infrared fluorescent dye-conjugated goat anti-rabbit antibodies were obtained from Biomol (Hamburg, Germany).

Techniques: Control, Comparison, Expressing, Agarose Gel Electrophoresis, Marker

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a WB analysis and quantification of CDK9 protein levels in MV4–11 cells treated with 200 nM DbTACs with different linker lengths (DbTACs-8, −11, −16, −21, −26, and −57 Å) and a positive compound B11 for 6 h. GAPDH was used as a loading control. An unpaired two-tailed t -test was used to evaluate statistical significance. **** P < 0.0001 (Control vs. DbTACs-26 Å), n.s. represents no significance. The error bars indicate the mean ± SD values; n = 3. b Immunofluorescence staining images of human hepatoma cells (HepG2) treated with 200 nM DbTACs with different linker lengths or control for 6 h. The nuclei were stained with DAPI in blue, and CDK9 protein was stained in green. The red dotted square in the merged layer indicates an individual cell at a higher magnification. Scale bars, 20 and 3 μm, respectively. c Concentration-dependent degradation and d time degradation characteristics of CDK9 by representative DbTACs-26 Å analyzed by WB. GAPDH was used as a loading control. An unpaired two-tailed t -test was used to evaluate statistical significance. ** P = 0.0083, *** P = 0.0009, **** P < 0.0001 (Control vs. 6 h+ and Control vs. 12h+). The error bars indicate the mean ± SD values; n = 3.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Control, Two Tailed Test, Immunofluorescence, Staining, Concentration Assay

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Live-cell imaging was performed to visualize the real-time localization of CDK9 in HEK293T cells and to track the decrease in CDK9 after treatment with DbTACs-26 Å for 6 h. The scale bars, 40 μm. b SEC-HPLC analysis of retention time of DbTACs-26 Å after incubation with human recombinant CDK9 or CRBN protein or both. c Molecular docking sites of the ternary complex in an all-atom model. SPR sensorgrams were employed to monitor the interaction between e DbTACs-26 Å (binary complexes) or d DbTACs-26 Å, f DbTACs-8 Å, and g DbTACs-57 Å preincubated with human recombinant CRBN protein (ternary complexes) and immobilized CDK9 protein. h WB analysis of the selective degradation potency of DbTACs-26 Å in MV4–11 cells. The cells were co-incubated with different treatments and collected after 6 h. i A ligand competition test for the degradation of the target protein by CDK9 degrader DbTACs-26 Å. Inhibitors of CDK9, CRBN, and proteasome (BAY., P.M., and MG132, respectively) were used, and all signals of each band were normalized successively to GAPDH.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Live Cell Imaging, Incubation, Recombinant

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Volcano plot showing fold changes of protein abundance from global proteomics analysis of MV4–11 cells treated with DbTACs-26 Å for 6 h at 200 nM. Statistical test ( t -test analysis). b Subcellular localization prediction of identified proteins using WoLFPSORT. The subcellular localization of identified proteins was predicated using the WoLFPSORT database with amino acid sequences of identified proteins. c Gene ontology (GO) analysis of a cellular component of samples between DbTACs-26 Å-treated group and control group. Statistical test (Fisher’s exact test). Molecular function analysis of compound DbTACs-26 Å-treated group and control group. Cluster analysis of d biological process, e molecular function, and f KEGG pathway of samples between DbTACs-26 Å-treated group and control group. n = 3. Statistical test (Fisher’s exact test). g Protein–protein interaction network analysis of CDK9 with other proteins.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Quantitative Proteomics, Control

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Schematic illustration of bis-DbTACs design, which is based on DbTACs. b Schematic illustration of three ligand covalent sites of bis-DbTACs equivalent to a DNA tetrahedral scaffold with three polyA domains. Au NPs (5, 10, and 15 nm) correspond to CRBN, CDK9, and CDK6 ligands, respectively. c Cartoon and representative TEM images of bis-DbTACs equivalents. Scale bars are 75 Å and 200 Å, respectively. d WB analysis of the selectively targeted degradation ability of bis-DbTACs at different concentrations and semiquantitative analysis of the grayscale. The error bars indicate the mean ± SD values; n = 3. e Immunofluorescence double-staining images of HepG2 cells treated with/without bis-DbTACs were recorded by confocal laser scanning microscopy. The cell nucleus was stained with DAPI. CDK6 and CDK9 proteins were labeled with anti-CDK6 and anti-CDK9 antibodies, respectively. Scale bars, 10 μm.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Immunofluorescence, Double Staining, Confocal Laser Scanning Microscopy, Staining, Labeling

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Strategy for designing Abs-DbTACs using CDK9 antibody as the POI ligand. b Self-assembly process of Abs-DbTACs was analyzed by agarose gel electrophoresis. The preparation of Abs-DbTACs was verified by c UV‒visible spectra and d SEC-HPLC. e WB analysis of the targeted CDK9 degradation ability of Abs-DbTACs at different concentrations in MOLM13 cells. A free CDK9 antibody was chosen as the negative control (NC). A semi-quantitative analysis of their grayscale values was performed. The error bars indicate the mean ± SD values; n = 3. An unpaired two-tailed t -test was used to evaluate statistical significance. ** P = 0.0016, *** P = 0.0002, **** P < 0.0001 (Control vs. 191 nM Abs-DbTACs and Control vs. 497 nM Abs-DbTACs), n.s. represents no significance.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Agarose Gel Electrophoresis, Negative Control, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: 3 channel colocalization images of a U2OS cell stained with Lysotracker blue, Magic-Red, and Oregon Green 488-dextran. Lysotracker blue and Magic Red stain cell’s acidic compartments and Cathepsin-B positive compartments, respectively.

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Staining

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: Lysosomes swollen by PIKfyve inhibition are hyperacidic. (A) Protocol timeline. U2OS cells were “lysosome-loaded” with Oregon Green 488 dextran (OG loading) and treated for 3h with PIKfyve inhibitor apilimod (100nM, red) or its vehicle (0.25% DMSO, control) before imaging. (B - E) A representative experiment. (B) Images of cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes in control (left) versus apilimod (right) conditions. Dotted lines delineate cell outlines. (C) pH calibration curves obtained in control (grey empty circle) or apilimod (red triangle) conditions. Each symbol represents the averaged lysosomal 488/445 ratio of one cell (4 cells per condition). (D) Lysosomal pH measured in control (left, grey empty circles, 16 cells, 4.40 ± 0.02) versus 100 μM apilimod (red triangles, 17 cells, 4.12 ± 0.03) for an individual experiment. Dark symbols represent averages over all cells in the experiment. Each pale symbol represents the averaged lysosomal pH of one cell. Unpaired t-test: p<0.0001. (E) Cumulative distribution of individual lysosomal pH for control (grey empty circle, 16 cells, 732 lysosomes) versus apilimod (red triangle, 17 cells, 628 lysosomes) conditions from the same experiment as in . Each symbol represents the fraction of lysosomes having a pH value below that represented in the abscissa. (F) Apilimod-induced pH shifts (−0.31 ± 0.04) from multiple independent experiments and corresponding fold changes in proton concentration (2.03 ± 0.17); 10 experiments (8-17 cells per condition in each experiment). Proton concentration change ([H + ] change ) was calculated from apilimod-induced pH shift (ΔpH) using the following relation: [H + ] change = 10 −ΔpH Paired t-test: p<0,0001. Dark symbols represent averages over all independent experiments. Each pale symbol represents the averaged lysosomal pH from multiple cells in one experiment. The dot delineated by a black box corresponds to the experiment presented in panels B-E. Data are displayed as mean ± SEM.

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Inhibition, Control, Imaging, Concentration Assay

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Protocol timeline to analyze the evolution of lysosomal pH and size during PIKfyve inhibition. U2OS cells were “lysosome-loaded” with OG then treated for 30min, 1h, 3h or 24h with apilimod (100nM, red) or its vehicle (0.25% DMSO, control) before imaging. ( B ) Representative images of cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes. Dotted lines delineate cell outlines. ( C - D ) Comparison of lysosomal pH ( C ) and size ( D ) in control (grey empty circle) and after different apilimod treatment times (red triangle). Each symbol represents the averaged lysosomal pH of one experiment (4 independent experiments, 10-15 cells per condition per experiment). Red dashed line in ( C ) represents the average pH value of 1h to 24h apilimod time points. P-values are obtained from one-way ANOVA Dunnett’s multiple comparisons test. ( E ) Comparison of lysosomal pH in control condition (grey empty circle) and after different treatment times with the PIKfyve inhibitor WX8 (1μM, blue triangle). Each symbol represents the averaged lysosomal pH of one experiment (2 experiments, 10-13 cells per condition per experiment). Blue dashed line represents the average value of 1h to 24h WX8 time points. ( F ) Protocol timeline to analyze the recovery of lysosomal pH and size after washout of apilimod. U2OS cells were “lysosome-loaded” with OG, treated 2h with 100nM apilimod or its vehicle (0.25% DMSO, control) to induce lysosomal hyperacidification and swelling. Apilimod was subsequently washed out with fresh media and imaged 30min, 1h, 3h, 6h and 24h after the washout. ( G ) Representative images of cells acquired using 445nm laser excitation. Bright objects represent OG positive lysosomes. Dotted lines delineate cell outlines. The orange box at 30min time point highlights lysosomal tubulation events. ( H - I ) Comparison of lysosomal pH ( H ) and size ( I ) in control condition (grey empty circle), after 2h apilimod treatment (black triangle) and at different times after apilimod washout (red triangle). Each symbol represents the average lysosomal pH of one experiment (3-7 experiments, 8-15 cells per condition per experiment).

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Inhibition, Control, Imaging, Comparison

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Protocol timeline. U2OS cells were “lysosome-loaded” with OG and pre-treated 30min with chloroquine (12μM, red), BafA1 (100nM, blue) or a vehicle (0.31% DMSO, grey), before 3h treatment with apilimod (100nM, bottom images) or a vehicle (control, DMSO 0.31%, upper images). ( B ) Representative images of cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes. Dotted lines delineate cells outlines. ( C , D ) Comparison of lysosomal pH ( C ) or size ( D ) in control (grey), chloroquine (chloro., red) and BafA1 (bafilo., blue) conditions during untreated (api. -, empty symbols) versus apilimod treated (api. +, filled symbols) conditions. Each symbol represents the averaged lysosomal pH or size of one experiment (4 experiments; 8-11 cells per condition). In (C) Apilimod induces a lysosomal pH shift to more acidic value in chloroquine condition (Paired t-test, p=0.02).

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Protocol timeline. U2OS cells were transfected with either mCherry alone, mCherry and PIKfyve, or mCherry and PIKfyve KYA and subsequently “lysosome-loaded” with OG. Cells were imaged after 24h expression to quantify lysosomal pH and size. ( B ) Western-blot indicating PIKfyve or PIKfyve KYA expression level for each condition after 24h expression. Note that PIKfyve endogenous level (control) was too low to be detected. ( C ) Representative cells imaged by 445nm laser excitation (OG channel, left image) and 640nm laser excitation (mCherry channel, right image). The red arrow indicates one cell containing lysosomes loaded with OG (bright dots in OG channel) and expressing mCherry (bright cell in mCherry channel). The white arrow indicates one cell from the same batch containing lysosomes filled with OG but not expressing mCherry. Dotted lines delineate cell shape. ( D , E ) Comparison of lysosomal pH ( D ) or size ( E ) in control (circle), PIKfyve-transfected (triangle) and PIKfyve KYA -transfected (diamond) conditions. For each condition, cells were separated into two populations based on the presence or absence of mCherry. Each symbol represents the averaged lysosomal pH or size of one experiment (6 experiments; 8-11 cells per condition per experiment). P-values: Paired t-test between PIKfyve-mCherry and PIKfyve KYA -mCherry conditions.

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Transfection, Expressing, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Agarose gel electrophoresis of PCR product amplified from ClCN7 deletion site of wild type (ClC7 Wt, left) and CLCN7 knock-out (ClC-7 KO, right) U2OS cells. ( B ) EXON1 sequence from CLCN7 WT (black) and KO (purple and teal) alleles. ( C ) Protocol timeline. Cells were “lysosome-loaded” with OG and treated for 3h with apilimod (100nM, red) or its vehicle (0.25% DMSO, control) before imaging. ( D ) Images from a representative experiment: ClC-7 WT (left) or ClC-7 KO (right) cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes in control (top) versus apilimod (bottom) conditions. Dotted lines delineate cell outlines. ( E , F ) Lysosomal pH ( E ) or size ( F ) from ClC-7 WT (grey circle) or ClC-7 KO (red triangle) cells in apilimod (filled symbols) versus control (empty symbols) in a representative experiment. Dark symbols are averages over all cells; each pale symbol represents the average lysosomal pH from one cell. P-values for apilimod effects are obtained from two-way ANOVA. ( G ) There is no significant difference in pH between untreated WT and untreated KO cells (P=0.5675, unpaired t-test). For G, H, I, dark symbols are averages over all experiments; each pale symbol represents the averaged lysosomal pH or size from one experiment (10 and 6 independent experiments for WT and KO conditions, respectively; each experiment represents 8-18 cells per condition). ( H , I ) Comparison of lysosomal pH-shift ( H ) or size-shift ( I ) induced by apilimod treatment in ClC-7 Wt (grey) versus ClC-7 KO (red) cells. Proton concentration change ( H , [H + ] change ) was calculated from apilimod-induced pH shift (ΔpH) using the following relation: [H + ] change = 10 −ΔpH . P-values from unpaired t-test.

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Agarose Gel Electrophoresis, Amplification, Knock-Out, Sequencing, Control, Imaging, Comparison, Concentration Assay

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) RT-qPCR amplification plot obtained from ClC-7 Wt (grey) and ClC-7 KO (red) U2OS cells whole RNA. GAPDH housekeeping RNA level (left-grey and left-red traces) is used as a control of RNA quantity. ClCN7 RNA level corresponds to right-grey and right-red traces. Δ = 3.5 cycles, indicating a 90% decrease in KO ClC-7 RNA level compared to Wt. ( B ) Agarose gel electrophoresis of qPCR product from ClC-7 Wt (grey) and ClC-7 KO (red) U2OS cells GAPDH or CLCN7 cDNA.

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Quantitative RT-PCR, Amplification, Control, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: Left: pH calibration curves of U2OS ClC-7 Wt (WT) or ClC-7 KO (KO) lysosomes loaded with OG in untreated (control) or 3h apilimod-treated conditions (apilimod). Each dot represents the averaged lysosomal 488/445 ratio of one cell (4 cells per condition). Right: Comparison of cumulative distribution of U2OS ClC-7 Wt (grey, control, 9 cells, 427 objects) versus ClC-7 KO (blue, 10 cells, 548 objects) lysosomal pH population. Each symbol represents the proportion of lysosomes having a pH value below the pH value represented in the abscissa axis.

Article Snippet: U2OS cells (HTB-96 TM ) were purchased from ATCC and cultured in McCoy’s 5A Modified Medium supplemented with 10% FBS and 100U.mL −1 Penicillin-Streptomycin (all from gibco) at 37 °C in 5 % CO 2 and 10 % humidity.

Techniques: Control, Comparison