Journal: Molecular oncology

Article Title: PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

doi: 10.1016/j.molonc.2014.09.009

Figure Lengend Snippet: Figure 1 e Experimental assays for characterization of PHF21B. (A) Scheme of the PHF21B gene and its promoter-associated CpG islands organized according to the physical location on 22q13.31. This gene contains 13 exons spanning approximately 128 Kb of DNA (UCSC Genome Browser on Human, assembly GRCh37/hg19, http://genome.ucsc.edu) and a highly dense promoter-associated CpG island. This island spans 3336 bp, including the 50 region, with the first two exons of this gene overlapping three alternative transcription start sites: (*) transcript variant 1, NM_138415.4, (**) transcript variant 2, NM_001135862.2, and (***) transcript variant 3, NM_001242450.1. The comprehensive analysis of the PHF21B gene in HNSCC patients included several target regions for PCR-based assays for detection of the DNA copy number (30UTR), gene expression (exons 5e7), and mutations (exons 3, 6, 7, 8, 9, and 11). Changes in DNA methylation of CpG Island 273 were screened for three regions in HNSCC-derived cell lines. (B) DNA copy number changes in the PHF21B gene in matched normal (peripheral blood) and tumoral samples obtained from HNSCC patients. Constitutive losses of PHF21B were detected in cases showing a positive family cancer history (case 04), in case 50 (negative for family cancer history), and in cases 35, 49, and 71 (unknown information). Tumor-specific acquired losses were detected in

Article Snippet: PHF21B overexpression The plasmid containing the human ORF for PHF21B (NM_138415) GFP-tagged was purchased from Origene.

Techniques: Variant Assay, Gene Expression, DNA Methylation Assay, Derivative Assay

Journal: Molecular oncology

Article Title: PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

doi: 10.1016/j.molonc.2014.09.009

Figure Lengend Snippet: Figure 2 e Clonogenesis and migration assays performed for PHF21B. (A) Amino acid sequence alignment between PHF21A and PHF21B reveals conservation of domains important for transcriptional repression. The sequences from the two proteins were aligned using BLASTP. Important functional domains from PHF21A were visualized by UNIPROT: a GLN-rich region is found between residues 4-108; a DNA binding domain A.T Hook is found between 425 and 437; a PHD-type zinc finger domain is found between 488 and 535; a region required for transcriptional repression is found between 486 and 680. (BeH) Subcellular distribution of endogenous and transfected PHF21B. FaDU (B and C) and SCC4 (D)

Article Snippet: PHF21B overexpression The plasmid containing the human ORF for PHF21B (NM_138415) GFP-tagged was purchased from Origene.

Techniques: Migration, Sequencing, Functional Assay, Binding Assay, Transfection

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: C3aR localization on mitochondria is increased by oxidative stress. Confocal imaging of C3aR immunolabeling (A) and western blot analysis (B) was performed on H 2 O 2 -treated ARPE-19 cell and isolated mitochondria. (A) ARPE-19 cells were treated with H 2 O 2 (0 as control, 0.2, 0.5 mM, 0.5 mM pre-treated with MnTABAP) and labeled with MTDR (red) for the identification of mitochondria and Hoechst33342 (blue) to identify the nucleus. C3aR were identified using Alexa-488 labeled secondary antibodies (green). (B) Quantitative analysis of C3aR relative fluorescence intensity on the entire cell (left graph), and normalized number of C3aR particles colocalized with MTDR fluorescence (identified as yellow particles) (right graph). (C) Western blot analysis for C3aR was performed in total cellular homogenates (C3aR and β-actin loading control) and mitochondrial fractions (mtC3aR and mitochondrial Cox IV protein loading control) of control and H 2 O 2 -treated ARPE-19 cells. (D) C3aR levels in total cellular homogenates (left graph) did not change with treatment; wherease mtC3aR were elevated with H 2 O 2 -treatment in a dose-dependent manner, an effect that could be reversed by pre-treatment with MnTBAP prior to the addtion of H 2 O 2 (right graph). (E) Orthogonal projections of C3aR colocalization on MTDR in 0.5 mM H 2 O 2 treated ARPE 19 cells and corresponding fluorescence intensity profile measurements of 3 different colocalized particles (#1–3). (F) Immunolabeling of C3aR on MTDR-labeled mitochondria isolated from control (left panel) and 0.5 mM H 2 O 2 -treated cells (middle), using no anti-C3aR antibody as a control (right), and its quantification. (G) HeLa cells are used as a positive control for C3aR receptor expression. Cells were stained with the antibody against C3aR (green) and colabeled with wheat germ agglutinin (WGA, red) to identify the plasma membrane, TMRM (magenta) for the identification of mitochondria and Hoechst 33,342 (blue) for the nucleus. Positive labeling was identified on the plasma membrane (left) and mitochondria (right); the lack of signal in the absence of the primary antibody documents the lack of non-specific binding of the secondary antibody (middle). (H) Hek293 cells are used as a negative control for C3aR receptor expression. Lack of C3aR was confirmed by immunolabeling on Hek293 cells (left) and western blot analysis on mitochondrial fractions (mtC3aR and mitochondrial Cox IV protein loading control), using control (left lane) and H 2 O 2 -treated ARPE-19 cells (middle lane) for comparison.Scale bar in (A) 10 μm (top row) and 5 μm (bottom row). n = 3 individual imaging experiments, multiple cells each, one-way ANOVA, T -test: * P < 0.05, ** P < 0.001. n = 3 samples for Western blotting, Kruskall–Wallis test, Mann–Whitney U -test: * P < 0.05.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Imaging, Immunolabeling, Western Blot, Isolation, Labeling, Fluorescence, Positive Control, Expressing, Staining, Binding Assay, Negative Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: Intracellular C3a-C3aR colocalization on mitochondria. (A) Confocal images of C3a and C3aR immunolabeling on mitochondria in ARPE-19. C3a- (red) and C3aR-fluorescent particles (green) distribute on mitochondria identified by MTDR (false-colored blue) in control and 0.5 mM H 2 O 2 -treated ARPE-19 cells. The insets provide higher magnification images. (B) Quantitative analysis of C3a-C3aR colocalization (yellow) demonstrates a dose-dependent increase. (C) Western blot analysis for C3a immunoreative contents in the cytoplasm of control cells and those treated with H 2 O 2 , or pre-treated with MnTBAP prior to the addition of H 2 O 2 . Anti-C3a antibody recognized 4 bands [~120, ~47, ~35, and 9–10 kDa (C3a fragment)]. (D) Quantitative analysis, calculating the ratio of the C3a fragment over the total protein content recognized by the anti-C3a antibody. Scale bar in (A) 10 μm. n = 3 individual imaging experiments, multiple cells each, one-way ANOVA, T -test: * P < 0.05. n = 3 samples for Western blotting, Kruskall–Wallis test, Mann–Whitney U -test.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Immunolabeling, Western Blot, Imaging, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: Live cell imaging of plasma membrane C3aR-GFP internalization. (A) ELISA assay documenting a dose-dependent increase of C3a in the culture medium 45 minutes after H 2 O 2 treatment, an effect that could be blunted by the cell-permeable superoxide dismutase mimetic and peroxynitrite scavenger Mn-TBAP. (B) Representative images captured from the time lapse analysis of C3aR-GFP internalization from the ARPE-19 plasma membrane to the intracellular compartment in response to 0.2 mM H 2 O 2 stimulation (top), no treatment (control, middle), and 0.2 mM H 2 O 2 in the presence of the endocytosis inhibitor, dynasore (bottom). The color image represents C3aR-GFP (green), WGA (red) to label plasma membranes and Hoechst (blue) to identify nuclei. The black and white (binary) image represents in the intracellular C3aR-GFP that is no longer colocalized with the WGA signal, which enables documentation of transfer into the intracellular compartment over time. (C) Quantitative analysis of normalized changes of C3aR-GFP fluorecence colocalized with WGA (red) (=C3a-GFPmem), and the internalized C3aR-GFP particles (=C3a-GFPin) over time derived from the time lapse data. In cells treated with H 2 O 2 -treated (left graph) the C3aR-GFP population on plasma mebrane remains mostly stable, wherease the levels in the intracellular compartment increase. In cells pre-treated with SB290157 prior to the addition of H 2 O 2 , no translocation was observed (right graph). (D) Quantitiative analysis of C3aR-GFP particles on the plasma membrane over time (left graph) and at the 60 minute time point, comparing membrane and intracellular localization (right graph). In cells treated with H 2 O 2 , membrane localization of C3aR-GFP is decreased, wherease levels in the intracellular compartment increase. In cells pre-treated with the endocytosis inhibitor dynasore or SB290157 prior to the addition of H 2 O 2 , no translocation was observed. Scale bar in (A) 10 μm. n =3. One-way ANOVA, T -test: * P < 0.05, ** P < 0.001.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Live Cell Imaging, Enzyme-linked Immunosorbent Assay, Derivative Assay, Translocation Assay

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: Live cell imaging analysis documenting C3aR-GFP trafficking to mitochondria. ARPE-19 cells were transiently transfected with C3aR-GFP (green) and the endosome marker Rab7-RFP (false colored magenta). For imaging, mitochondria were labeled with MTDR (red) and nuclei were identified with Hoechst (blue). (A) Representative time lapse images of 0.2 mM H 2 O 2 -treated cells are indicated, allowing for analysis of C3aR-GFP and MTDR as well as C3aR-GFP and Rab7-RFP colocalization. (B–F) Quantitative analysis of colocalization of respective markers over time, documenting the transfer of C3aR-GFP to mitochondrial via endosomes. (B) C3aR-GFP colocalization with MTDR increased over time in H 2 O 2 but not control cells; an effect that could be inhibited by CID1067700. (C) C3aR-GFP colocalization with Rab7-RFP-positive endosomes (<2.05 μm 2 , see for rationale) increased transiently in H 2 O 2 but not control cells; an effect that could be inhibited by CID1067700. (D) Finally, Rab7-RFP and MTDR colocalization increased over time in H 2 O 2 but not control cells; an effect that could be inhibited by CID1067700. Together the data in B-D show the transient localization of C3aR to endosomes, the increase in contact between mitochondria and endosomes and the accumulation of C3aR in mitochondria over time, an effect that is dependent on Rab7 signaling. (E) In cells transiently transfected with C3aR-GFP and the lysosome marker Lamp1-RFP , contacts between lysosomes and mitochondria increased over time in H 2 O 2 but not control cells; an effect that could be inhibited by CID1067700. (F) However, colocalization between lysosomes and C3aR-GFP was not affected by H 2 O 2 exposure. Scale bar in (A) 10 μm. n = 3 individual imaging experiments, multiple cells each, one-way ANOVA, T -test: * P < 0.05, ** P < 0.001, *** P < 0.0001.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Live Cell Imaging, Transfection, Marker, Imaging, Labeling

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: mtC3aR activation enhances mitochondrial Ca 2+ uptake. MTDR and Fluo-8 AM pre-labeled mitochondria were isolated under caclium free conditions to enable calcium imaging in MTDR-positive organelles. Mitochondria were isolated from control cells and cells exposed to 0.2 and 0.5 mM H 2 O 2 to enable mitochondrial transfer of C3aR. In all experiments, C3aR activation was triggered by 260 nM C3a, unless otherwise noted. The Fluo-8 AM signal was analyzed at 2 time points after the addition of calcium, the early [100–200 s, (E) top graph] and late phase [1,000–1,100 s, (E) bottom graph]. (A) A representative color image of calcium flux measured with Fluo-8 AM on MTDR-labeled isolated mitochondria from 0.5 mM H 2 O 2 -treated cells which time point?. C3aR activation was mediated by 260 nM C3a, calcium uptake was triggered by exposure to 10 μM Ca 2+ . (B) Fluo-8 AM fluorescence intensity changes measured over time in mitochondria isolated from no-treatment (control, gray), 0.2 mM H 2 O 2 -treated (purple) and 0.5 mM H 2 O 2 -treated (black and yellow) ARPE-19 cell. In the absence of C3a, calcium exposure triggered a slow wave in control mitochondria, compared to a fast wave in 0.5 mM H 2 O 2 mitochondria. In the presence of C3a, calcium exposure triggered an early and late calcium response in 0.5 mM H 2 O 2 mitochondria, but only the early response in 0.2 mM H 2 O 2 mitochondria. (C) Both the early and late phase of the calcium response triggered in H 2 O 2 mitochondria triggered by C3a and Ca 2+ could be inhibited by the C3aR antagonist SB290157 (blue) and the Gαi G-protein inhibitor PTX (pink). (D) Fluo-8 AM fluorescence intensity changes measured over time in the presence of inhibitors known to block the mitochondrial calcium uniporter (mCU, Ru360) and the mitochondrial permeability transition pore (mPTP, cyclosporin A, CsA). Ru360 blocked the Ca 2+ -induced calcium uptake, CsA had no effect on the rapid Ca 2+ elevation, but blocked the late phase of the response. (E) The normalized Fluo-8 AM intensity changes of the early phase (100–200 s) and late phase (1,000–1,100 s) are analyzed and presented for comparison. Scale bar in (A) 10 μm. n = 3 individual imaging experiments, multiple cells each, one-way ANOVA, T -test: * P < 0.05, ** P < 0.001.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Activation Assay, Labeling, Isolation, Imaging, Fluorescence, Blocking Assay, Permeability

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: mtC3aR activation inhibited mitochondrial electron transport chain activiy. High resolution respirometric XFe96 Seahorse assays were utilized on mitochondria isolated from control cells and cells exposed to 0.2 and 0.5 mM H 2 O 2 to drive C3aR to the mitochondria. In all experiments, C3aR activation was triggered by 260 nM 30 min prior to the assay, unless otherwise noted. Oxygen consumption rates (OCR) are recorded every 5 min to determine ADP driven state III respiration and the maximal respiratory capacity. Those 2 parameters were analyzed upon injection of ADP (4 mM) and the protonophore FCCP (6 μM), respectively. All data was normalized to the mean OCR value of the control at peak state III or FCCP response. (A) Examples of state III respiration (left) and FCCP response (right) are presented from mitochondria isolated from H 2 O 2 -treated ARPE-19 and treated with C3a and C3a + C3a antagonist, respectively. Quantitative OCR results of (B) state III respiration and the (C) FCCP response of isolated mitochondria (control, 0.2, 0.5 mM H 2 O 2 ) stimulated with C3a and/or C3a and C3aR antagonist SB290157 are presented. C3a inhibited ADP drive (state III respiration) and FCCP response in stressed mitochondria, an effect that could be reversed by C3aR antagonism. To examine the involvement of G-protein signaling and the known downstream signaling molecule of Gαi in mitochondria, the soluble adenylyl cyclase, (D) state III respiration and the (E) FCCP response of isolated mitochondria (0.5 mM H 2 O 2 ) stimulated with C3a was determined. Normalized OCR results demonstrated that the Gαi inhibitor PTX and the soluble adenylyl cyclase activator HCO 3 - reversed the inhibtory effect of C3a on ADP drive as well as on maximal respiratory capacity. Data from 9 to 25 wells in 3–5 independent experiments are reported. One-way ANOVA, T -test: * P < 0.05, ** P < 0.001, *** P < 0.0001.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Activation Assay, Isolation, Injection

Journal: Frontiers in Immunology

Article Title: Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism

doi: 10.3389/fimmu.2021.628062

Figure Lengend Snippet: ARPE-19 cell cybrids that confer disease risk exhibit high levels of mtC3aR and their activation ATP synthesis. Transmitochondrial cybrid ARPE-19 cells contain identical nuclear but different mitochondrial DNA. The J-haplotype is associated with reduced metabolism and increased disease risk when compared to the H-haplotype. (A) Confocal microscopy was used to analyze the distribution of C3aR by immunohistochemistry (green). Mitochondria were identified by MTDR (red), the nucleus by Hoechst 33,342 (blue). Images in cells exposed to vehicle (left column) and H 2 O 2 (right hand column) are provided. The inset highlights the mitochondrial localization (yellow). (B) Quantitative analysis of C3aR particle counts on mitochondria normalized to that of identified in H-cybrids exposed to vehicle. mtC3aR levels in vehicle treated J-cybrids are 3-fold higher than those of H-cybrids. And while mtC3aR levels could be increased upon 0.5 mM H 2 O 2 exposure in H-cybrids, levels in J-cybrids could not be elevated any further. (C) Western Blot analysis of C3aR in mitochondrial protein fractions isolated from J- and H-cybrids confirmed the results of the imaging analysis. (D,E) Mitochondrial respiration was analyzed in isolated mitochondria harvested from J- and H-cybrids as well as control and H 2 O 2 treated ARPE-19 cell. XFe96 Seahorse assays were utilized as in , determining OCR levels for state III respiration (D) and maximal respiratory capacity [FCCP response, (E)] . All values are reported as OCR %, representing the measured value normalized to its own baseline. mtC3aR were activated by the addition of C3a prior to the respirometric analysis. The FCCP response in mitochondria from J-cybrids and H 2 O 2 treated ARPE-19 cells was susceptible to C3a inhibition, whereas those from control and H-cybrids were not. State III respiration was not affected by any of the parameters. Scale bar in (A) 10 μm. n = 3 individual imaging experiments, multiple cells each, one-way ANOVA, T -test. n = 3 samples for Western blotting, Kruskall–Wallis test, Mann–Whitney U -test. Data for Seahorse from 8 wells, one-way ANOVA, T -test: * P < 0.05, ** P < 0.001.

Article Snippet: Plasmids for C3aR-GFP (human C3a Receptor cDNA ORF Clone, C-GFPSpark ® , tag; Sino Biological, Wayne PA), RFP-Rab7 [canine Rab7, N-terminal fusion with red fluorescent protein, RFP; Addgene, Watertown MA; ( ) and Lamp1-RFP [rat Lamp1, C-terminal fusion with RFP; Addgene ( ) were used for transient transfections.

Techniques: Activation Assay, Confocal Microscopy, Immunohistochemistry, Western Blot, Isolation, Imaging, Inhibition, MANN-WHITNEY

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