Journal: Cell reports

Article Title: Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

doi: 10.1016/j.celrep.2018.12.014

Figure Lengend Snippet: Figure 1. MTDs Impair Cell Surface Expression of PD-1 by T Lymphocytes (A) Drug screen for inhibitors of cell surface PD-1 expression by human T cells gated from activated PBMCs. Microtubule-unrelated drugs (gray) and significant hits after Bonferroni correction for multiple comparisons versus untreated stimulated cells are shown as colored bars. CPT, camptothecin. n = 6, means ± SDs. (B) Representative contour plots of PD-1 and CD69 expression by resting and stimulated T lymphocytes treated or not treated with CI-980 (10 nM); gated from PBMC; representative of 6 experiments; percentages of cells in gate are shown. (C) Cell surface PD-1 expression by activated CD4+, CD8+, and TCR gd T lymphocytes (gated from activated PBMCs), treated or not treated with CI-980 (10 nM); representative of 6 experiments; percentages of cells in gate are shown. (D) CD3 and CD4, CD8, and TCR Vg9 expression by activated CD3+CD4+, CD3+CD8+, and CD3+TCR Vg9+ T cells, respectively, treated or not treated with CI-980 (10 nM); representative of 6 experiments. (E) CD3+ T cell differentiation, defined by the CD45RA and CCR7 markers, in treated (CI-980, 10 nM) or untreated conditions; representative of 6 experiments. (F–H) Quantification of the effect of CI-980 (10 nM) on CD3+ T cell differentiation (F), and on CD69 (G) and PD-1 (H) expression by the naive (CD45RA+CCR7+), central memory (CM) (CD45RACCR7+), effector memory (EM) (CD45RACCR7), and terminally differentiated effector memory (EMRA) (CD45RA+CCR7) T cells (n = 6, means ± SDs). Wilcoxon test; *p < 0.05, ***p < 0.001, ****p < 0.0001. (I) PD-1 inhibition level on activated T lymphocytes (gated from activated PBMCs) treated with MTDs including colchicin-binding site drugs (range of reds), vinca alkaloids (range of oranges), and taxane (green). CBT-A4, combretastatin-A4; PPTx, podophyllotoxin; b-Lumi, b-lumicolchicine (n = 6, means ± SDs).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited Data 30-UTR length (Grillo et al., 2010) http://utrdb.ba.itb.cnr.it Kinesin gene expression in immune cells NCBI GEO dataset repository GSE12195, GSE13738, GSE14879, GSE27291, GSE28490, GSE28491, GSE28726, GSE43769, GSE45535, GSE56314, GSE56464, GSE61697, GSE65010, GSE66384, GSE71566, GSE72642, GSE8059 Experimental Models: Cell Lines Human: HeLa cells ATCC CCL-2; RRID:CVCL_0030 Oligonucleotides ON-TARGET plus Non-targeting Pool (siScramble) Dharmacon D-001810 ON-TARGET plus Human KIF1B siRNA Dharmacon L-009317 ON-TARGET plus Human KIF3A siRNA Dharmacon L-004964 ON-TARGET plus Human KIF5B siRNA Dharmacon L-008867 ON-TARGET plus Human KLC1 siRNA Dharmacon L-019482 ON-TARGET plus Human DYNC1H1 siRNA Dharmacon L-006828 See Table S4 for the list of Oligos N/A N/A Recombinant DNA PDCD1 cDNA coding vector Origene SC117011 PDCD1 cDNA coding vector (p1 - full 50UTR) This paper N/A CD3E cDNA coding vector (p13) This paper N/A Software and Algorithms Cytobank Stanford University, Stanford, California (Kotecha et al., 2010) https://www.cytobank.org/ ImageJ NIH https://imagej.nih.gov/ij/ Icy France-BioImaging, Institut Pasteur, France (de Chaumont et al., 2012) http://icy.bioimageanalysis.org/ Find Fasta max length This paper https://sites.google.com/site/fredsoftwares/ products/find-fasta-max-length Zen Blue Carl Zeiss Microscopy GmbH N/A Harmony 4.6 Perkin Elmer http://www.perkinelmer.com:80/product/ harmony-4-8-office-hh17000001 Vigibase World Health Organization’s (WHO) https://www.who-umc.org/vigibase/vigibase/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific- software/prism/

Techniques: Expressing, Cell Differentiation, Inhibition, Binding Assay

Journal: Cell reports

Article Title: Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

doi: 10.1016/j.celrep.2018.12.014

Figure Lengend Snippet: Figure 2. PDCD1 mRNA Binds to Tubulin in Activated T Cells (A) Projection of confocal microscopy images of stimulated T lymphocytes treated or not treated with CI-980 (white lines, cell contour). Scale bar: 2 mm. Representative of 4 experiments. (B) Automated quantification of T cell staining for the specified marker in CI-980-treated cells (red, n = 73) and untreated control cells (purple, n = 53). Bars: group medians; Mann-Whitney test; ****p < 0.0001. (C) qRT-PCR quantification of PDCD1, CD69, and CD3E mRNAs immunoprecipitated from the lysates of activated T lymphocytes by anti-tubulin or isotype (Ig) control antibodies (n = 5, means ± SDs). t test; **p < 0.01. (D) RNA affinity chromatography of T cell lysates using biotinylated RNA fragments, as indicated, followed by western blot. FL, full length. Representative of 2 experiments. (E and F) Surface plasmon resonance sensorgrams of (E) T cell lysates on sensor chips coated with the specified PDCD1 mRNA fragment and (F) after the addition of antibodies to tubulin. Representative of 2 experiments. RU, resonance unit.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited Data 30-UTR length (Grillo et al., 2010) http://utrdb.ba.itb.cnr.it Kinesin gene expression in immune cells NCBI GEO dataset repository GSE12195, GSE13738, GSE14879, GSE27291, GSE28490, GSE28491, GSE28726, GSE43769, GSE45535, GSE56314, GSE56464, GSE61697, GSE65010, GSE66384, GSE71566, GSE72642, GSE8059 Experimental Models: Cell Lines Human: HeLa cells ATCC CCL-2; RRID:CVCL_0030 Oligonucleotides ON-TARGET plus Non-targeting Pool (siScramble) Dharmacon D-001810 ON-TARGET plus Human KIF1B siRNA Dharmacon L-009317 ON-TARGET plus Human KIF3A siRNA Dharmacon L-004964 ON-TARGET plus Human KIF5B siRNA Dharmacon L-008867 ON-TARGET plus Human KLC1 siRNA Dharmacon L-019482 ON-TARGET plus Human DYNC1H1 siRNA Dharmacon L-006828 See Table S4 for the list of Oligos N/A N/A Recombinant DNA PDCD1 cDNA coding vector Origene SC117011 PDCD1 cDNA coding vector (p1 - full 50UTR) This paper N/A CD3E cDNA coding vector (p13) This paper N/A Software and Algorithms Cytobank Stanford University, Stanford, California (Kotecha et al., 2010) https://www.cytobank.org/ ImageJ NIH https://imagej.nih.gov/ij/ Icy France-BioImaging, Institut Pasteur, France (de Chaumont et al., 2012) http://icy.bioimageanalysis.org/ Find Fasta max length This paper https://sites.google.com/site/fredsoftwares/ products/find-fasta-max-length Zen Blue Carl Zeiss Microscopy GmbH N/A Harmony 4.6 Perkin Elmer http://www.perkinelmer.com:80/product/ harmony-4-8-office-hh17000001 Vigibase World Health Organization’s (WHO) https://www.who-umc.org/vigibase/vigibase/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific- software/prism/

Techniques: Confocal Microscopy, Staining, Marker, Control, MANN-WHITNEY, Quantitative RT-PCR, Immunoprecipitation, Chromatography, Western Blot, SPR Assay

Journal: Cell reports

Article Title: Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

doi: 10.1016/j.celrep.2018.12.014

Figure Lengend Snippet: Figure 3. PDCD1 30 UTR and ORF Are Important Regulatory Elements of PDCD1 mRNA Regulation (A) Schematic of the different constructs used for the transfection experiments in HeLa cells. (B) Representative western blots of PD-1 in lysates from HeLa cells transfected by the specified construct and treated or not treated with CI-980 (10 nM). Representative of 5 experiments. (C and D) PD-1 protein (C) and PDCD1 mRNA (D) levels in HeLa cells transfected with the specified construct and treated or not treated with CI-980 (10 nM), relative to the untreated p1 construct (n = 5, means ± SDs). t test; *p < 0.05, **p < 0.01, ***p < 0.001. (E) Representative western blots of CD3ε in the lysates from HeLa cells transfected with the p13 construct and treated or not treated with CI-980 (10 nM). Representative of 5 experiments. (F and G) CD3ε protein (F) and CD3E mRNA (G) levels in HeLa cells transfected with p13, and treated or not treated with CI-980 (10 nM), relative to the untreated construct (n = 5, means ± SDs). (H and I) PD-1 and CD3ε protein (H) and PDCD1 and CD3E mRNA (I) levels in HeLa cells transfected with the specified construct, treated or not treated with CI-980 (10 nM), relative to the untreated and native construct (p1 or p13, respectively); n = 3, means ± SDs; t test; *p < 0.05.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited Data 30-UTR length (Grillo et al., 2010) http://utrdb.ba.itb.cnr.it Kinesin gene expression in immune cells NCBI GEO dataset repository GSE12195, GSE13738, GSE14879, GSE27291, GSE28490, GSE28491, GSE28726, GSE43769, GSE45535, GSE56314, GSE56464, GSE61697, GSE65010, GSE66384, GSE71566, GSE72642, GSE8059 Experimental Models: Cell Lines Human: HeLa cells ATCC CCL-2; RRID:CVCL_0030 Oligonucleotides ON-TARGET plus Non-targeting Pool (siScramble) Dharmacon D-001810 ON-TARGET plus Human KIF1B siRNA Dharmacon L-009317 ON-TARGET plus Human KIF3A siRNA Dharmacon L-004964 ON-TARGET plus Human KIF5B siRNA Dharmacon L-008867 ON-TARGET plus Human KLC1 siRNA Dharmacon L-019482 ON-TARGET plus Human DYNC1H1 siRNA Dharmacon L-006828 See Table S4 for the list of Oligos N/A N/A Recombinant DNA PDCD1 cDNA coding vector Origene SC117011 PDCD1 cDNA coding vector (p1 - full 50UTR) This paper N/A CD3E cDNA coding vector (p13) This paper N/A Software and Algorithms Cytobank Stanford University, Stanford, California (Kotecha et al., 2010) https://www.cytobank.org/ ImageJ NIH https://imagej.nih.gov/ij/ Icy France-BioImaging, Institut Pasteur, France (de Chaumont et al., 2012) http://icy.bioimageanalysis.org/ Find Fasta max length This paper https://sites.google.com/site/fredsoftwares/ products/find-fasta-max-length Zen Blue Carl Zeiss Microscopy GmbH N/A Harmony 4.6 Perkin Elmer http://www.perkinelmer.com:80/product/ harmony-4-8-office-hh17000001 Vigibase World Health Organization’s (WHO) https://www.who-umc.org/vigibase/vigibase/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific- software/prism/

Techniques: Construct, Transfection, Western Blot

Journal: Cell reports

Article Title: Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

doi: 10.1016/j.celrep.2018.12.014

Figure Lengend Snippet: Figure 4. Microtubule Motors Kinesin 1 Convey PDCD1 mRNA to Ribosomes (A) High-resolution microscopy of a-tubulin (green) and PDCD1 mRNA (orange) in HeLa cells transfected with full-length PDCD1. (B) Western blot (WB) of PD-1, CD3ε, and the indicated motor proteins in PDCD1- or CD3E-HeLa transfectants silenced by the specified small interfering RNA (siRNA) or by a scramble siRNA control (Scr). Representative of 3 experiments. (C) PD-1 and CD3ε protein levels in the specified siRNA-mediated knockdown experiment. Percentage of the level in transfectants silenced by Scr control (n = 3, means ± SDs). t test; *p < 0.05. (D) qRT-PCR quantification of PDCD1, CD69, and CD3E mRNAs after immunoprecipitation with an anti-KIF5B, anti-KLC1, anti-KIF1B, or isotype (Ig) control antibody from the lysates of activated T lymphocytes (n = 3, means ± SDs). (E) Polysomal profiling of HeLa cells treated or not treated with CI-980 (10 nM). Representative of 4 experiments. (F) PDCD1, CD3E, and GAPDH mRNA levels by qRT-PCR in non-polysomal (NP) and polysomal (P) fractions from HeLa cells transfected with the PDCD1 and CD3E plasmid reporters and treated or not treated with CI-980 (n = 4, means ± SDs). Wilcoxon test; **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited Data 30-UTR length (Grillo et al., 2010) http://utrdb.ba.itb.cnr.it Kinesin gene expression in immune cells NCBI GEO dataset repository GSE12195, GSE13738, GSE14879, GSE27291, GSE28490, GSE28491, GSE28726, GSE43769, GSE45535, GSE56314, GSE56464, GSE61697, GSE65010, GSE66384, GSE71566, GSE72642, GSE8059 Experimental Models: Cell Lines Human: HeLa cells ATCC CCL-2; RRID:CVCL_0030 Oligonucleotides ON-TARGET plus Non-targeting Pool (siScramble) Dharmacon D-001810 ON-TARGET plus Human KIF1B siRNA Dharmacon L-009317 ON-TARGET plus Human KIF3A siRNA Dharmacon L-004964 ON-TARGET plus Human KIF5B siRNA Dharmacon L-008867 ON-TARGET plus Human KLC1 siRNA Dharmacon L-019482 ON-TARGET plus Human DYNC1H1 siRNA Dharmacon L-006828 See Table S4 for the list of Oligos N/A N/A Recombinant DNA PDCD1 cDNA coding vector Origene SC117011 PDCD1 cDNA coding vector (p1 - full 50UTR) This paper N/A CD3E cDNA coding vector (p13) This paper N/A Software and Algorithms Cytobank Stanford University, Stanford, California (Kotecha et al., 2010) https://www.cytobank.org/ ImageJ NIH https://imagej.nih.gov/ij/ Icy France-BioImaging, Institut Pasteur, France (de Chaumont et al., 2012) http://icy.bioimageanalysis.org/ Find Fasta max length This paper https://sites.google.com/site/fredsoftwares/ products/find-fasta-max-length Zen Blue Carl Zeiss Microscopy GmbH N/A Harmony 4.6 Perkin Elmer http://www.perkinelmer.com:80/product/ harmony-4-8-office-hh17000001 Vigibase World Health Organization’s (WHO) https://www.who-umc.org/vigibase/vigibase/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific- software/prism/

Techniques: Microscopy, Transfection, Western Blot, Small Interfering RNA, Control, Knockdown, Quantitative RT-PCR, Immunoprecipitation, Plasmid Preparation

Journal: Cell reports

Article Title: Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

doi: 10.1016/j.celrep.2018.12.014

Figure Lengend Snippet: Figure 5. Activation-Induced SGs Regulate PD-1 Expression (A) qRT-PCR quantification of SG component mRNAs at different time points after T cell activation, in treated (CI-980, 10 nM) or untreated conditions (n = 3, means ± SDs). (B) qRT-PCR quantification of P-body component mRNAs at different time points after T cell activation, in treated (CI-980, 10 nM) or untreated conditions (n = 3, means ± SDs). (C) Protein quantification of SGs and P-body components at different time points after T cell activation, in treated (CI-980, 10 nM) or untreated conditions (n = 3, means ± SDs). A representative western blot is shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited Data 30-UTR length (Grillo et al., 2010) http://utrdb.ba.itb.cnr.it Kinesin gene expression in immune cells NCBI GEO dataset repository GSE12195, GSE13738, GSE14879, GSE27291, GSE28490, GSE28491, GSE28726, GSE43769, GSE45535, GSE56314, GSE56464, GSE61697, GSE65010, GSE66384, GSE71566, GSE72642, GSE8059 Experimental Models: Cell Lines Human: HeLa cells ATCC CCL-2; RRID:CVCL_0030 Oligonucleotides ON-TARGET plus Non-targeting Pool (siScramble) Dharmacon D-001810 ON-TARGET plus Human KIF1B siRNA Dharmacon L-009317 ON-TARGET plus Human KIF3A siRNA Dharmacon L-004964 ON-TARGET plus Human KIF5B siRNA Dharmacon L-008867 ON-TARGET plus Human KLC1 siRNA Dharmacon L-019482 ON-TARGET plus Human DYNC1H1 siRNA Dharmacon L-006828 See Table S4 for the list of Oligos N/A N/A Recombinant DNA PDCD1 cDNA coding vector Origene SC117011 PDCD1 cDNA coding vector (p1 - full 50UTR) This paper N/A CD3E cDNA coding vector (p13) This paper N/A Software and Algorithms Cytobank Stanford University, Stanford, California (Kotecha et al., 2010) https://www.cytobank.org/ ImageJ NIH https://imagej.nih.gov/ij/ Icy France-BioImaging, Institut Pasteur, France (de Chaumont et al., 2012) http://icy.bioimageanalysis.org/ Find Fasta max length This paper https://sites.google.com/site/fredsoftwares/ products/find-fasta-max-length Zen Blue Carl Zeiss Microscopy GmbH N/A Harmony 4.6 Perkin Elmer http://www.perkinelmer.com:80/product/ harmony-4-8-office-hh17000001 Vigibase World Health Organization’s (WHO) https://www.who-umc.org/vigibase/vigibase/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific- software/prism/

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell reports

Article Title: Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

doi: 10.1016/j.celrep.2018.12.014

Figure Lengend Snippet: Figure 7. MTDs Impair Inhibitory Checkpoint Expression by T Cells from Cancer Patients and Increase Autoimmunity Risk (A) Immunohistochemistry (IHC) staining for PD-1 expression by TILs (CD3) in biopsies from 2 Hodgkin lymphoma (HL) patients after chemotherapy including vinblastine. Magnification insert: 3400, bars: 500 mm. Results representative of 6 different HL patients. (B) Disproportionality analysis of immune-related adverse events (IrAE-ROR) for microtubule inhibitors (from VigiBase). Means and 95% confidence intervals are shown for each drug, including ICB (range of blues), MTDs of CBS type (range of reds), vinca alkaloid MTDs (range of oranges), taxane MTDs (range of greens), and control drugs for unrelated targets (range of purples). Total population size of N = 202,693 patients. (C) Association of in vitro IC50 for PD-1 expression and IrAE-RORs in 202,693 patients.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited Data 30-UTR length (Grillo et al., 2010) http://utrdb.ba.itb.cnr.it Kinesin gene expression in immune cells NCBI GEO dataset repository GSE12195, GSE13738, GSE14879, GSE27291, GSE28490, GSE28491, GSE28726, GSE43769, GSE45535, GSE56314, GSE56464, GSE61697, GSE65010, GSE66384, GSE71566, GSE72642, GSE8059 Experimental Models: Cell Lines Human: HeLa cells ATCC CCL-2; RRID:CVCL_0030 Oligonucleotides ON-TARGET plus Non-targeting Pool (siScramble) Dharmacon D-001810 ON-TARGET plus Human KIF1B siRNA Dharmacon L-009317 ON-TARGET plus Human KIF3A siRNA Dharmacon L-004964 ON-TARGET plus Human KIF5B siRNA Dharmacon L-008867 ON-TARGET plus Human KLC1 siRNA Dharmacon L-019482 ON-TARGET plus Human DYNC1H1 siRNA Dharmacon L-006828 See Table S4 for the list of Oligos N/A N/A Recombinant DNA PDCD1 cDNA coding vector Origene SC117011 PDCD1 cDNA coding vector (p1 - full 50UTR) This paper N/A CD3E cDNA coding vector (p13) This paper N/A Software and Algorithms Cytobank Stanford University, Stanford, California (Kotecha et al., 2010) https://www.cytobank.org/ ImageJ NIH https://imagej.nih.gov/ij/ Icy France-BioImaging, Institut Pasteur, France (de Chaumont et al., 2012) http://icy.bioimageanalysis.org/ Find Fasta max length This paper https://sites.google.com/site/fredsoftwares/ products/find-fasta-max-length Zen Blue Carl Zeiss Microscopy GmbH N/A Harmony 4.6 Perkin Elmer http://www.perkinelmer.com:80/product/ harmony-4-8-office-hh17000001 Vigibase World Health Organization’s (WHO) https://www.who-umc.org/vigibase/vigibase/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific- software/prism/

Techniques: Expressing, Immunohistochemistry, Control, In Vitro

Journal: Nature metabolism

Article Title: A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation.

doi: 10.1038/s42255-021-00443-2

Figure Lengend Snippet: Fig. 6. α-Syn Expression or Frataxin Deficiency Impacts the DMiro-dMIC60 Complex. (a, g) Representative confocal stack images of MitoSox staining of fly brains as indicated. The total MitoSox intensity is measured for each fly brain and normalized to the background intensity. (a) n=4 (Elav-GAL4) and 5 (Elav>SNCA-A53T) fly brains. p=0.0317. (g) n=5 (Actin>Fh RNAi) and 7 (Actin-GAL4) fly brains. p=0.0313. (b) Whole-body lysates of 5- day-old α-syn-A53T-expressing flies (Actin>UAS-SNCAA53T) and controls (Actin-GAL4) were IPed with anti-DMiro and blotted as indicated. The band intensity of dMIC60 in IP is normalized to that of DMiro. n=4 independent experiments. p=0.0286. (c) Whole-body lysates of 15-day-old flies were immunoblotted. The DMiro band intensity is normalized to that of β-actin from the same blot. n=4. p=0.0286. (d) Confocal stack images show DA neurons immunostained with anti-TH in the PPL1 clusters of 40-day-old flies. The DA neuron number in the PPM1/2 and PPL1 clusters is quantified. n=14, 12, 13, 15 brains for PPL1, and 19, 12, 12, 20 for PPM1/2 (from left to right). p<0.0001, =0.0027, 0.0018. Details of comparisons and p values are in Source Data. (e) The DA neuron number in the PPL1 clusters is quantified. n=6 (TH>dMIC60-WT), 8 (TH>dMIC60-CS), 10 (THSNCAA53T, dMIC60-CS) brains. p<0.0001. (f) Locomotor ability shown as Performance Index of 42-day-old flies. n=25 (TH>dMIC60- WT), 40 (TH>dMIC60-CS), 45 (THSNCAA53T, dMIC60-CS); 3 independent experiments. p=0.009, 0.0007, 0.003. The rest of the precise p

Article Snippet: Constructs pJFRC8–40×UAS-SNCA was generated by PCR amplifying the human SNCA cDNA from the pCMV6-XL5 vector (#SC119919, OriGene), engineered with the unique Xhol/Xbal restriction sites at either side, and cloning it into a pJFRC8–40×UAS-IVS-mCD8::GFP plasmid (a gift from Gerald Rubin, #26221, Addgene) 59.

Techniques: Expressing, 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: 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

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