Journal: International Journal of Biological Sciences

Article Title: Hypericin maintians PDX1 expression via the Erk pathway and protects islet β-cells against glucotoxicity and lipotoxicity

doi: 10.7150/ijbs.33817

Figure Lengend Snippet: Hypericin protects INS-1 cells from glucotoxicity and lipotoxicity via Erk signaling and maintianing PDX1 expression. (A) Effects of hypericin on the PDX1 mRNA level in INS-1 cells under glucotoxicity. INS-1 cells were treated with high (33 mM) glucose, 200 nM hypericin or a combination of the two for 72 h. Total RNA was extracted, and PDX1 mRNA was amplified by conventional SYBR Green real-time PCR analysis. Data are presented as the mean ± S.D. (n = 3). (B) Effects of hypericin on the protein levels of PDX1, Erk1/2 and p-Erk in INS-1 cells under glucotoxicity. INS-1 cells were treated as in (A). Cell lysates were prepared and subjected to Western blots using the indicated antibodies. GAPDH was used as a loading control. The density ratios of PDX1 to GAPDH or p-Erk to Erk were measured by ImageJ as shown in the right panel. The experiment was repeated three times. *** p<0.001 versus the 33 mM glucose-treated group. (C) Blockade of hypericin-mediated effects by U0126 in INS-1 cells under glucotoxicity. INS-1 cells were treated with different combinations of high (33 mM) glucose, 200 nM hypericin and U0126 as indicated for 72 h. Then, target proteins were detected by Western blot using the indicated antibodies. GAPDH was used as a loading control. The density ratios of PDX1 to GAPDH, p-Erk to Erk or CC3 to GAPDH were measured by ImageJ as shown in the right-hand panel. The experiment was repeated three times. **p<0.01, ***p<0.001 versus the 33 mM glucose-treated group; ##p<0.01, ###p<0.001 versus the 33 mM glucose+200 nM hypericin-treated group. (D) Effects of hypericin on the Erk pathway in INS-1 cells under lipotoxicity. INS-1 cells were treated with 200 μM PA, 200 nM hypericin or a combination of the two for 24 h. Cell lysates were prepared and subjected to Western blots using anti-Erk and anti-p-Erk antibodies. GAPDH was used as a loading control. The density ratios of p-Erk to Erk were measured by ImageJ as shown in the right panel. The experiment was repeated three times. **p<0.01, *** p<0.001 versus the 200 μM PA-treated group. (E) Blockade of hypericin-mediated effects by U0126 in INS-1 cells under lipotoxicity. INS-1 cells were treated with different combinations of 200 μM PA, 200 nM hypericin and U0126 as indicated for 24 h. Then, target proteins were detected and analysed as in (c). *** p<0.001 versus the 200 μM PA-treated group; ## p<0.01 versus the 200 μM PA+200 nM hypericin-treated group.

Article Snippet: Anti-GAPDH antibodies (Cat#60004-1-Ig), PDX1 (Cat#10951-1-AP) and goat anti-rabbit (Cat#SA00001-2) or anti-mouse IgG-horseradish peroxidase (IgG-HRP, Cat#SA00001-1) secondary antibodies were purchased from Proteintech (Proteintech, Wuhan, China).

Techniques: Expressing, Amplification, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Hypericin maintians PDX1 expression via the Erk pathway and protects islet β-cells against glucotoxicity and lipotoxicity

doi: 10.7150/ijbs.33817

Figure Lengend Snippet: Prophylactic use of hypericin decreases β-cell loss and maintains islet mass in HFHS-fed mice. (A) Histological sections of mouse pancreatic tissue. After sacrifice, the mouse pancreases were removed and weighed. Portions of the mouse pancreases from (A) were fixed and subjected to HE staining. The scale bar represents 100 μm. Arrows indicate pancreatic islets. (B) IHC analysis of the mouse pancreas using anti-C-peptide antibodies. Portions of the mouse pancreases from (A) were fixed and subjected to IHC analysis. The scale bar represents 100 μm. Arrows indicate positively stained cells. (C) Measurement of islet area in the mouse pancreas. Pancreatic sections subjected to IHC staining with an anti-C-peptide antibody in (B) were used to measure the islet area of the pancreas. Data are presented as the mean ± S.D. (n = 8). (D) Calculation of β-cell mass of the pancreas. Pancreatic sections that were IHC stained with an anti-C-peptide antibody in (B) were used to calculate the β-cell mass of the pancreas. Data are presented as the mean ± S.D. (n = 8). (E) PDX1 protein levels in the mouse pancreas. Portions of the mouse pancreases from (A) were homogenized, and total cellular lysates were prepared and subjected to Western blots using anti-PDX1 antibodies. GAPDH was used as a loading control. The density ratios of PDX1 to GAPDH were measured by ImageJ, and the fold change relative to the normal group is shown in the right-hand panel. Data are presented as the mean ± S.D. (n = 6). * p< 0.05, **p<0.01, ***p<0.001 versus the HFHS group.

Article Snippet: Anti-GAPDH antibodies (Cat#60004-1-Ig), PDX1 (Cat#10951-1-AP) and goat anti-rabbit (Cat#SA00001-2) or anti-mouse IgG-horseradish peroxidase (IgG-HRP, Cat#SA00001-1) secondary antibodies were purchased from Proteintech (Proteintech, Wuhan, China).

Techniques: Staining, Immunohistochemistry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Hypericin maintians PDX1 expression via the Erk pathway and protects islet β-cells against glucotoxicity and lipotoxicity

doi: 10.7150/ijbs.33817

Figure Lengend Snippet: Therapeutic use of hypericin decreases β-cell loss and maintains islet mass in mice with HFHS-induced diabetes. (A) Histological sections of mouse pancreatic tissue. The mice in Fig. were sacrificed, and the pancreases were removed and weighed. Portions of the mouse pancreases were fixed and subjected to HE staining as in Fig. A. Scale bar represents 100 μm. Arrows indicate pancreatic islets. (B-D) Fixed pancreas tissue from A was subjected to IHC analysis using anti-C-peptide antibodies (B) as shown in Fig. B, after which IHC-stained pancreatic sections were used to calculate the islet area (C) and β-cell mass (D) of the pancreas. The scale bar represents 100 μm. Arrows indicate positively stained cells. Data are presented as the mean ± S.D. (n = 8). (E) PDX1 protein levels in the mouse pancreas. Portions of the mouse pancreases from (A) were homogenized. PDX1 levels in the homogenate were detected by Western blot, and the density ratios of PDX1 to GAPDH were measured as in Fig. E. Fold change relative to the normal group is shown in the right-hand panel. Data are presented as the mean ± S.D. (n = 6). *p<0.05, **p<0.01 ***p<0.001, versus the HFHS group.

Article Snippet: Anti-GAPDH antibodies (Cat#60004-1-Ig), PDX1 (Cat#10951-1-AP) and goat anti-rabbit (Cat#SA00001-2) or anti-mouse IgG-horseradish peroxidase (IgG-HRP, Cat#SA00001-1) secondary antibodies were purchased from Proteintech (Proteintech, Wuhan, China).

Techniques: Staining, Western Blot

Journal: PLoS ONE

Article Title: PD-1 Blockade in Chronically HIV-1-Infected Humanized Mice Suppresses Viral Loads

doi: 10.1371/journal.pone.0077780

Figure Lengend Snippet: A ) Percentages of human CD8 + and CD4 + T cells from lymphocyte gate at 13, 16–18 and 26–30 weeks post infection (p.i.), times corresponding to 0, 3–5 and 13–17 weeks after start of anti-PD-1 mAb treatment (a.t.), respectively. Horizontal lines within data points depict mean values. * P = 0.0007, Mann-Whitney test; ** P = 0.047, Wilcoxon paired test. B ) Western blot of plasma samples taken from 2 mice before and after anti-PD-1 mAb treatment showing anti-HIV IgG Abs and anti-HIV IgM Abs. *'s depict positive bands indicating the presence of antibodies to HIV proteins listed at left. Negative Controls, from the same blots as the data, were “cut-and-pasted” for image layout purposes. HIV-specific binding assays were performed using ELISA to measure IgG titers against C ) p24 and D ) gp120. Human plasma samples were collected 6-25 weeks post diagnosis of HIV-1 infection. BLT plasma samples were collected 26 weeks post infection.

Article Snippet: HIV-specific IgM and IgG human antibodies were detected in plasma samples from HIV-infected BLT mice using Genetic Systems (GS) HIV-1 Western Blot kits (Bio-Rad) according to the manufacturer's instructions, substituting mouse anti-human IgM and anti-human IgG antibodies conjugated to horseradish peroxidase (Southern Biotech, AL) for the anti-human Ig antibody supplied.

Techniques: Infection, MANN-WHITNEY, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies

doi: 10.1016/j.celrep.2022.110841

Figure Lengend Snippet: (A) Superimposition of the GP1 subunit of LASV GP in the pre-fusion (gray cartoon) and primed (orange) conformation highlights structural rearrangements that may occur upon LAMP1 binding. The loop harboring H230 is central to the GPC-A epitope and its location and its location shifts ~90° relative to the pre-fusion state. (B) ELISA analysis of LAMP1 association with LASV pfGP-TD alone and in complex with the indicated IgG. Error bars indicate the mean ± SD of two independent experiments, each performed in duplicate.

Article Snippet: Mouse anti-Human CD107a-UNLB (LAMP1) , Southern Biotech , Cat# 9835–01; RRID: AB_2797102.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies

doi: 10.1016/j.celrep.2022.110841

Figure Lengend Snippet: (A) Schematic depicting native entry and fusion of LASV into cells. GP interacts with α-DG at the cell surface and undergoes a pH-dependent receptor shift to LAMP-1 inside the endosome. The pH 5.0 environment of the late endosome promotes membrane fusion and subsequent release of the viral genome. (Middle) Neutralization of rVSV-LASV by the GPC-A mAbs 25.10C and 36.1F and the GPC-B mAb 18.5C in wild-type Vero cells under native entry conditions. (B) (Left) Schematic depicting entry of LASV at the cell surface in wild-type Vero cells. In this entry pathway, exposure to acidic pH, or acid bypass, forces fusion of target cell and LASV membranes; this fusion occurs in a LAMP1-independent manner. (Middle) Fusogenic profile of rVSV-LASV at the indicated pH. (Right) Neutralization of rVSV-LASV by the GPC-A mAbs 25.10C and 36.1F and the GPC-B mAb 18.5C in wild-type Vero cells, at the cell surface at pH 4.0. (C) (Left) Schematic depicting entry of LASV at the cell surface in Vero cells expressing LAMP1Δ384 with cell-surface localization of LAMP1. In this entry pathway, LASV first binds to cell-surface receptors, such as matriglycosylated α-DG and then, upon exposure to lower pH, switches to cell-surface LAMP1Δ384 before fusion. (Middle) The fusogenic profile of rVSV-LASV in LAMP1Δ384-Vero cells at the indicated pH is shown. (Right) Neutralization of rVSV-LASV by the GPC-A mAbs 25.10C and 36.1F and the GPC-B mAb 18.5C in Vero cells stably expressing LAMP1Δ384 at pH 5.0. (D) (Left, middle, right) Binding of the indicated antibody to LASV pfGP-TD at varying pHs as determined by ELISA. For (B-middle) and (C-middle), data were normalized to the total infectivity of virus under native entry conditions (i.e., endosomal dependent, and not exposed to either acid or lysosomotropic agents). Error bars indicate the mean ± SD of two independent experiments, each performed in quadruplicate. For (A-middle), (B-right), and (C-right), data are displayed as the relative neutralization as compared with a PBS control. Error bars indicate the SD of the mean of three independent experiments, each performed in duplicate. Schematics were created with BioRender.com . See also .

Article Snippet: Mouse anti-Human CD107a-UNLB (LAMP1) , Southern Biotech , Cat# 9835–01; RRID: AB_2797102.

Techniques: Neutralization, Expressing, Stable Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: Cell reports

Article Title: Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies

doi: 10.1016/j.celrep.2022.110841

Figure Lengend Snippet: (A) (Left) Schematic depicting the native entry pathway for rVSV-LASV-H230E. (Middle) Neutralization of rVSV-LASV-H230E by the indicated mAb under native entry conditions. (B) (Left) Schematic depicting entry of rVSV-LASV-H230E at the cell surface in wild-type Vero cells. In this entry pathway, viral fusion occurs in a LAMP1-independent manner. (Middle) The fusogenic profile of rVSV-LASV-H230E at the indicated pH is shown. Data were normalized to the total infectivity of virus under native entry conditions (i.e., endosomal dependent, and not exposed to either acid or lysosomotropic agents). Error bars indicate the mean± SD of two independent experiments, each performed in quadruplicate. (Right) Neutralization of rVSV-LASV-H230E by the indicated mAb at pH 5.0. For (A-middle and B-right), data are displayed as the relative neutralization as compared with a PBS control. Error bars indicate the SD of the mean of three independent experiments, each performed in duplicate.

Article Snippet: Mouse anti-Human CD107a-UNLB (LAMP1) , Southern Biotech , Cat# 9835–01; RRID: AB_2797102.

Techniques: Neutralization, Infection

Journal: Cell reports

Article Title: Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies

doi: 10.1016/j.celrep.2022.110841

Figure Lengend Snippet:

Article Snippet: Mouse anti-Human CD107a-UNLB (LAMP1) , Southern Biotech , Cat# 9835–01; RRID: AB_2797102.

Techniques: Purification, Mutagenesis, Recombinant, Expressing, Electron Microscopy, Transfection, Plasmid Preparation, Software, Crystallization Assay, High Content Screening