Journal: IUBMB life

Article Title: Expression and functional characterization of NPA motif-null aquaporin-1 mutations.

doi: 10.1002/iub.203

Figure Lengend Snippet: Figure 2. Expression of the DNPA mutant and wild type human AQP1 protein. A: Immunoblot analysis of wild type and mutant hAQP1 protein in stably transfected CHO cell clones (CHO/ hAQP1 wild type, CHO/hAQP1DNPA1, CHO/hAQP1D NPA2, CHO/hAQP1DNPA1,2). B: Immunofluorescence showing AQP1 expression in plasma membrane. CHO cells nontransfected with hAQP1 served as control. Double labeling of AQP1DNPA1,2 mutant protein and ER marker GRP94 showing membrane expression pattern of AQP1DNPA1,2. AQP1DNPA1, and AQP1DNPA2 double labeling showed similar results (not shown). Bar: 10 lm.

Article Snippet: In some experiments, dual immunofluorescence labeling was performed using a monoclonal AQP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1:100) and a rabbit polyclonal antibody against ERspecific marker glucose-regulated protein (GRP94) (Boster, Wuhan, China, 1:200) simultaneously and detected with a mixture of Cy3coupled sheep anti-mouse IgG secondary antibody and a FITCcoupled goat anti-rabbit IgG secondary antibody (Sigma).

Techniques: Expressing, Mutagenesis, Western Blot, Stable Transfection, Transfection, Clone Assay, Clinical Proteomics, Membrane, Control, Labeling, Marker

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc increase S protein specific IgG responses in vivo. a) C57Bl6 mice were vaccinated at day 0 and 14 with different concentrations of cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S and OX40L-Fc or with AD100 or PBS (controls). b) Mice were vaccinated at day 0 and 14 with 1μg/ml of ZVX-55 and ZVX-60 or with AD100 and PBS (controls). Serum was collected 5 days after last vaccination, and S protein specific IgG response was analyzed by ELISA. c) Mice were vaccinated at day 0 and 14 with 1 μg/ml ZVX-55 or ZVX-60 and S protein specific IgG response in serum was analyzed at day 5, 14 and 19. Data represent 3 independent biological replicates per group and mean ± standard error. To compare control (ZVX55) with experimental (ZVX60) group (alpha level of 0.05) unpaired t-test (two-tailed) was applied, *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc induce B cells responses. C57Bl6 mice were vaccinated at day 0 and 14 with a cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S (ZVX-55, 1ug gp96-Ig) and OX40L-Fc (ZVX-60,1 ug gp96-Ig) or with AD100 or PBS (controls). a) Spleen cells (SPL) were isolated from vaccinated and control mice 5 days after last vaccination, stained for CD45, CD3, CD19, IgM, CD21, CD23, CD49, CD93. Frequency of CD19+IgM+ (activated B cells) and CD21+CD23- (marginal zone, MZ), CD21+CD23+ (follicular, FO) and CD21-CD23- (double negative or ABC cells) CD19+IgM+ cells was determined by flow cytometry. b) SPL were isolated from unvaccinated mice and co-cultured with vaccine cells (ZVX55 or ZVX60) and control cells AD100 at 5:1 ratio for 96h. Frequency of activated B cells (CD19+IgM+) within total CD45+ T cells and frequency of FO (CD21+CD23+) within CD19+IgM+ cells was determined by flow cytometry. Data represent 3 independent biological replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Isolation, Staining, Flow Cytometry, Cell Culture

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc induce T follicular helper (TFH) cell responses. C57Bl6 mice were vaccinated at day 0 and 14 with a cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S (ZVX-55, 1ug gp96-Ig) and OX40L-Fc (ZVX-60,1 ug gp96-Ig) or with AD100 or PBS (controls). a) Spleen cells (SPL) were isolated from vaccinated and control mice 5 days after last vaccination, stained for CD45, CD3, CD4, PD1 and CXCR5. Frequency of PD1+CXCR5+ (TFH cells) within CD4+ T cells was determined by flow cytometry. b) SPL were isolated from unvaccinated mice and co-cultured with vaccine cells (ZVX55 or ZVX60) and control cells AD100 at 5:1 ratio for 96h. Frequency of TFH cells (PD1+CXCR5+) within total CD4+ T cells was determined by flow cytometry. Data represent 3 independent biological replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Isolation, Staining, Flow Cytometry, Cell Culture

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Characteristics of the cell line expressing gp96-Ig, SARS-CoV-2 Spike (S) protein and OX40L-Fc. Cell line (AD100) was transfected with plasmids encoding a) gp96-Ig and full length protein S and b) gp96-Ig, full length protein S and OX40L-Fc. c) Secreted gp96-Ig was measured in the cell supernatant by ELISA. One million cells were plated in 1 ml for 24 h. Purified gp96-Ig was used as standard d) Secreted OX40L-Fc was measured in the cell supernatant by ELISA. One million cells were plated in 1 ml for 24 h. Purified OX40L-Fc was used as standard e) SARS-CoV2 protein S expression was analyzed by immunofluorescence f) SARS-CoV2 protein S expression in supernatant was measured by ELISA. One million cells were plated in 1 ml for 48 h and purified SARS-CoV2 protein S was used as standard.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Purification, Immunofluorescence

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Enhancement of S1- specific CD8+ T cell responses by Gp96-Ig-S-OX40L-Fc in the spleen, lung tissue, and BAL . a) 5 days after the vaccination of HLA-A2 transgenic mice (n=3-5) with the ZVX-55, ZVX-60 vaccine cells (1ug secreted gp96-Ig) or AD100 or PBS (controls), splenocytes (SPL), lung cells and bronchioalveolar lavage (BAL) cells were isolated from vaccinated and control mice (PBS). Cells were stained with HLA-A2 02-01 pentamer containing YLQPRTFLL peptides, followed by surface staining for CD45, CD3, CD4, CD8, CD69, CXCR6. Bar graphs represent percentage of the pentamer positive cells within CD8+ T cells. b) 5 days after the vaccination of C57Bl6 mice (n=3), splenocytes and lung cells were isolated from vaccinated and control mice (PBS and AD100) and in vitro restimulated with S1 and S2 overlapping peptides in the presence of protein transport inhibitor, brefeldin A for the last 5 hours of culture. After 20 hours of culture, intracellular cytokine (IFNg, TNFa and IL-2) staining was preformed to quantify protein S-specific CD8+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. Data represent at least 2 technical replicates with 3–5 independent biologic replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Transgenic Assay, Isolation, Staining, In Vitro, Expressing

Journal: Molecular medicine reports

Article Title: A hyposensitive anticancer drug induces higher surface expression and release of heat shock proteins in a human hepatocellular carcinoma cell line.

doi: 10.3892/mmr.2015.3727

Figure Lengend Snippet: Figure 2. Alterations in the mRNA levels and quantity of secretory HSP70 and gp96 in etoposide‑ or carboplatin‑treated HepG2 cells. (A and B) HepG2 cells were treated with etoposide for 24 h or carboplatin for 72 h and the HSP70/gp96 mRNA levels were determined by reverse transcription quan titative polymerase chain reaction. (C and D) Cell culture supernatants were collected and the HSP70/gp96 protein levels were quantified by ELISA. The data shown are representative of four independent experiments for each drug. HSP70, heat shock protein 70.

Article Snippet: Secondary antibodies used for the visualization of HSP70 or gp96 were FITC-conjugated goat anti-mouse IgG (Wuhan Boster Biological Technology, Ltd.) and Cy3-conjugated sheep anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine reports

Article Title: A hyposensitive anticancer drug induces higher surface expression and release of heat shock proteins in a human hepatocellular carcinoma cell line.

doi: 10.3892/mmr.2015.3727

Figure Lengend Snippet: Figure 3. Alterations in cytoplasmic and surface HSP70/gp96 expression in etoposide‑ or carboplatin‑treated HepG2 cells. HepG2 cells were treated with etoposide or carboplatin for the indicated time intervals. (A) Cytoplasmic and surface HSP70/gp96 alterations were determined by flow cytometry. (B) Cytoplasmic and surface gp96 alterations. The data shown are representa tive of more than four independent experiments. Cyto‑etoposide, cytoplasmic HSP70/gp96 expression in etoposide‑treated HepG2 cells; cyto‑carboplatin, cytoplasmic HSP70/gp96 expression in carboplatin‑treated HepG2 cells; sur‑etoposide, surface HSP70/gp96 expression in etoposide‑treated HepG2 cells; sur‑carboplatin, surface HSP70/gp96 expression in carboplatin‑treated HepG2 cells. HSP70, heat shock protein 70; MFI, mean fluorescence inten sity.

Article Snippet: Secondary antibodies used for the visualization of HSP70 or gp96 were FITC-conjugated goat anti-mouse IgG (Wuhan Boster Biological Technology, Ltd.) and Cy3-conjugated sheep anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Molecular medicine reports

Article Title: A hyposensitive anticancer drug induces higher surface expression and release of heat shock proteins in a human hepatocellular carcinoma cell line.

doi: 10.3892/mmr.2015.3727

Figure Lengend Snippet: Figure 4. Representative images of cytoplasmic and surface HSP70/gp96 expression in etoposide‑ or carboplatin‑treated HepG2 cells. HepG2 cells were treated with etoposide for 24 h or carboplatin for 72 h. HSP70/gp96 expression was determined using immunofluorescence staining and confocal images were captured. (A) Cytoplasmic HSP70/gp96 expression. (B) Surface gp96 expression. The images are shown at x400 magnification. FITC, fluorescein isothiocyanate; HSP70, heat shock protein 70.

Article Snippet: Secondary antibodies used for the visualization of HSP70 or gp96 were FITC-conjugated goat anti-mouse IgG (Wuhan Boster Biological Technology, Ltd.) and Cy3-conjugated sheep anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Immunofluorescence, Staining