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Addgene inc 65974 vsv g expressing pmd2 g plasmid didier trono lab
65974 Vsv G Expressing Pmd2 G Plasmid Didier Trono Lab, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcaggs+expression+plasmids/pm41348541-758-252-262?v=Addgene+inc
Average 93 stars, based on 37 article reviews

65974 vsv g expressing pmd2 g plasmid didier trono lab - by Bioz Stars, 2026-06

93/100 stars

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(A) KALRN-depleted TM cells transfected with c-Myc–tagged KALRN-7 or KALRN-12 (TM219) showed successful isoform expression (green: c-Myc, red: KALRN). Scale bar, 50μm. (B) ZO-1 immunostaining (green) decreased at cell-cell junctions in KALRN-7 and KALRN-12 restored cells (c-Myc–positive cells, red). Scale bars, 50μm. (C) Quantification confirmed reduction in cell area after re-expression of KALRN-7 and −12 isoforms (n=30-50 cells per group; Kruskal–Wallis test; Mean areas: si-Control: 1868 pixel², si-KALRN: 7242 pixel², si-KALRN + plasmid KALRN-7 (pK7): 3716 pixel², si-KALRN + plasmid KALRN-12 (pK12): 4603 pixel²). (D) Chi-square analysis showed the changed junctional ZO-1 patterns in the cells with re-expression of KALRN-7 and −12 isoforms. TM219; si-KALRN=76 cells; si-KALRN + pK7=18 cells; si-KALRN + pK12=10 cells. (E-H) FLIM–FRET analysis showed reduced Rac1 activity and increased RhoA activation in KALRN-depleted cells (TM219). In Rac1 biosensor–expressing cells, warmer colours (shorter lifetimes) indicate higher activity. KALRN depletion reduced Rac1 activity in the basal half of the cells, as shown by cooler colour shifts. RhoA activity increased at the basal side, indicated by warmer colours (Kruskal-Wallis tests; Rac1, n=13 per group; RhoA, n=10 per group). (I) Inhibition of Rac using NSC23766 in TM cells reproduced features of KALRN loss, including decreased pp-MLC2 staining (red), thicker F-actin filaments (green), and increased ZO-1 staining at cell-cell junction area. TM219. Scale bar, 50μm. (J) Rac inhibition increased cell area (TM219; control: n=76 cells, NSC: n=55 cells from 5 images; Mann–Whitney test). (K) Percentage of TM cells with ZO-1 type II distribution were elevated following Rac inhibition (n=4 images, TM219; unpaired t-test).

Journal: bioRxiv

Article Title: Interdependent regulation of trabecular meshwork cell physiology and intraocular pressure by KALRN and TMCO1

doi: 10.1101/2025.09.19.677310

Figure Lengend Snippet: (A) KALRN-depleted TM cells transfected with c-Myc–tagged KALRN-7 or KALRN-12 (TM219) showed successful isoform expression (green: c-Myc, red: KALRN). Scale bar, 50μm. (B) ZO-1 immunostaining (green) decreased at cell-cell junctions in KALRN-7 and KALRN-12 restored cells (c-Myc–positive cells, red). Scale bars, 50μm. (C) Quantification confirmed reduction in cell area after re-expression of KALRN-7 and −12 isoforms (n=30-50 cells per group; Kruskal–Wallis test; Mean areas: si-Control: 1868 pixel², si-KALRN: 7242 pixel², si-KALRN + plasmid KALRN-7 (pK7): 3716 pixel², si-KALRN + plasmid KALRN-12 (pK12): 4603 pixel²). (D) Chi-square analysis showed the changed junctional ZO-1 patterns in the cells with re-expression of KALRN-7 and −12 isoforms. TM219; si-KALRN=76 cells; si-KALRN + pK7=18 cells; si-KALRN + pK12=10 cells. (E-H) FLIM–FRET analysis showed reduced Rac1 activity and increased RhoA activation in KALRN-depleted cells (TM219). In Rac1 biosensor–expressing cells, warmer colours (shorter lifetimes) indicate higher activity. KALRN depletion reduced Rac1 activity in the basal half of the cells, as shown by cooler colour shifts. RhoA activity increased at the basal side, indicated by warmer colours (Kruskal-Wallis tests; Rac1, n=13 per group; RhoA, n=10 per group). (I) Inhibition of Rac using NSC23766 in TM cells reproduced features of KALRN loss, including decreased pp-MLC2 staining (red), thicker F-actin filaments (green), and increased ZO-1 staining at cell-cell junction area. TM219. Scale bar, 50μm. (J) Rac inhibition increased cell area (TM219; control: n=76 cells, NSC: n=55 cells from 5 images; Mann–Whitney test). (K) Percentage of TM cells with ZO-1 type II distribution were elevated following Rac inhibition (n=4 images, TM219; unpaired t-test).

Article Snippet: The RhoA biosensor expression plasmid pCAGGS-Raichu-RhoA-CR (Addgene plasmid # 40258; http://n2t.net/addgene:40258 ; RRID:Addgene 40258) and pcDNA3-Clover (Addgene plasmid # 40259; http://n2t.net/addgene:40259 ; RRID: Addgene_40259) were gifts from Michael Lin . pCAGGS-Raichu-Rac1-CR was constructed by replacing the sequence between Clover and mRuby2 in pCAGGS-Raichu-RhoA-CR with the corresponding sequence of pCAGGS-Raichu-Rac1 (kindly provided by Michiyuki Matsuda; Department of Pathology and Biology of Diseases, Kyoto University, Japan).

Techniques: Transfection, Expressing, Immunostaining, Control, Plasmid Preparation, Activity Assay, Activation Assay, Inhibition, Staining, MANN-WHITNEY

Cell–cell fusion assays and recombinant envelope and receptor constructs. ( A ). Cell–cell fusion assays involved transfection of one set of cells with an Env expression construct (in red) plus a PM-targeted split GFP helix 1–10 expression construct (pcDNA3.1-PLCdeltaPH-GFP-H1-10; green), and transfection of a separate set of cells with expression vectors for CD4 (blue) and CXCR4 (pink) variants, plus a PM-targeted split GFP helix 11 expression construct (For pGFP-H11-PLCdeltaPH; green). One day post-transfection cells were seeded together onto coverslips, and scored for GFP+ syncytia two days later. ( B ). HIV-1 Env expression constructs included one that produced the WT HIV-1 NL4-3 envelope protein (Env-WT: pMDG-NL43-Env-WT or SVIII-Env-WT) and one that produced an Env protein (Env-753stop: pMDG-NL43-Env-753stop), truncated at Env residue 753, before the CT amphipathic helices. CD4 expression constructs included one that expressed a Myc-tagged WT human CD4 protein (CD4: pCAGGS-CD4-Myc) or a variant in which the CD4 transmembrane and cytoplasmic tail (TMCT) domains were replaced with a GPI anchor (CD4-GPI: pCAGGS-CD4-GPI). CXCR4 expression constructs included a WT human CXCR4 expression construct (CXCR4: pcDNA3-CXCR4), constructs with partial (CXCR4-318: pcDNA3-CXCR4-318Stop) or complete (CXCR4-309: pcDNA3-CXCR4-309Stop) CXCR4 cytoplasmic tail (CT) deletions, and constructs in which CT truncations were fused to the PLCδPH that preferentially binds to PI(4,5)P2 (CXCR4-318-PH and CXCR4-309-PH).

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Cell–cell fusion assays and recombinant envelope and receptor constructs. ( A ). Cell–cell fusion assays involved transfection of one set of cells with an Env expression construct (in red) plus a PM-targeted split GFP helix 1–10 expression construct (pcDNA3.1-PLCdeltaPH-GFP-H1-10; green), and transfection of a separate set of cells with expression vectors for CD4 (blue) and CXCR4 (pink) variants, plus a PM-targeted split GFP helix 11 expression construct (For pGFP-H11-PLCdeltaPH; green). One day post-transfection cells were seeded together onto coverslips, and scored for GFP+ syncytia two days later. ( B ). HIV-1 Env expression constructs included one that produced the WT HIV-1 NL4-3 envelope protein (Env-WT: pMDG-NL43-Env-WT or SVIII-Env-WT) and one that produced an Env protein (Env-753stop: pMDG-NL43-Env-753stop), truncated at Env residue 753, before the CT amphipathic helices. CD4 expression constructs included one that expressed a Myc-tagged WT human CD4 protein (CD4: pCAGGS-CD4-Myc) or a variant in which the CD4 transmembrane and cytoplasmic tail (TMCT) domains were replaced with a GPI anchor (CD4-GPI: pCAGGS-CD4-GPI). CXCR4 expression constructs included a WT human CXCR4 expression construct (CXCR4: pcDNA3-CXCR4), constructs with partial (CXCR4-318: pcDNA3-CXCR4-318Stop) or complete (CXCR4-309: pcDNA3-CXCR4-309Stop) CXCR4 cytoplasmic tail (CT) deletions, and constructs in which CT truncations were fused to the PLCδPH that preferentially binds to PI(4,5)P2 (CXCR4-318-PH and CXCR4-309-PH).

Article Snippet: The lentivirus GagPol packaging plasmid (psPAX2), the GFP expression plasmids (GFP-PS, GFP-NoPS, GFP-C1-PLCdelta-PH, pEGFP-GFP11-ClathrinLightChain, pcDNA3.1-GFP [ , , , , , , , , , ]), the VSV G expression vector pMD.G, the CD4 expression plasmid pCAGGS-CD4-Myc, and the CXCR4 expression plasmid pcDNA3-CXCR4 were all obtained from Addgene, and were the gifts of Drs. Didier Trono, Jennifer Doudna, Tobias Meyer, Bo Huang, Simon Davis, Erik Procko, and Jacob Yount [ , , , , , , ].

Techniques: Recombinant, Construct, Transfection, Expressing, Produced, Residue, Variant Assay

HIV-1 Env cell–cell fusion assays. Cell–cell fusion assays were performed with 293 cells as illustrated in . Panels ( A – F ) show coincubations of cells transfected with pGFP-H11-PLCdeltaPH plus CD4-GPI plus CXCR4-318 mixed with cells transfected with pcDNA3.1-PLCdeltaPH-GFP-H1-10 plus either Env-WT ( A , B ), Env-753Stop ( C , D ), or a mock ( E , F ). Panels ( A – F ) are matched photos imaged for DAPI nuclear stain ( A , C , E ) and GFP ( B , D , F ). Note that the size bar (panel ( F )) pertains to all fluorescent images ( A – F ). In panel ( G ), numbers of syncytia per 0.4 mm 2 are averaged with standard deviations from five images each of coincubations of cells co-transfected with split GFP constructs, plus the indicated Env or CD4 plus CXCR4 expression construct variants. Note that numbers of syncytia for Env-753 were approximately tenfold higher than syncytia for Env-WT, and that syncytia for Env-WT were approximately twenty-fold higher than syncytia for the Env-minus (No Env) control. Differences between the sets were highly significant ( p < 0.001), and were obtained by conversion of means and standard deviations to Z values for probability calculations.

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: HIV-1 Env cell–cell fusion assays. Cell–cell fusion assays were performed with 293 cells as illustrated in . Panels ( A – F ) show coincubations of cells transfected with pGFP-H11-PLCdeltaPH plus CD4-GPI plus CXCR4-318 mixed with cells transfected with pcDNA3.1-PLCdeltaPH-GFP-H1-10 plus either Env-WT ( A , B ), Env-753Stop ( C , D ), or a mock ( E , F ). Panels ( A – F ) are matched photos imaged for DAPI nuclear stain ( A , C , E ) and GFP ( B , D , F ). Note that the size bar (panel ( F )) pertains to all fluorescent images ( A – F ). In panel ( G ), numbers of syncytia per 0.4 mm 2 are averaged with standard deviations from five images each of coincubations of cells co-transfected with split GFP constructs, plus the indicated Env or CD4 plus CXCR4 expression construct variants. Note that numbers of syncytia for Env-753 were approximately tenfold higher than syncytia for Env-WT, and that syncytia for Env-WT were approximately twenty-fold higher than syncytia for the Env-minus (No Env) control. Differences between the sets were highly significant ( p < 0.001), and were obtained by conversion of means and standard deviations to Z values for probability calculations.

Techniques: Transfection, Staining, Construct, Expressing, Control

Infection of Env-expressing cells via fusion in reverse. ( A ) Target 293 cells expressing different Env variants are infected with a β-gal-transducing, HIV-1-based, lentivirus vector pseudotyped with a GPI-anchored CD4 variant (CD4-GPI) plus a C-terminally-truncated CXCR4 protein (CXCR4-318). Receptor plus co-receptor binding to cellularly expressed Env activate the Env fusion machinery, resulting in virus infection. ( B ) Shown are β-gal activity-derived infectivities of cells expressing Env-753Stop, Env-WT, or no Env (mock), normalized to the Env-753Stop cells. Note that results are averaged from five independent experiments each, are plotted on a log scale graph to facilitate comparison, and that standard deviations for the Env-753Stop and mock cells were too small to be observed on the graph. ( C ) Cells expressing the indicated Env proteins were infected with CD4-GPI plus CXCR4-318 pseudotyped β-gal-transducing HIV-1 lentivirus vectors in the absence (753, WT) or presence (753 + T20, WT + T20) of HIV-1 Env fusion inhibitor T20. Results for pairs of infections are normalized to the infectivities of viruses in the absence of T20. Averages and standard deviations derive from three separate infections, and differences between 753 and WT and WT and mock were highly significant ( p < 0.001), as were differences between untreated and treated samples. Probabilities were obtained by conversion of means and standard deviations to Z values, which were used for calculations.

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of Env-expressing cells via fusion in reverse. ( A ) Target 293 cells expressing different Env variants are infected with a β-gal-transducing, HIV-1-based, lentivirus vector pseudotyped with a GPI-anchored CD4 variant (CD4-GPI) plus a C-terminally-truncated CXCR4 protein (CXCR4-318). Receptor plus co-receptor binding to cellularly expressed Env activate the Env fusion machinery, resulting in virus infection. ( B ) Shown are β-gal activity-derived infectivities of cells expressing Env-753Stop, Env-WT, or no Env (mock), normalized to the Env-753Stop cells. Note that results are averaged from five independent experiments each, are plotted on a log scale graph to facilitate comparison, and that standard deviations for the Env-753Stop and mock cells were too small to be observed on the graph. ( C ) Cells expressing the indicated Env proteins were infected with CD4-GPI plus CXCR4-318 pseudotyped β-gal-transducing HIV-1 lentivirus vectors in the absence (753, WT) or presence (753 + T20, WT + T20) of HIV-1 Env fusion inhibitor T20. Results for pairs of infections are normalized to the infectivities of viruses in the absence of T20. Averages and standard deviations derive from three separate infections, and differences between 753 and WT and WT and mock were highly significant ( p < 0.001), as were differences between untreated and treated samples. Probabilities were obtained by conversion of means and standard deviations to Z values, which were used for calculations.

Techniques: Infection, Expressing, Plasmid Preparation, Variant Assay, Binding Assay, Virus, Activity Assay, Derivative Assay, Comparison

Fusion in reverse infectivities for different Env, receptor, and co-receptor combinations. 293 cells expressing the 753Stop ( A ) or WT HIV-1 Env ( B ) were infected with equivalent volumes of β-gal-transducing HIV-1 particles carrying the indicated receptor plus co-receptor combinations (CD4, CD4-GPI [gpi],—[mock], CXCR4, 318 [CXCR4-318], 309 [CXCR4-309], 318PH [CXCR4-318-PH], 309PH [CXCR4-309-PH]). At 72 h post-infection, cells were subjected to β-gal assays and β-gal activities are plotted as infectivities relative to viruses carrying the CD4-GPI/CXCR4-318 receptor/co-receptor combinations. Averages and standard deviations derive from the following numbers of experiments: 753/gpi/318, 9; 753/gpi/-, 2; 753/-/318, 2; 753/CD4/325, 2; 753/gpi/CXCR4, 4; 753/gpi/309, 3; 753/gpi/318PH, 5; 753/gpi/309PH, 5; WT/gpi/318, 9; WT/gpi/-, 2; WT/-/318, 2; WT/CD4/318, 3; WT/gpi/CXCR4, 6; WT/gpi/309, 4; WT/gpi/318PH, 5; WT/gpi/309PH, 5. Note that differences between the gpi/318 samples versus the gpi/-, -/318, and CD4/318 samples had probabilities of p < 0.001, as determined by conversion of means and standard deviations to Z values for probability calculations.

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Fusion in reverse infectivities for different Env, receptor, and co-receptor combinations. 293 cells expressing the 753Stop ( A ) or WT HIV-1 Env ( B ) were infected with equivalent volumes of β-gal-transducing HIV-1 particles carrying the indicated receptor plus co-receptor combinations (CD4, CD4-GPI [gpi],—[mock], CXCR4, 318 [CXCR4-318], 309 [CXCR4-309], 318PH [CXCR4-318-PH], 309PH [CXCR4-309-PH]). At 72 h post-infection, cells were subjected to β-gal assays and β-gal activities are plotted as infectivities relative to viruses carrying the CD4-GPI/CXCR4-318 receptor/co-receptor combinations. Averages and standard deviations derive from the following numbers of experiments: 753/gpi/318, 9; 753/gpi/-, 2; 753/-/318, 2; 753/CD4/325, 2; 753/gpi/CXCR4, 4; 753/gpi/309, 3; 753/gpi/318PH, 5; 753/gpi/309PH, 5; WT/gpi/318, 9; WT/gpi/-, 2; WT/-/318, 2; WT/CD4/318, 3; WT/gpi/CXCR4, 6; WT/gpi/309, 4; WT/gpi/318PH, 5; WT/gpi/309PH, 5. Note that differences between the gpi/318 samples versus the gpi/-, -/318, and CD4/318 samples had probabilities of p < 0.001, as determined by conversion of means and standard deviations to Z values for probability calculations.

Techniques: Expressing, Infection

CD4 incorporation into virions. ( A ). Media samples from 293 cells transfected with psPAX2 (psPAX) plus the CD4 or CD4-gpi expression constructs were pelleted through 20% sucrose cushions, separated by electrophoresis and immunoblotted for detection of CA and CD4 immunoreactive proteins. PrGag, p41 (Gag intermediate), CA, and CD4 proteins were identified based on their immunoreactivities and migration mobilities relative to protein molecular weight standards run in parallel. ( B , C ). Cell samples (top panels) and media samples that were pelleted through 30% sucrose cushions (bottom panels) were collected from cells transfected with the indicated constructs, separated by electrophoresis, and immunoblotted for detection of PrGag, p41 (Gag intermediate) and CA ( B ) or CD4 and CD4-gpi ( C ). Note that viral CD4-to-CA ratios (expressed as percentages) were 33% for ( A ), and 25% for ( B , C ); while viral CD4-gpi-to-CA ratios were 26% for ( A ), and 34% for ( B , C ).

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: CD4 incorporation into virions. ( A ). Media samples from 293 cells transfected with psPAX2 (psPAX) plus the CD4 or CD4-gpi expression constructs were pelleted through 20% sucrose cushions, separated by electrophoresis and immunoblotted for detection of CA and CD4 immunoreactive proteins. PrGag, p41 (Gag intermediate), CA, and CD4 proteins were identified based on their immunoreactivities and migration mobilities relative to protein molecular weight standards run in parallel. ( B , C ). Cell samples (top panels) and media samples that were pelleted through 30% sucrose cushions (bottom panels) were collected from cells transfected with the indicated constructs, separated by electrophoresis, and immunoblotted for detection of PrGag, p41 (Gag intermediate) and CA ( B ) or CD4 and CD4-gpi ( C ). Note that viral CD4-to-CA ratios (expressed as percentages) were 33% for ( A ), and 25% for ( B , C ); while viral CD4-gpi-to-CA ratios were 26% for ( A ), and 34% for ( B , C ).

Techniques: Transfection, Expressing, Construct, Electrophoresis, Migration, Molecular Weight

Infection of HIV-1-infected cells. Jurkat T cells, HIV-1-infected J1.1 Jurkat cells, and J1.1 cells induced with tumor necrosis factor alpha (J1.1 + TNFa) were subjected to immunoblot detection of Gag proteins ( A ) with an anti-CA antibody, immunoblot detection of Env proteins ( B ) with an anti-gp41(TM) antibody, and infection with a lentivirus vector ( C ) generated by transfection of 293 cells with psPAX2, pLVX-puro-Xho-ATG-β-gal, CD4-GPI, and CXCR4-328 plasmids. ( A , B ) Migration positions of PrGag, CA, gp160 and gp41 proteins are indicated, as are molecular weight standards (in kDa) that were electrophoresced in parallel. ( C ) Infectivity results represent normalized transduced β-gal activity readings normalized to results for induced J1.1 cells. Averages and standard deviations derive from duplicate (Jurkat, J1.1) or triplicate (J1.1 + TNFα) infections, and differences between Jurkat and J1.1, as well as J1.1 versus J1.1 + TNFα were highly significant ( p < 0.001), as determined by conversion of means and standard deviations to Z values that were used in probability calculations.

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of HIV-1-infected cells. Jurkat T cells, HIV-1-infected J1.1 Jurkat cells, and J1.1 cells induced with tumor necrosis factor alpha (J1.1 + TNFa) were subjected to immunoblot detection of Gag proteins ( A ) with an anti-CA antibody, immunoblot detection of Env proteins ( B ) with an anti-gp41(TM) antibody, and infection with a lentivirus vector ( C ) generated by transfection of 293 cells with psPAX2, pLVX-puro-Xho-ATG-β-gal, CD4-GPI, and CXCR4-328 plasmids. ( A , B ) Migration positions of PrGag, CA, gp160 and gp41 proteins are indicated, as are molecular weight standards (in kDa) that were electrophoresced in parallel. ( C ) Infectivity results represent normalized transduced β-gal activity readings normalized to results for induced J1.1 cells. Averages and standard deviations derive from duplicate (Jurkat, J1.1) or triplicate (J1.1 + TNFα) infections, and differences between Jurkat and J1.1, as well as J1.1 versus J1.1 + TNFα were highly significant ( p < 0.001), as determined by conversion of means and standard deviations to Z values that were used in probability calculations.

Techniques: Infection, Western Blot, Plasmid Preparation, Generated, Transfection, Migration, Molecular Weight, Activity Assay

Effects of lipid variations on fusion in reverse infections. ( A ) The HIV-1 viral membrane is highly enriched in cholesterol (CHOL), and has substantial amounts of ceramide (CER). The inner leaflets are enriched for phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2; PIP2), and phosphatidylethanolamine (PE), while the outer leaflet is enriched in sphingomyelin (SM), hexosylceramides (HEX), and saturated phosphatidylcholines (PCs). ( B – D ) Infectivities of equivalent volumes of virus were scored via β-gal activities, and were normalized either to untreated (−) samples, or for convenience in the panel ( D ) CD/CX infections, to viruses from CerS−/− cells. In all panels, CD/CX viruses refer to lentivirus vectors generated by co-transfection of 293 cells with a GagPol expression vector (psPAX2), a β-gal-transducing vector (pLVX-puro-Xho-ATG-β-gal), as well as CD4-GPI and CXCR4-318 plasmids. Also, in all panels, Env-WT and Env-753 targets cells refer to 293 cells transfected to express those proteins. For panels ( B , C ), WT and 753 viruses are the NL4-3 variants and were used to infect TZM-bl reporter cells; for panel ( D ), WT refers to viruses produced from cells transfected with psPAX2, pLVX-puro-Xho-ATG-β-gal, plus a WT Env expression vector, while targets were 293 cells transfected to express the CD4-GPI and CXCR4-318 proteins. In panel ( B ), AME refers to treatment of viruses with 10 mM AME prior to infections. In panel ( C ), TMEM refers to co-transfection of virus producing cells with (+) or without (−) the mTMEM-GY scramblase expression vector. In panel ( D ), CerS2−/− pertains to producing viruses either from 293 cells (−) or from 293-CerS2−/− knockout cells (+). For panels ( B – D ), averages and standard deviations are as indicated, noting that where error bars are missing, it is because the standard deviations were too small to be depicted. The following significant differences were obtained by conversion of means and standard deviations to Z values for probability calculations: Panel ( B ) WT/TZMBL, AME −/+, p < 0.001; Panel ( B ) CDCX/Env-WT, AME −/+, p < 0.001; Panel C all pairs p < 0.001; Panel ( D ) CDCX/Env-WT, CerS2−/−, −/+, p < 0.01; all other Panel D pairs, p < 0.001. Note that in panel ( D ), if normalizations were based on CDCX viruses produced from WT 293 cells and infections of Env-753Stop-expressing cells, infectivities of CDCX viruses from virus–cell combinations would be as follows: from WT 293 cells into Env-753Stop cells, 100%; from CerS2−/− cells into Env-753Stop cells 500%; from WT 293 cells into Env-WT cells, 8%; from CerS2−/− cells into Env-WT cells, 19%.

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Effects of lipid variations on fusion in reverse infections. ( A ) The HIV-1 viral membrane is highly enriched in cholesterol (CHOL), and has substantial amounts of ceramide (CER). The inner leaflets are enriched for phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2; PIP2), and phosphatidylethanolamine (PE), while the outer leaflet is enriched in sphingomyelin (SM), hexosylceramides (HEX), and saturated phosphatidylcholines (PCs). ( B – D ) Infectivities of equivalent volumes of virus were scored via β-gal activities, and were normalized either to untreated (−) samples, or for convenience in the panel ( D ) CD/CX infections, to viruses from CerS−/− cells. In all panels, CD/CX viruses refer to lentivirus vectors generated by co-transfection of 293 cells with a GagPol expression vector (psPAX2), a β-gal-transducing vector (pLVX-puro-Xho-ATG-β-gal), as well as CD4-GPI and CXCR4-318 plasmids. Also, in all panels, Env-WT and Env-753 targets cells refer to 293 cells transfected to express those proteins. For panels ( B , C ), WT and 753 viruses are the NL4-3 variants and were used to infect TZM-bl reporter cells; for panel ( D ), WT refers to viruses produced from cells transfected with psPAX2, pLVX-puro-Xho-ATG-β-gal, plus a WT Env expression vector, while targets were 293 cells transfected to express the CD4-GPI and CXCR4-318 proteins. In panel ( B ), AME refers to treatment of viruses with 10 mM AME prior to infections. In panel ( C ), TMEM refers to co-transfection of virus producing cells with (+) or without (−) the mTMEM-GY scramblase expression vector. In panel ( D ), CerS2−/− pertains to producing viruses either from 293 cells (−) or from 293-CerS2−/− knockout cells (+). For panels ( B – D ), averages and standard deviations are as indicated, noting that where error bars are missing, it is because the standard deviations were too small to be depicted. The following significant differences were obtained by conversion of means and standard deviations to Z values for probability calculations: Panel ( B ) WT/TZMBL, AME −/+, p < 0.001; Panel ( B ) CDCX/Env-WT, AME −/+, p < 0.001; Panel C all pairs p < 0.001; Panel ( D ) CDCX/Env-WT, CerS2−/−, −/+, p < 0.01; all other Panel D pairs, p < 0.001. Note that in panel ( D ), if normalizations were based on CDCX viruses produced from WT 293 cells and infections of Env-753Stop-expressing cells, infectivities of CDCX viruses from virus–cell combinations would be as follows: from WT 293 cells into Env-753Stop cells, 100%; from CerS2−/− cells into Env-753Stop cells 500%; from WT 293 cells into Env-WT cells, 8%; from CerS2−/− cells into Env-WT cells, 19%.

Techniques: Membrane, Virus, Generated, Cotransfection, Expressing, Plasmid Preparation, Transfection, Produced, Knock-Out

(A) 293T cells were transfected with nsp5 expression plasmids with FLAG. After 24 h, the subcellular localization of the expressed SARS-CoV-2 nsp5 was examined using anti-FLAG and anti-mouse IgG (H+L)-CF488 as primary and secondary antibodies, respectively. Scale bar = 10 mm. (B, C) Total intracellular proteins were extracted 24 post-transfection, and western blot analysis was performed using anti-FLAG and anti-beta actin antibodies. M represent molecular weight markers. pCAGGS-MCS DNA-transfected samples were used as controls.

Journal: PLOS ONE

Article Title: Amino acid T25 in the substrate-binding domain of SARS-CoV-2 nsp5 is involved in viral replication in the mouse lung

doi: 10.1371/journal.pone.0312800

Figure Lengend Snippet: (A) 293T cells were transfected with nsp5 expression plasmids with FLAG. After 24 h, the subcellular localization of the expressed SARS-CoV-2 nsp5 was examined using anti-FLAG and anti-mouse IgG (H+L)-CF488 as primary and secondary antibodies, respectively. Scale bar = 10 mm. (B, C) Total intracellular proteins were extracted 24 post-transfection, and western blot analysis was performed using anti-FLAG and anti-beta actin antibodies. M represent molecular weight markers. pCAGGS-MCS DNA-transfected samples were used as controls.

Article Snippet: After overnight incubation at 37°C, cells were transfected with 2.0 μg of pCAGGS nsp5 expression plasmid using TransIT-LT1 Transfection Reagent (Mirus Bio).

Techniques: Transfection, Expressing, Western Blot, Molecular Weight

(A) Schematic of the reporter assay to detect the protease activity of SARS-CoV-2 nsp5. The nsp5 cleavage sequence, VTFQS, was inserted between the luciferase genes; in the presence of nsp5, Q and S in the VTFQS sequence are cleaved and luciferase luminescence is detected. (B) nsp5 mutant protease activity detected using the split luciferase assay. 293T cells were transfected with nsp5 expression plasmids with pGlo-VTFQS and pNL1.1 (nanoluciferase-expressing plasmid). Intracellular luciferase activity was measured after 48 h. The luciferase activity in each sample was normalized to the nanoluciferase activity. The values represent the means ±SD derived from three independent experiments. *** P < 0.001. (C) 293T cells were transfected with nsp5-C145A expression plasmid along with pGlo-VTFQS and pNL1.1. Intracellular luciferase activity was measured after 48 h. Each sample’s luciferase activity was normalized against the nanoluciferase activity. The values represent the means ±SD from three independent experiments. **** P < 0.0001, *** P < 0.001. (D) Total intracellular proteins were isolated 24 post-transfection, and subjected to western blot analysis using anti-FLAG and anti-beta actin antibodies. M represents molecular weight markers.

Journal: PLOS ONE

Article Title: Amino acid T25 in the substrate-binding domain of SARS-CoV-2 nsp5 is involved in viral replication in the mouse lung

doi: 10.1371/journal.pone.0312800

Figure Lengend Snippet: (A) Schematic of the reporter assay to detect the protease activity of SARS-CoV-2 nsp5. The nsp5 cleavage sequence, VTFQS, was inserted between the luciferase genes; in the presence of nsp5, Q and S in the VTFQS sequence are cleaved and luciferase luminescence is detected. (B) nsp5 mutant protease activity detected using the split luciferase assay. 293T cells were transfected with nsp5 expression plasmids with pGlo-VTFQS and pNL1.1 (nanoluciferase-expressing plasmid). Intracellular luciferase activity was measured after 48 h. The luciferase activity in each sample was normalized to the nanoluciferase activity. The values represent the means ±SD derived from three independent experiments. *** P < 0.001. (C) 293T cells were transfected with nsp5-C145A expression plasmid along with pGlo-VTFQS and pNL1.1. Intracellular luciferase activity was measured after 48 h. Each sample’s luciferase activity was normalized against the nanoluciferase activity. The values represent the means ±SD from three independent experiments. **** P < 0.0001, *** P < 0.001. (D) Total intracellular proteins were isolated 24 post-transfection, and subjected to western blot analysis using anti-FLAG and anti-beta actin antibodies. M represents molecular weight markers.

Article Snippet: After overnight incubation at 37°C, cells were transfected with 2.0 μg of pCAGGS nsp5 expression plasmid using TransIT-LT1 Transfection Reagent (Mirus Bio).

Techniques: Reporter Assay, Activity Assay, Sequencing, Luciferase, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Derivative Assay, Isolation, Western Blot, Molecular Weight

Growth kinetics of recombinant viruses in VeroE6/hTMPRSS2 cells (A) and 293T/hACE2 cells (B). VeroE6/hTMPRSS2 and 293T/hACE2 cells were inoculated with the recombinant viruses at MOI of 0.0001 and 0.01, respectively. Cells infected with rSARS2-QHmusX-WT or rSARS2-QHmusX-nsp5-T25I were cultured for the indicated time periods. Viral titers in the culture supernatants were determined using the TCID 50 assay. The values represent the means ±SD from three independent experiments. ** P < 0.01. Expression of N RNA from recombinant viruses in VeroE6/hTMPRSS2 cells (C) and 293T/hACE2 cells (D) measured using one-step real-time PCR. ns, no statistically significant difference; h.p.i., hours post-infection. The values represent the means ±SD from three independent experiments. ** P < 0.01.

Journal: PLOS ONE

Article Title: Amino acid T25 in the substrate-binding domain of SARS-CoV-2 nsp5 is involved in viral replication in the mouse lung

doi: 10.1371/journal.pone.0312800

Figure Lengend Snippet: Growth kinetics of recombinant viruses in VeroE6/hTMPRSS2 cells (A) and 293T/hACE2 cells (B). VeroE6/hTMPRSS2 and 293T/hACE2 cells were inoculated with the recombinant viruses at MOI of 0.0001 and 0.01, respectively. Cells infected with rSARS2-QHmusX-WT or rSARS2-QHmusX-nsp5-T25I were cultured for the indicated time periods. Viral titers in the culture supernatants were determined using the TCID 50 assay. The values represent the means ±SD from three independent experiments. ** P < 0.01. Expression of N RNA from recombinant viruses in VeroE6/hTMPRSS2 cells (C) and 293T/hACE2 cells (D) measured using one-step real-time PCR. ns, no statistically significant difference; h.p.i., hours post-infection. The values represent the means ±SD from three independent experiments. ** P < 0.01.

Article Snippet: After overnight incubation at 37°C, cells were transfected with 2.0 μg of pCAGGS nsp5 expression plasmid using TransIT-LT1 Transfection Reagent (Mirus Bio).

Techniques: Recombinant, Infection, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

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