sac51 5ʹ leader-gfp transcriptional fusion construct containing an 860-bp cdna fragment of the sac51 5ʹ leader region in pt7 blue-2  (Promega)

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: Schematic illustration of engineering PD-1 NVs carrying GEM for the treatment of triple-negative breast cancer.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques:

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: Schematic illustration and characterization of HEK293T/3T3L1 stably overexpressing PD-1. ( A , B ) Confocal images indicated the establishment of HEK293T cell line stably expressing GFP and human PD-1. WGA Alexa-Fluor 594 dye was used to label cell membrane. Scale bar: 5 µm. ( C ) Western blot assay verified the expression of PD-1 receptors on the whole cell lysate of the overexpressing PD-1 cell line. β-actin was used as a loading control. ( D , E ) RT-qPCR assay exhibited the expression of PD-1 on the whole cell lysate of the stable cell line including human cell (HEK293T) and mouse cell (3T3L1). Data were expressed as mean ± SEM, n = 3. *** p < 0.001.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: The preparation and characterization of PD-1 NVs and PD-1&GEM NVs. ( A ) The TEM image showed the shape and size of PD-1 NVs and PD-1&GEM NVs. Scale bar: 100 nm. ( B ) Western blot assay exhibited the expression of PD-1 receptors on the NVs of the stable cell line. Na, K-ATPase was used as a loading control. ( C , D ) The size distribution and zeta potentials of PD-1 NVs and PD-1&GEM NVs measured by dynamic light scattering (DLS) analysis. Data were expressed as mean ± SEM, n = 3.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: Western Blot, Expressing, Stable Transfection

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: In vitro PD-1 NVs interacted with MDA-MB-231 cell. ( A ) FACS indicated the cell surface expression of PD-L1 in MDA-MB-231 cell line. ( B ) RT-qPCR assay exhibited the expression of PD-L1 in the MDA-MB-231 cell line and MCF-7 (non-TNBC cells). Data were expressed as mean ± SEM, n = 3. **** p < 0.0001. ( C ) GFP-PD-1 NVs bound with the cell membrane of MDA-MB-231 cancer cell. PD-1 NVs (50 µg/mL, protein weight) were incubated with MDA-MB-231 cancer cell for 2 h. Arrows pointed to PD-1 (on NVs), the MDA-MB-231 cell membrane (expressing PD-L1) and colocalization, respectively. WGA Alexa-Fluor 594 dye was used to detect MDA-MB-231 cell membrane (Scar bar: 10 µm).

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Incubation

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: GEM inhibited the proliferation of tumor cells and PD-1&GEM NVs promoted cell apoptosis. ( A ) Cell cytotoxicity of gemcitabine and olaparib on MDA-MB-231 breast cancer cell line. ( B ) The cell clone estimated the inhibition of gemcitabine on MDA-MB-231 cell line. ( C ) FACS assay exhibited PD-1&GEM NVs induced cell apoptosis. ( D ) Corresponding statistic data measured the proportion of PI + /PI − apoptosis cell. Data were expressed as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: Inhibition

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: PD-1&GEM NVs promoted the apoptosis of MDA-MB-231 and activated the proliferation of PBMC cells in vitro. ( A ) Flow cytometry analysis of the proliferation of PBMC when co-cultured with MDA-MB-231 in groups received different treatments (Ctrl, NC, PD-1 NVs, and PD-1&GEM NVs) for 3 days. Control group: PBMC at day 0, NC group: free NVs. ( B ) The corresponding quantitative analysis of PBMC from different treatment groups ( n = 3). ( C ) Microscopic examination estimated that the proliferation of MDA-MB-231 in groups received different treatments (NC, PD-1 NVs and PD-1&GEM NVs together with PBMC). ( D ) Flow cytometry analysis of the apoptosis of MDA-MB-231, which were co-cultured with PBMC. ( E ) Column data estimated the proportion of viable cell, apoptosis cell, necrotic cell, and dead cell. ( F , G ) Representative western blot plots and quantitative analysis of the effect of GEM at different time points on the expression of γ-H2AX, β-actin was used as a loading control. Data were expressed as mean ± SEM, n = 3. NS: no significant, * p < 0.05, *** p < 0.001.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: In Vitro, Flow Cytometry, Cell Culture, Western Blot, Expressing

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: In vivo targeting ability and antitumor effect of PD-1&GEM NVs. ( A ) In vivo biodistribution imaging of PD-1 NVs that accumulate on the tumor compared to Free NVs. ( B ) Survival curves for the BALB/c mouse inoculated with TNBC received treatment of different groups ( n = 5). Saline (Group 1), Free NVs (Group 2), GEM (Group 3), PD-1 NVs (Group 4), PD-1&GEM NVs (Group 5). ( C ) Body weight of the BALB/c mouse inoculated with TNBC received treatment of different groups ( n = 5). Saline, Free NVs, GEM, PD-1 NVs, and PD-1&GEM NVs. ( D ) Average tumor volumes of mice inoculated with TNBC in different groups ( n = 5). ( E ) Representational tumor image collected from euthanized mice after different treatments. Saline, Free NVs, GEM, PD-1 NVs, and PD-1&GEM NVs. ( F ) Quantitative analysis of tumor weight of different groups ( n = 3). ( G , H ) Representative plots and quantitative analysis of CD8 + T cells (gated on positive CD3 + cells) in tumor in differently treated mice groups by flow cytometry ( n = 3). Error bar, mean ± SEM. ( I ) Histological images for H&E staining obtained from the tumor of mice treated in different group. Data were expressed as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: In Vivo, Imaging, Flow Cytometry, Staining

Journal: Pharmaceutics

Article Title: PD-1 Cellular Nanovesicles Carrying Gemcitabine to Inhibit the Proliferation of Triple Negative Breast Cancer Cell

doi: 10.3390/pharmaceutics14061263

Figure Lengend Snippet: In vivo PD-1&GEM NVs promoted the density of CD8 + T cells in spleens and lymph nodes. ( A , B ) Representative plots and quantitative analysis of CD8 + T cells (gated on positive CD3 + cells) in spleens in different treated mice groups by flow cytometry ( n = 3). Error bar, mean ± SEM. ( C , D ) Representative plots and quantitative analysis of CD8 + T cells (gated on positive CD3 + cells) in lymph nodes in different treated mice groups by flow cytometry ( n = 3). Error bar, mean ± SEM. ( E ) Histological images for H&E staining obtained from the spleen of mice treated in different group. Data were expressed as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. scale bar = 100 µm.

Article Snippet: Human PD-1 lentivirus open reading fragment (ORF) cDNA expression plasmid with green fluorescent protein (C-GFP Spark tag) and mouse PD-1 lentivirus ORF cDNA expression plasmid (C-GFP Spark tag) were supplied by Sino Biological Inc (Sino Biological, Beijing, China).

Techniques: In Vivo, Flow Cytometry, Staining