Journal: mBio

Article Title: Guanylate-binding protein 5 antagonizes viral glycoproteins independently of furin processing

doi: 10.1128/mbio.02086-24

Figure Lengend Snippet: GBP5 expression increases the electrophoretic mobility of cell-associated HIV-1 Env, MLV Env, and SARS-CoV-2 S and reduces their incorporation into virions. HEK293T cells were co-transfected with varying quantities of a GBP5 expression vector, with total DNA held constant (at 0.5 µg) with empty vector control and ( A ) the pNL4-3 molecular clone (4 µg), ( B ) the Env-defective pNL4-3 derivative pNL4-3/KFS (3.3 µg) and MLV Env expression plasmid (0.6 µg), or ( C ) the Env(−) luciferase-encoding NL4-3-derived vector pNL4-3.Luc.R-E- (pNL4-3/Luc) (3 µg) and SARS-CoV-2 S expression plasmid (1 µg). Two days post-transfection, cell and virus lysates were prepared and subjected to Western blot analysis with anti-HIV-1 Ig to detect Gag proteins, anti-HIV-1 gp120, anti-HIV-1 gp41, anti-MLV gp70, anti-MLV p15(E), anti-SARS-CoV/SARS-CoV-2 S, anti-GBP5, or anti-alpha-tubulin. Positions of the HIV-1 Env glycoprotein precursor gp160, HIV-1 surface glycoprotein gp120, HIV-1 transmembrane glycoprotein gp41, Gag precursor Pr55Gag, p24 CA protein, MLV Env precursor Pr85Env, MLV surface Env glycoprotein gp70, MLV transmembrane protein p15(E), S precursor S0, and transmembrane spike glycoprotein S2 are indicated. The mobility of molecular mass standards is shown on the left of each blot in kilodaltons. The virion-associated levels of ( D ) HIV-1 gp120, ( E ) MLV gp70, and ( F ) SARS-CoV-2 S were quantified and normalized to virion-associated p24; values were set at 100% in the absence of GBP5. Analysis was performed with ImageJ 1.53k software. Data shown are means ± SDs from three to four independent experiments. Statistical significance (two-tailed unpaired t -test): * P < 0.05, ** P < 0.02, *** P < 0.01. ns, not significant.

Article Snippet: The following antibodies were used in this study: anti-HIV-1 Ig, anti-HIV-1 gp120 (16H3, catalog no. 12559), and anti-HIV-1 gp41 (clone Chessie 8, catalog no. ARP-13049) (obtained from the NIH ARP); anti-SARS-CoV/SARS-CoV-2 S (GeneTex, catalog no. GTX632604); anti-VSV-G (Cell Signaling, catalog no. 93372); anti-GBP5 (Cell Signaling, catalog no. 67798); anti-STAT-1 (Cell Signaling, catalog no. 14994); anti-alpha-tubulin (Sigma-Aldrich, clone B-5-1-2, catalog no. T5168); anti-CD4 (Santa Cruz, catalog no. sc-19641); goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Invitrogen, catalog no. 31446); goat anti-rabbit IgG conjugated to HRP (Invitrogen, catalog no. 65-6120); and anti-human IgG conjugated to HRP (Sigma-Aldrich, catalog no. GENA933).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Luciferase, Derivative Assay, Virus, Western Blot, Software, Two Tailed Test

Journal: mBio

Article Title: Guanylate-binding protein 5 antagonizes viral glycoproteins independently of furin processing

doi: 10.1128/mbio.02086-24

Figure Lengend Snippet: GBP5 reduces cell-surface HIV-1 Env and SARS-CoV-2 S expression. HEK293T cells were co-transfected with either wild-type (WT) GBP5 expression plasmid (0.5 µg) or the isoprenylation-deficient GBP5 mutant C583A (0.5 µg) and ( A ) the pNL4-3 molecular clone (4 µg) or ( B ) the Env(−) pNL4-3 derivative pNL4-3/KFS (3 µg) and SARS-CoV-2 S expression plasmid (1 µg). Two days post-transfection, cells were collected, incubated with anti-HIV-1 gp120 or Alexa Fluor 647-conjugated SARS-CoV-2 spike S1 subunit antibody, fixed, and permeabilized. After permeabilization, cells were incubated with goat anti-human antibody conjugated to Alexa Fluor 647 to stain HIV-1 gp120 and with PE-conjugated anti-HA.11 epitope tag antibody to detect GBP5. Histograms of HIV-1 Env- and SARS-CoV-2 S-positive Alexa Fluor 647 (AF647) expression were plotted in the absence of GBP5 (green) or in the presence of either C583A GBP5 (orange) or WT GBP5 (gray). Histogram of AF647 positivity of unstained cells (blue) was included as a control. Mean and median fluorescence intensity (MFI) of HIV-1 Env and SARS-CoV-2 S cell-surface expression was calculated with FlowJo version 10.9.0 software. The MFI in the absence of GBP5 is set at 100%. Data shown are means ± SDs from three independent experiments. Statistical significance (two-tailed unpaired t -test): *** P < 0.01; **** P < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used in this study: anti-HIV-1 Ig, anti-HIV-1 gp120 (16H3, catalog no. 12559), and anti-HIV-1 gp41 (clone Chessie 8, catalog no. ARP-13049) (obtained from the NIH ARP); anti-SARS-CoV/SARS-CoV-2 S (GeneTex, catalog no. GTX632604); anti-VSV-G (Cell Signaling, catalog no. 93372); anti-GBP5 (Cell Signaling, catalog no. 67798); anti-STAT-1 (Cell Signaling, catalog no. 14994); anti-alpha-tubulin (Sigma-Aldrich, clone B-5-1-2, catalog no. T5168); anti-CD4 (Santa Cruz, catalog no. sc-19641); goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Invitrogen, catalog no. 31446); goat anti-rabbit IgG conjugated to HRP (Invitrogen, catalog no. 65-6120); and anti-human IgG conjugated to HRP (Sigma-Aldrich, catalog no. GENA933).

Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Staining, Control, Fluorescence, Software, Two Tailed Test

Journal: mBio

Article Title: Guanylate-binding protein 5 antagonizes viral glycoproteins independently of furin processing

doi: 10.1128/mbio.02086-24

Figure Lengend Snippet: Overexpression of HIV-1 Env and SARS-CoV-2 S confers GBP5 resistance. HEK293T cells were co-transfected with either WT GBP5 expression plasmid (0.5 µg) or the isoprenylation-deficient GBP5 mutant C583A (0.5 µg) and the Env-defective pNL4-3 derivative pNL4-3/KFS and HIV-1 Env expression plasmid at a ratio of 1:10 ( A and B ) or 1:3 ( C and D ), or Env(−) luciferase-encoding NL4-3-derived vector pNL4-3.Luc.R-E- (pNL4-3/Luc) and SARS-CoV-2 S expression plasmid at a ratio of 1:5 ( E and F ) or 1:1 ( G and H ). Two days post-transfection, cells were collected, incubated with anti-HIV-1 gp120 or AF647-conjugated SARS-CoV-2 spike S1 subunit antibody, fixed, and permeabilized. After permeabilization, cells were incubated with goat anti-human antibody conjugated to AF647 to stain HIV-1 gp120 and with PE-conjugated anti-HA.11 epitope tag antibody to detect GBP5. Histograms of HIV-1 Env- ( A and C ) and SARS-CoV-2 S- ( E and G ) positive expression were plotted in the absence of GBP5 (green) or in the presence of either C583A GBP5 (orange) or WT GBP5 (gray). Histogram of AF647 positivity of unstained cells (blue) was included as a control. Mean and median fluorescence intensity (MFI) of HIV-1 Env and SARS-CoV-2 S cell-surface expression was calculated with FlowJo 10.9.0 software. The MFI in the absence of GBP5 is set at 100%. Data shown are means ± SDs from three independent experiments. Statistical significance (two-tailed unpaired t -test): * P < 0.05; ** P < 0.02; **** P < 0.0001. Virus supernatants were collected, normalized for RT, and used to infect TZM-bl cells ( B and D ) or HEK293T cells stably expressing hACE2 and transfected with TMPRSS2 expression plasmid ( F and H ). The infectivity in the absence of GBP5 is set at 100%. Data shown are means ± SDs from three independent experiments. Statistical significance (two-tailed unpaired t -test): *** P < 0.01; **** P < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used in this study: anti-HIV-1 Ig, anti-HIV-1 gp120 (16H3, catalog no. 12559), and anti-HIV-1 gp41 (clone Chessie 8, catalog no. ARP-13049) (obtained from the NIH ARP); anti-SARS-CoV/SARS-CoV-2 S (GeneTex, catalog no. GTX632604); anti-VSV-G (Cell Signaling, catalog no. 93372); anti-GBP5 (Cell Signaling, catalog no. 67798); anti-STAT-1 (Cell Signaling, catalog no. 14994); anti-alpha-tubulin (Sigma-Aldrich, clone B-5-1-2, catalog no. T5168); anti-CD4 (Santa Cruz, catalog no. sc-19641); goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Invitrogen, catalog no. 31446); goat anti-rabbit IgG conjugated to HRP (Invitrogen, catalog no. 65-6120); and anti-human IgG conjugated to HRP (Sigma-Aldrich, catalog no. GENA933).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Derivative Assay, Incubation, Staining, Control, Fluorescence, Software, Two Tailed Test, Virus, Stable Transfection, Infection

Journal: mBio

Article Title: Guanylate-binding protein 5 antagonizes viral glycoproteins independently of furin processing

doi: 10.1128/mbio.02086-24

Figure Lengend Snippet: GBP5 reduces the glycosylation of HIV-1 Env, MLV Env, SARS-CoV-2 S, SARS-CoV S, and VSV-G. HEK293T cells were co-transfected with varying amounts of a GBP5 expression vector, with the total DNA held constant with empty vector (0.5 µg total), and ( A ) pNL4-3 (4 µg), ( B ) the Env(−) pNL4-3 derivative pNL4-3/KFS (3 µg) and MLV Env expression plasmid (0.6 µg), ( C ) Env(−) luciferase-encoding NL4-3-derived vector pNL4-3.Luc.R-E- (pNL4-3/Luc) (3.3 µg) and SARS-CoV-2 S expression plasmid (1 µg), ( D ) Env(−) luciferase-encoding NL4-3-derived vector pNL4-3.Luc.R-E- (pNL4-3/Luc) (3.3 µg) and SARS-CoV S expression plasmid (1 µg), or ( E ) the Env(−) pNL4-3 derivative pNL4-3/KFS (4 µg) and VSV-G expression plasmid (0.5 µg). Two days post-transfection, cell lysates were prepared and treated with PNGase F before being subjected to Western blot analysis with anti-HIV-1 gp120, anti-HIV-1 gp41, anti-MLV gp70, anti-SARS-CoV/SARS-CoV-2 S, anti-VSV-G, anti-GBP5, or anti-alpha-tubulin. The positions of the glycan-stripped viral glycoproteins in the PNGaseF-treated samples are indicated. The mobility of molecular mass standards is shown on the left of each blot in kilodalton.

Article Snippet: The following antibodies were used in this study: anti-HIV-1 Ig, anti-HIV-1 gp120 (16H3, catalog no. 12559), and anti-HIV-1 gp41 (clone Chessie 8, catalog no. ARP-13049) (obtained from the NIH ARP); anti-SARS-CoV/SARS-CoV-2 S (GeneTex, catalog no. GTX632604); anti-VSV-G (Cell Signaling, catalog no. 93372); anti-GBP5 (Cell Signaling, catalog no. 67798); anti-STAT-1 (Cell Signaling, catalog no. 14994); anti-alpha-tubulin (Sigma-Aldrich, clone B-5-1-2, catalog no. T5168); anti-CD4 (Santa Cruz, catalog no. sc-19641); goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Invitrogen, catalog no. 31446); goat anti-rabbit IgG conjugated to HRP (Invitrogen, catalog no. 65-6120); and anti-human IgG conjugated to HRP (Sigma-Aldrich, catalog no. GENA933).

Techniques: Glycoproteomics, Transfection, Expressing, Plasmid Preparation, Luciferase, Derivative Assay, Western Blot

Journal: mBio

Article Title: Guanylate-binding protein 5 antagonizes viral glycoproteins independently of furin processing

doi: 10.1128/mbio.02086-24

Figure Lengend Snippet: GBP5 does not impair the processing of HIV-1 Env, MLV Env, or SARS-CoV-2 S glycoproteins under the conditions tested. Quantification of the ratios of cell-associated (A) HIV-1 gp160/gp120, (B) MLV Pr85Env/gp70, and (C) SARS-CoV-2 S0/S2 in the presence of varying amounts of GBP5 expression plasmid (0–0.5 µg) is shown. Quantification included only fully glycosylated forms of gp120, gp70, or S2 (dark bars) or fully glycosylated plus the more rapidly migrating, less-glycosylated forms (light bars). Blots below the graphs are reproduced from for ease of viewing; fully glycosylated forms of gp120, gp70, and S2 are denoted by #; lower-molecular-weight (less glycosylated) species are indicated by ##. Quantification of the ratios of cell-associated ( D ) HIV-1 gp160/gp120, ( E ) MLV Pr85Env/gp70, and ( F ) SARS-CoV-2 S0/S2 from PNGaseF-treated samples in the absence or presence of GBP5 expression plasmid (0.5 µg). Blots are reproduced from for ease of viewing, with the bands quantified in the graphs boxed in the blots. Analysis was performed with ImageJ 1.53k software. Glycoprotein processing in the absence of GBP5 is set at 100%. Data shown are means ± SDs from three to four independent experiments. Statistical significance (two-tailed unpaired t -test): * P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used in this study: anti-HIV-1 Ig, anti-HIV-1 gp120 (16H3, catalog no. 12559), and anti-HIV-1 gp41 (clone Chessie 8, catalog no. ARP-13049) (obtained from the NIH ARP); anti-SARS-CoV/SARS-CoV-2 S (GeneTex, catalog no. GTX632604); anti-VSV-G (Cell Signaling, catalog no. 93372); anti-GBP5 (Cell Signaling, catalog no. 67798); anti-STAT-1 (Cell Signaling, catalog no. 14994); anti-alpha-tubulin (Sigma-Aldrich, clone B-5-1-2, catalog no. T5168); anti-CD4 (Santa Cruz, catalog no. sc-19641); goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Invitrogen, catalog no. 31446); goat anti-rabbit IgG conjugated to HRP (Invitrogen, catalog no. 65-6120); and anti-human IgG conjugated to HRP (Sigma-Aldrich, catalog no. GENA933).

Techniques: Expressing, Plasmid Preparation, Molecular Weight, Software, Two Tailed Test

Journal: Neurotoxicology

Article Title: Contribution of child ABC-transporter genetics to prenatal MeHg exposure and neurodevelopment

doi: 10.1016/j.neuro.2022.05.019

Figure Lengend Snippet: Summary of current knowledge and distribution in different populations for the six SNPs evaluated in the study.

Article Snippet: Genotyping was performed by TaqMan real-time PCR on the ABI 7900HT Fast Real Time PCR System (Applied Biosystems, Thermo Fisher, Waltham, USA), using manufacturer’s recommended standard conditions and the following custom genotyping assays from Thermo Scientific: C__11711720C_30 and C__11711720D_40 (rs2032582), C__29490882_10 (rs10276499), C__2982705_20 (rs1202169), C__3188828_10 (rs212093), C__27065543_10 (rs215088) C__2814642_10 (rs717620).

Techniques: Expressing, Membrane

Journal: Neurotoxicology

Article Title: Contribution of child ABC-transporter genetics to prenatal MeHg exposure and neurodevelopment

doi: 10.1016/j.neuro.2022.05.019

Figure Lengend Snippet: Mean cord blood (prenatal) MeHg concentrations by child and maternal genotypes of ABC transporter SNP. For each SNP, the p-value for the test comparing the means in the three groups is reported.

Article Snippet: Genotyping was performed by TaqMan real-time PCR on the ABI 7900HT Fast Real Time PCR System (Applied Biosystems, Thermo Fisher, Waltham, USA), using manufacturer’s recommended standard conditions and the following custom genotyping assays from Thermo Scientific: C__11711720C_30 and C__11711720D_40 (rs2032582), C__29490882_10 (rs10276499), C__2982705_20 (rs1202169), C__3188828_10 (rs212093), C__27065543_10 (rs215088) C__2814642_10 (rs717620).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing

doi: 10.3390/ijms231911787

Figure Lengend Snippet: List of genes and their main function selected for this study.

Article Snippet: ABCC2_rs3740066 , C__11214910_20 , DME , G.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing

doi: 10.3390/ijms231911787

Figure Lengend Snippet: Variants of 28 polymorphisms identified in 18 human osteosarcoma cell lines by multimodal targeted next generation sequencing and TaqMan genotyping analysis.

Article Snippet: ABCC2_rs3740066 , C__11214910_20 , DME , G.

Techniques: Next-Generation Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing

doi: 10.3390/ijms231911787

Figure Lengend Snippet: Assays used for TaqMan genotyping and reference allele of multimodal targeted next generation sequencing (mmNGS) for variant interpretation.

Article Snippet: ABCC2_rs3740066 , C__11214910_20 , DME , G.

Techniques: Next-Generation Sequencing, Variant Assay

Journal: Liver Transplantation

Article Title: Development of Ectopic Livers by Hepatocyte Transplantation Into Swine Lymph Nodes

doi: 10.1002/lt.25872

Figure Lengend Snippet: Definition of Genes Investigated

Article Snippet: ABCB1 , ATP binding cassette subfamily B member 1 , Ss03373436_m1.

Techniques: Binding Assay

Journal: Liver Transplantation

Article Title: Development of Ectopic Livers by Hepatocyte Transplantation Into Swine Lymph Nodes

doi: 10.1002/lt.25872

Figure Lengend Snippet: Auxiliary liver and biliary system. (A) Whole liver tissues engrafted in a LN of animal P147‐13. Multiple pictures of H & E staining were taken to present the whole engrafted liver tissue in LN. The black rectangle indicates the area of magnification presented in the middle panel and the location where small bile ducts were identified, outside of the hepatic engraftment. In the left panel, the inset is a CK19 staining (brown) on a serial paraffin‐embedded section identifying bile ducts. (B) Immunostaining of frozen sections from ectopic and control liver showing the presence of CK7+ bile duct cells, as well as the presence of intrahepatic ducts. (C) Tissues bile acid analyses. Bile acids were quantified in the normal (native) liver, ectopic liver, engrafted LN (without ectopic liver tissue collected), nonengrafted LN (in the experimental animals), and native liver (in the experimental animals). (D) The mRNA expression by quantitative polymerase chain reaction of ALB, CYP1A2, ASGR1, OCT, CYP7A1, CYP27A1, ABCB1, ABCC2, DPP4, LYVE1, EpCAM, ACTB, and PPIA; n = 3 per group, presented as mean ± SD. (E) Immunostaining of CYP7A1, CYP27A1, BSEP, and MRP2 (ABCC2) on frozen sections of the ectopic liver (red). All immunostainings were counterstained with Hoechst 33342 dye. Scale bar, 100 μm or as indicated.

Article Snippet: ABCB1 , ATP binding cassette subfamily B member 1 , Ss03373436_m1.

Techniques: Staining, Immunostaining, Control, Expressing, Real-time Polymerase Chain Reaction