Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza modulated the neutrophil-associated TIME in the lungs. (A) Experimental setup. The mice were constructed as experimental metastasis models after a period of acclimatization. The mice were prepared as experimental metastasis models following an acclimatization period. Ginseng and Salvia miltiorrhiza extracts were administered daily to the relevant groups according to the schematic diagram, and lung tissues were collected on day 28. (B) Representative bioluminescence images of metastasis in the intravenously implanted LLC mice. (C) Representative H&E images of the tumor area in the lungs of the mice (scale bar: 50 μm). (D) Quantitative analysis of the lung tumor area ( n = 3). (E) Quantitative analysis of the number of lung metastatic nodules in tumor-bearing mice( n = 15). (F) Metastasis rates of LLC tumor-bearing mice ( n = 15). (G) GOBP analysis of intersecting genes in the control, model and G + S groups. (H) GSEA of genes related to neutrophil migration. (I) Representative images of immunohistochemical staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 50 μm). (J) Representative images of immunofluorescence staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 100 μm). All the data are presented as mean ± SEM. #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Construct, Control, Migration, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: CPT inhibited CD62E–mediated neutrophil adhesion and recruitment to metastases. (A) Representative images and quantification of HL–60 cell adhesion to HUVECs (scale bar: 200 μm). (B) Schematic diagram of the parallel plate flow chamber combined with the microscope. (C) Number of neutrophils firmly adhered to HUVECs. (D) Representative immunofluorescence images and quantification of CD62E in the lungs ( n = 3; scale bar: 50 μm). (E) Representative western blot images of CD62E levels in the lungs and quantitative data ( n = 3). GAPDH was used as a loading control. (F) The mRNA expression levels of TNF–α, ICAM1, and VCAM1 in lung tissues were determined by qRT–PCR ( n = 3). (G) The correlation between CD62E expression and neutrophil infiltration in LUAD and LUSC was analyzed via TIMER. (H) Representative images and quantification of Ly6G + neutrophils in lungs detected by flow cytometry ( n = 4). (I) Representative immunofluorescence images and quantification of Ly6G + neutrophils in the lungs (n = 3; scale bar: 50 μm). All the data are presented as mean ± SEM. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Microscopy, Immunofluorescence, Western Blot, Control, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Neutrophil RNA sequencing analysis and correlation analysis of NETs and lung cancer. (A) Schematic diagram of neutrophil isolation and RNA sequencing from mouse lung tissues. (B-C) Heatmap analysis of neutrophil-related genes and KEGG analysis of altered genes. (D) Results of partial GOBP analysis of intersecting genes in the control, model and G + S groups. (E) The expression of HIST3H3 in different cancers and adjacent normal tissues was analyzed via TIMER. (F–H) The correlation between copy number alterations in HIST3H3, ELANE, and MPO and neutrophil infiltration was analyzed via TIMER. (I) The correlations between cumulative survival and HIST3H3, ELANE, MPO and neutrophil infiltration in lung cancer patients were analyzed via TIMER. A two-sided Wilcoxon rank-sum test was used. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: RNA Sequencing, Isolation, Control, Expressing

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza reduced the generation of NETs in lung tissues, and effective substances from ginseng were screened. (A) Representative immunohistochemical images of MPO expression in the lungs (scale bar: 100 μm). (B) Representative immunofluorescence images of H3cit and NE in the lungs (scale bar: 100 μm). (C–E) Quantitative analysis of MPO, H3cit and NE expression in the lungs ( n = 3). (F) Neutrophil MPO release was detected by ELISA. The chemical structure of Rg1 is shown on the right. (G) Representative images and quantification of extracellular DNA release after Sytox™ Green staining (scale bar: 50 μm). All the data are presented as mean ± SEM. ####p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reduced NET formation by inhibiting ROS production and ERK1/2 and MAPK phosphorylation. (A) Representative immunofluorescence images of the NET components H3cit and NE in neutrophils ( n = 3; scale bar: 50 μm). (B–C) Representative western blot images of H3cit expression in neutrophils and quantitative data. GAPDH was used as a loading control ( n = 3). (D-E) ROS production in dHL-60 cells (D) and neutrophils (E) was assessed by flow cytometry. The median fluorescence intensity of ROS was used for quantification. (F-G) Representative western blot images and quantification of p-ERK1/2, ERK1/2, p-MAPK and MAPK expression in dHL-60 cells (F) and neutrophils (G). GAPDH was used as a loading control. All the data are presented as mean ± SEM. # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reversed NET-induced invasion, EMT, and adhesion of LLC cells and increased vascular permeability. (A) LLC cell proliferation was detected via a CCK8 assay. (B) Schematic diagram of NET isolation and coculture with LLC cells in the transwell system. (C) Images of the transwell invasion assay (scale bar, 200 μm). The number of infiltrated cells and the OD values were used for quantification. (D-E) Representative immunofluorescence images and quantification of MMP9 in the lungs (n = 3, scale bar 50 μm). (F) Representative immunofluorescence images of E-cadherin and N-cadherin in LLC cells treated with or without Rg1 or NETs. (G-I) Representative immunofluorescence images and quantification of E-cadherin and N-cadherin in the lungs ( n = 3, scale bar 50 μm). (J) Schematic diagram of the adhesion assay of LLC cells by NETs. (K) Representative fluorescence images of the adhesion assay for DiI-labeled LLC cells trapped within PMA- or Rg1-treated neutrophils. (L) RNA-seq analysis of neutrophils. GSEA of genes related to the regulation of vascular permeability. (M) Schematic diagram of the endothelial permeability assay. (N) The effects of Rg1 or NETs on vascular permeability were tested via an endothelial permeability assay. (O-P) Representative immunofluorescence images and quantification of CD31 and FITC-dextran in the lungs ( n = 3; scale bar, 50 μm). All the data are presented as mean ± SEM. # p < 0.05, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Permeability, CCK-8 Assay, Isolation, Transwell Invasion Assay, Immunofluorescence, Cell Adhesion Assay, Fluorescence, Labeling, RNA Sequencing, Control

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Representative flow cytometry graphs of CD11b and GFP in peripheral blood of KDRGFP mice bearing gliomas. Astrocytic tumors were generated by RCAS/TVA-mediated overexpression of PDGF as described in Methods. Low-grade gliomas were observed by week 5 and high grade by weeks 7–8. Controls (CTL) were mice bearing an intact Kdr locus without GFP knockin. (B) Quantification of CD11b+KDRGFP+ cell frequency in peripheral blood of RCAS/TVA tumor mice at low-grade and high-grade stages. ***P < 0.001, Student’s t test. LEU, Leukocytes. (C) Further characterization of murine CD11b+KDRGFP+ cells in peripheral blood by Ly6C and Ly6G staining. (D) Quantification of Ly6G+ and Ly6C+ frequency out of total CD11b+ cells in the CD11b+KDRGFP+ and CD11b+KDRGFP– populations in peripheral blood at high-grade stage. **P < 0.01, for Ly6Chi cells, Student’s t test. (E) CFU assays were performed on Lin–KDRGFP+ hematopoietic cells from BM, and macrophage (M) colonies and macrophage/granulocyte (GM) colonies, but not granulocyte (G) colonies, were observed. Quantification of various colony types formed from lineage-negative KDRGFP– or KDRGFP+ cells. ***P < 0.001, in granulocytes, Student’s t test. (F) Gene expression (quantitative RT-PCR) of various markers, including Kdr in in vitro–cultured BM Lin– cells exposed to GL261-conditioned medium at different time points. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Flow Cytometry, Generated, Over Expression, Knock-In, Staining, Gene Expression, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: Lethal dose–irradiated C57BL/6 mice were transplanted with Ubc-GFP Rosa26-CreERT2Kdrfl/fl and Rosa26-CreERT2KDRfl/+ BM cells, and GL261 tumors were implanted after BM engraftment. (A) Peripheral white blood cells were analyzed on side scatter (SSC) and GFP by flow cytometry. The GFP+ and GFP– populations were gated for further analysis. CD11b versus GFP (B), Ly6C versus Ly6G (C), and CD3 versus B220 (D) are shown on both GFP+ and GFP– populations. (E) BM cells were also analyzed on side scatter and GFP. (F) Lineage-negative cells were gated for analysis of HSCs and HPCs by c-Kit versus Sca-1 (G). (H) HPCs (Lin–c-Kit+Sca-1–) were further characterized by FcγR versus CD34 within both GFP+ and GFP– populations. The experiment was conducted 5 times.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Irradiation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) The unsupervised principal component analysis of significantly altered genes in Lin–c-Kit+Sca-1– from naive mice, Lin–c-Kit+Sca-1– KDR-GFP+ cells, and Lin–c-Kit+Sca-1–KDR-GFP– cells from BM of tumor-bearing mice. (B) Lin–c-Kit+Sca-1– cells from naive mice (black), Lin–c-Kit+Sca-1– KDR-GFP+ cells (red), and Lin–c-Kit+Sca-1–KDR-GFP– cells (green) from BM of tumor-bearing mice, based on similarity of gene profiles. PC1, principal component 1. (C) The candidate genes (P < 0.05, >1.5-fold change; Lin–c-Kit+Sca-1– KDR-GFP+ versus Lin–c-Kit+Sca-1–KDR-GFP–) were divided according to the subset with the highest expression and analyzed for categories with significant enrichment (P < 0.05) of categories in GO biologic processes using DAVID tools. Similar categories were grouped accordingly. The presence of association between functions and genes was color highlighted (black [negative] versus green [positive]). (D) Expression of Id2 in various lineages of hematopoietic cells. ***P < 0.001, 1-way ANOVA. (E) Expression of ID2 in HPCs (CD45–CD34+) from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 21. (F) Expression of ID2 in CD11b+ blood cells from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 20. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Chimeric C57BL/6 mice transplanted with Id2–/– BM cells (Id2+/+ BM cells as control) were implanted with luciferase-labeled GL261 tumors intracranially. Tamoxifen was applied at day 3 after implantation. The tumors were monitored by bioluminescence. Representative images were taken at day 14. The color bar on the right represents photon intensity. The experiments had 2 replicates. The tumor growth curve is based on bioluminescence. n = 10. **P < 0.01, 1-way ANOVA. (B) Flow cytometry analysis of peripheral blood cells on CD11b, Ly6C, and Ly6G in Id2+/+ BMT and Id2–/– BMT groups. Quantification of CD11b+ cell frequency out of total white blood cells and of Ly6Chi cell frequency out of CD11b+ cells. **P < 0.01, 1-way ANOVA, for each group. n = 7. (C) Tubule formations of HCMEC/D3 (GFP) cocultured with lineage-negative Id2+/+, Id2–/–, Id2–/– scrambled sequence (SC) control, or Id2–/– KDR overexpression (OE) HPCs pretreated with TGF-β/GM-CSF. Quantifications of tubule lengths in the indicated group. ***P < 0.0001, 1-way ANOVA. n = 6. (D) Growth factor–reduced Matrigel plugs of lineage-negative Id2+/+, Id2–/–, Id2–/– SC CTL, or Id2–/– KDR overexpression HPCs pretreated with TGF-β/GM-CSF. Blood vessels (red) were perfused with rhodamine-dextran. Lower panels show magnified views to highlight vascular permeability. Quantifications of based blood vessels density (BVD) in each group. **P < 0.01, 1-way ANOVA. n = 6. Data are represented as mean ± SD. Scale bars: 50 μm (D, upper panels); 20 μm (C, D, lower panels).

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Control, Luciferase, Labeling, Flow Cytometry, Sequencing, Over Expression, Permeability

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 1. Schematic representation of experimental protocol and workflow of this present work. The top panel is the schematic illustration of the SNA substrates inserts into commercial dishes for CD34+HSPCs enrichment. Below which is the schematic representation of SMNP delivery CBE- and sgRNA-plasmids into commercial dishes with SNA substrates inserts. Then, after obtaining umbilical cord blood cells, SNA substrate and SMNP delivery mediated CBE base editing (SNA⋅SMNP⋅CBE)— enable efficiently and precisely modify BCL11A promoter to achieve C-T conversion and HBG elevation in CD34+HSPCs. The edited human CD34+HSPC was successively transplanted into SCID mouse by intraosseous injection to detect the CD34 engraftment.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 3. supramolecular nanoparticle (SMNP) enables delivery of CBE and sgRNA plasmids to modify BCL11A to achieve HBG over expression in vitro. (A), Schematic representation of base-editing experiments in human umbilical cord blood derived CD34+HSPCs enriched by SNA substrate CBE-, sgRNA-(with a red fluorescent label (mCherry) expressing plasmid were co-delivered by SMNP in human CD34+HSPCs enriched by SNA substrate. (B), Representative images of mCherry + cells at 2 h post-transfection following SMNP delivery CBE-, sgRNA-plasmids. (C), Representative flow cytometry results of edited CD34 positive cells ratio after SMNP delivery CBE- and sgRNA- plasmids. (D), Editing efficiency of targeted BCL11A PCR products was detected by Sanger sequencing, with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2.(E), Real-time PCR analysis of mCherry (left panel), BCL11A (middle panel) and HBG (right panel) expression in CD34+HSPCs subjected with/without SMNP delivering co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids (edited CD34+HSPCs/ naïve CD34+HSPCs). CD34+HSPCs delivered by SMNP co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids was designated as edited group, and unedited group. Results are normalized to GAPDH and shown as mean±SEM. (n=3 biological replicates). Statistical tests compare edited samples and unedited control group (**P＜ 0.01). (F), Western blots were used to detect HBG expression at protein level in edited CD34+HSPCs and naïve CD34+HSPCs. Results are representative of three biological replicates.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Over Expression, In Vitro, Derivative Assay, Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Sequencing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 4. Comparison of our novel system and conventional strategy in the aspect of editing efficiency and CD34+HSPCs property. (A), Editing efficiency of targeted BCL11A PCR products from CD34+HSPCs subjected to different strategies. The top panel (SNA⋅SMNP group) is CD34+HSPCs was enriched by our SNA substrate and transfected with CBE- and sgRNA targeting BCL11A- plasmids by SMNP delivery (with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2). The middle panel (IMS⋅EP group) is CD34+HSPCs was isolated by conventional immunomagnetic bead method (IMS) and transfected CBE- and sgRNA targeting BCL11A- plasmids with electroporation (EP) (with C-T conversion value of 33.7 % base edits at position C1, and 10.2 % at position C2). The bottom panel (control group) was CD34+HSPCs without edited treatment. (B), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups: SNA⋅SMNP treated CD34+HSPCs, IMS⋅EP treated CD34+HSPCs and naïve CD34+ (right panel). (C), CD34 mRNA expression in three groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates). (D) mRNA expression of hematopoietic stem cell marker (CD133, CD90) and cell differentiation markers (GATA-1, SPI1, CD235a and CD33) in four groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs, naïve CD34+HSPCs, and PMBC. CD133 and CD90 were shown in left panel, while differ entiation markers including GATA-1, SPI1, CD235a and CD33 were presented in right panel. Results shown as mean±SEM (n=3 biological replicates).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, Transfection, Isolation, Electroporation, Control, Quantitative Proteomics, Expressing, Marker, Cell Differentiation

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 5. Tracks of editing feasibility of SNA to maintain CD34 property by Intraosseous injection of transplanted edited CD34+HSPCs into SCID mouse. (A), Gene edited human CD34+HSPCs were transplanted into SCD mouse with two different injection strategies: intraosseous injection into the bone marrow of the mouse tibia (left panel) and tail vein injection (right panel). (B), Representative immunofluorescence images (scale bar=8μm) of CD34 in the bone marrow (left panel) and peripheral blood of mice at 14 weeks of intraosseous injection (top) and tail vein injection (below) strategy, respectively. (C), Representative flow cytometry results of CD45+ proportion in the bone marrow (left panel) and peripheral blood of transplanted mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. (D), Real-time PCR analysis of CD34 mRNA expression in the bone marrow (left panel) and peripheral blood of mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. Results shown as mean±SEM (n=3 biological replicates, *P<0.05, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection, Immunofluorescence, Flow Cytometry, Labeling, Transplantation Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 6. Detection of BCL11A gene modification and HBG/HBB expression after transplantation of edited human CD34+HSPCs. (A), Representative Sanger sequencing of BCL11A from bone marrow (left panel) and peripheral blood (right panel) of transplanted mice, evidenced from C-T conversions. (B), (C), Real-time PCR was used to detect human HBG and HBB expression in the bone marrow (B) and peripheral blood (C) of SCID mice at 10, 12, 14weeks after transplantation of edited human CD34+HSPCs.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Modification, Expressing, Transplantation Assay, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 7. Comparison of our novel strategy and conventional strategy in maintain CD34 property in vivo. (A), Schematic illustration of protocol. The top panel is the process of conventional strategy IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection, while the below panel is the process of our novel strategy SNA⋅SMNP⋅CBE3 ⋅intraosseous injection. After transplantation edited human CD34+HSPCs with different treatment at 14 weeks, SCID mice from the two different groups were euthanatized, mononuclear cells were resorted and enriched from the bone marrow of mice in both groups. Then, transcriptome analysis was performed to detect and compare the gene expression profiles in each group with naïve CD34 controls. (B), Representative flow cytometry results of CD45+ proportion in the bone marrow of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection counterparts. (C), Real-time PCR analysis of human HBG expression in the bone marrow (left panel) and peripheral blood (right panel) of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅ intravenous (tail vein) injection counterparts. (D), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: IMS⋅EP⋅CBE3⋅intravenous, SNA⋅SMNP⋅CBE3⋅intraosseous injection and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups (right panel). (E), Representative CD34 expression by Real-time PCR in the three groups: SNA⋅SMNP⋅CBE3⋅intraosseous group, IMS⋅EP⋅CBE3⋅tail vein injection counterparts, as well as naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, In Vivo, Injection, Transplantation Assay, Gene Expression, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Control, Quantitative Proteomics

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: Chronic stress strengthens neutrophil self‐recruitment via CXCL2. Representative images of flow cytometry and quantification of A) PB‐infiltrating and B) lung‐infiltrating neutrophils in 4‐week tumor‐bearing mice from the control group ( n = 6), chronic stress group ( n = 6), and chronic stress and propranolol co‐treated group ( n = 6). C) Representative lung immunofluorescence staining of mice injected with DiI + vivo S‐exo. Red indicates DiI + exosomes, blue indicates DAPI, and green indicates MPO. Scale bar: 10 µm (bottom) and 40 µm (up). D) Representative images of flow cytometry and quantification of lung‐infiltrating neutrophils 4 h after injection with DiI + vivo C‐exo ( n = 5) and vivo S‐exo ( n = 5). E) Representative image of DiI‐labeled neutrophils absorbing DiO‐labeled TDEs after 4 h of co‐culture. 488 nm, TDE; 405 nm, Hoechst; 555 nm, neutrophils. The arrows indicate neutrophils that have taken up DiO‐labeled exosomes. F) Representative images of Bouin's and H&E staining for lungs from the 4‐week tumor‐bearing mice. Quantification of the number of lung metastatic nodules and burden of lung metastases ( n = 5 mice/group). G,H) Transwell migration assay to detect the neutrophil recruitment ability of the conditioned medium ( n = 4). I) qRT‐PCR analysis of chemokine expression in naïve neutrophils treated alone ( n = 3), with vivo C‐exo ( n = 3), and vivo S‐exo ( n = 3), respectively for 4 h. J) ELISA analysis of CXCL2 concentrations in the supernatant of naïve neutrophils treated alone ( n = 5), with vivo C‐exo ( n = 5), and vivo S‐exo ( n = 5), respectively for 4 h. K) qRT‐PCR analysis of Cxcl2 expression in naïve neutrophils treated with vivo C‐exo ( n = 3), vivo S‐exo ( n = 3), and vivo S+P‐exo ( n = 3) for 4 h. L) ELISA analysis of CXCL2 concentrations in the lung supernatant of 4‐week tumor‐bearing mice in the control group ( n = 7), chronic stress group ( n = 7), and chronic stress and propranolol co‐treated group ( n = 7). M,N) Transwell migration assay to detect the neutrophil recruitment ability of N+vivo S‐exo CM ( n = 6) and N+vitro ISO‐exo CM ( n = 6) with or without the addition of anti‐CXCL2 antibody. The data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques: Flow Cytometry, Control, Immunofluorescence, Staining, Injection, Labeling, Co-Culture Assay, Transwell Migration Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: TDEs under chronic stress polarize IL‐1β + neutrophils through the TLR4‐NFκβ pathway. A) qRT‐PCR analysis of Il‐1β expression in naïve neutrophils treated alone ( n = 3), with vivo C‐exo ( n = 3), and vivo S‐exo ( n = 3), respectively for 4 h. B) qRT‐PCR analysis of Il‐1β expression of naïve neutrophils treated alone ( n = 3), with vitro C‐exo ( n = 3), and vitro ISO‐exo ( n = 3), respectively for 4 h. C) WB analysis of IL‐1β ( n = 11) and NFκβ pp65 ( n = 5) expression in naïve neutrophils treated alone, with vivo C‐exo, and vivo S‐exo, respectively for 4 h. D) WB analysis of IL‐1β ( n = 6) and NFκβ pp65 ( n = 3) expression in naïve neutrophils treated alone, with vitro C‐exo, and vitro ISO‐exo, respectively for 4 h. E) ELISA analysis of IL‐1β concentrations in the supernatant of naïve neutrophils treated alone ( n = 6), with vivo C‐exo ( n = 6), vivo S‐exo ( n = 6), and ISO ( n = 6), respectively for 4 h. F) IL‐1β + neutrophils quantification by flow cytometry of naïve neutrophils treated alone ( n = 6), with vivo C‐exo ( n = 6), vivo S‐exo ( n = 6), and vivo S+P‐exo ( n = 6), respectively for 4 h. G) Flow cytometry quantification of lung‐infiltrating IL‐1β + neutrophils in 4‐week tumor‐bearing mice in the control group ( n = 8), chronic stress group ( n = 8), and chronic stress & propranolol co‐treated group ( n = 8). H) ELISA analysis of IL‐1β concentrations in the lung supernatant of 4‐week tumor‐bearing mice in the control group ( n = 24), chronic stress group ( n = 24), and chronic stress & propranolol co‐treated group ( n = 24). I) qRT‐PCR analysis of Nfκβ1 , Tlr2 , and Tlr4 expression of naïve neutrophils treated alone ( n = 3), with vivo C‐exo ( n = 3), and vivo S‐exo ( n = 3), respectively for 4 h. J) qRT‐PCR analysis of Nfκβ1 , Tlr2 , and Tlr4 expression of naïve neutrophils treated alone ( n = 3), with vitro C‐exo ( n = 3), and vitro ISO‐exo ( n = 3), respectively for 4 h. K) Flow cytometry quantification of lung‐infiltrating TLR4 + neutrophils in 4‐week tumor‐bearing mice of the control group ( n = 8) and chronic stress group ( n = 8). L) Quantification of IL‐1β + neutrophils by flow cytometry in naïve neutrophils treated alone ( n = 5), with vivo C‐exo ( n = 5), vivo S‐exo ( n = 5), vivo S‐exo and TLR2 inhibitor ( n = 5), vivo S‐exo and TLR4 inhibitor ( n = 5), respectively for 4 h. M) Quantification of IL‐1β + neutrophils by flow cytometry in naïve neutrophils treated alone, with vivo C‐exo ( n = 6), vitro C‐exo ( n = 6), vivo C‐exo and a TLR4 agonist ( n = 6), vitro C‐exo and a TLR4 agonist ( n = 5), vivo C‐exo and a TLR4 agonist and a NFκβ inhibitor ( n = 6), and vitro C‐exo and a TLR4 agonist and a NFκβ inhibitor ( n = 6), respectively for 4 h. The data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: Exosomal SP1 activates the neutrophil TLR4‐NFκβ‐IL‐1β pathway, leading to lung metastasis. A) WB analysis of SP1 expression in vivo C‐exo ( n = 3) and vivo S‐exo ( n = 3). B) WB analysis of SP1 expression in BM‐PMNs treated alone ( n = 6), with vivo C‐exo ( n = 6), or with vivo S‐exo ( n = 6) for 4 hours. C) WB analysis of SP1 expression of BM‐PMNs treated alone ( n = 5), with vitro C‐exo ( n = 5), and vitro ISO‐exo ( n = 5), respectively for 4 h. D) Flow cytometry quantification of IL‐1β + neutrophils in BM‐PMNs treated with vivo C‐exo ( n = 6), vivo S‐exo ( n = 6), vivo S‐exo and the 500 n m SP1 inhibitor mithramycin A ( n = 6) for 4 h. E) Flow cytometry quantification of IL‐1β + neutrophils of BM‐PMNs treated alone ( n = 3), with 500 n m mithramycin A ( n = 3), vitro C‐exo ( n = 3), vitro ISO‐exo ( n = 3), and vitro ISO‐exo and 500 n m mithramycin A ( n = 3), respectively for 4h. F) Representative H&E staining and quantification of lung metastases in 4‐week tumor‐bearing mice of the chronic stress + PBS injection group ( n = 5) and the chronic stress + 1 mg kg −1 mithramycin A injection group ( n = 5). Scale bar: 1 mm. G,H) Flow cytometry quantification of lung‐infiltrating neutrophils and lung‐infiltrating IL‐1β + neutrophils in 4‐week tumor‐bearing mice from the chronic stress + PBS injection group ( n = 5) and the chronic stress + 1 mg kg −1 mithramycin A injection group ( n = 5). I) Diagram illustrating the injection of SP1 control exosomes (4T1‐shCTRL C‐exo) and SP1 knockdown exosomes (4T1‐shSP1#2 C‐exo) into mouse via the tail vein. Exosomes were injected every 3 days into BALB/C mice that had received a subcutaneous injection of 10 5 4T1 tumor cells 7 days earlier. J) Representative images of Bouin's and H&E staining for lungs from the 4‐week tumor‐bearing mice injected with 4T1‐shCTRL C‐exo ( n = 5) or 4T1‐shSP1#2 C‐exo ( n = 5). Quantification of the number of lung metastatic nodules and burden of lung metastases are shown. K) Flow cytometry quantification of lung‐infiltrating neutrophils and lung‐infiltrating IL‐1β + neutrophils in 4‐week tumor‐bearing mice injected with 4T1‐shCTRL C‐exo ( n = 5) or 4T1‐shSP1#2 C‐exo ( n = 5). The data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques: Expressing, In Vivo, Flow Cytometry, Staining, Injection, Control, Knockdown

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: Chronic stress significantly promotes the secretion of TDEs and alters the contents of exosomes by stimulating the adrenergic β receptor. Chronic stress strengthens neutrophil self‐recruitment through the CXCL2 autocrine. Upon exposure to chronic stress, the level of SP1 in TDEs increases, which promotes neutrophil secretion of IL‐1β through the TLR4‐NFκβ pathway, thereby remodeling the lung microenvironment and contributing to the promotion of lung metastasis of breast cancer.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques:

Journal: Genome Biology

Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

doi: 10.1186/gb-2007-8-7-r148

Figure Lengend Snippet: The BBB neocentromere contains a major and a minor centromere chromatin domain. DNA obtained from chromatin immunoprecipitation (ChIP) using antibodies to CENP-A, CENP-C, and CENP-H from cell line BBB was hybridized to a custom made microarray containing 257 unique polymerase chain reaction (PCR) fragments. Three independent biological replicates were performed for each antibody, and the scale normalized mean log 2 Cy-5:Cy-3 intensity ratios (ChIP to input), were plotted on the y-axis with the standard error (SE) for each PCR fragment. Intensity ratios at least three times the standard deviation (SD) from the background mean (dashed line) were considered positives (see Materials and methods). An alpha satellite containing plasmid was included as a positive control (far right). (a) Centromere protein (CENP)-A ChIP. The major CENP-A domain was about 80.3 kilobases (kb; shaded region), with positive intensity ratios 1.17 to 2.46. The minor domain was about 8.5 kb (shaded region) and was approximately 162 kb downstream from the major domain; intensity ratios were 1.14 to 1.33. Background experimental mean was -0.39 ± 0.47 SD, one-tailed distribution cut-off was ≤ 0.68, positive values were ≥ 1.02 (dashed line). Alpha satellite = 1.63 ± 0.18 SE. (b) CENP-C ChIP. Major CENP-C domain was 87.8 kb (shaded region). Intensity ratios were 0.67 to 3.41. Minor domain was 8.5 kb; intensity ratios were 0.65 to 1.07 (shaded region). Background experimental mean was -0.37 ± 0.34 SD, one-tailed distribution cut-off was ≤ 0.31, positive values were ≥ 0.65 (dashed line). Alpha satellite = 2.36 ± 0.70 SE. (c) CENP-H ChIP. Major CENP-H domain was about 86.3 kb (shaded region), and positive intensity ratios were 0.64 to 3.35. Minor domain was about 1.9 kb (shaded region), and intensity ratios were 0.82 and 1.14. Background experimental mean was -0.33 ± 0.32 SD, one-tailed distribution cutoff was ≤ 0.56, positive values were ≥ 0.63 (dashed lines). Alpha sat = 2.06 ± 0.59 SE. (d) The 2.3 megabase (Mb) region included in the PCR CHIP. The central 350 kb region, covered by PCR fragments at high density. The adjacent megabase on either side of the central region, shown at a 10 fold reduced scale, was covered by PCR fragments at decreasing density. PCR microarray fragments listed in Table 1, found at the edges of CENP-A, CENP-C and CENP-H domains, and the negative values within the first domain, are shown. The major and minor chromatin domains are shown by the rectangles. The tiling path of the unique sequenced regions of each bacterial artificial chromosome (BAC) and their overlaps are shown within the 350 kb region. The corresponding Repeat Masker data from the Human Genome Browser at UCSC and thegenes in the area are indicated [50].

Article Snippet: Each qRT-PRC was performed in a 10 μl volume using equal amounts of input and ChIP DNA (2 to 5 ng), 20 mmol/l Tris HCl (pH 8.4), 5 mmol/l MgCl 2 , 0.2 mmol/l dNTPs, 0.2 μmol/l of each primer, and 0.025U of Platinum Taq DNA polymerase (Invitrogen).

Techniques: Chromatin Immunoprecipitation, Microarray, Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation, Positive Control, One-tailed Test

Journal: Genome Biology

Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

doi: 10.1186/gb-2007-8-7-r148

Figure Lengend Snippet: qRT-PCR confirms two separate CenpA domains in the neocentromeric cell line BBB. (a) Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on equal amounts of total DNA obtained from centromere protein (CENP)-A chromatin immunoprecipitation (ChIP) DNA and Input DNA from BBB cell line. The thirty-four PCR primer pairs used (shown as black lines in the x-axis) amplified fragments from 150 to 250 base pairs contained within the 350 kb neocentromere region (see Figure 2). Each primer pair was assayed in at least three independent CENP-A ChIP experiments. The qRT-PCR results for each primer pair were expressed on the y-axis as the fold enhancement between the CENP-A ChIP DNA and input DNA (= 1.93 ΔCt(CENP-A-Input) ) normalized to the value obtained for the positive control alpha satellite DNA primer pair (far right). The shaded region indicates the area determined to be the CENP-A domain in Figure 2. (b) The 34 qRT-PCR primer pairs and the 133 PCR products from this region on the PCR microarray (Figure 2) are shown. qRT-PCR primers that amplified products wholly contained within a PCR microarray fragment are indicated by numbers in parentheses; the rest are labeled alphabetically. Only qRT-PCR fragments shown in Table 1 are indicated; information for all other primers can be found in the Additional data file 3. CENP-A domains derived from the PCR microarray data are indicated. Genome coordinates correspond to the region of chr13 from the Human Genome Browser at UCSC (hg17) [50].

Article Snippet: Each qRT-PRC was performed in a 10 μl volume using equal amounts of input and ChIP DNA (2 to 5 ng), 20 mmol/l Tris HCl (pH 8.4), 5 mmol/l MgCl 2 , 0.2 mmol/l dNTPs, 0.2 μmol/l of each primer, and 0.025U of Platinum Taq DNA polymerase (Invitrogen).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification, Positive Control, Microarray, Labeling, Derivative Assay

Journal: Genome Biology

Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

doi: 10.1186/gb-2007-8-7-r148

Figure Lengend Snippet: CENP-A nucleosomes are interspersed at variable densities throughout the core centromeric domain. (a) The 87.8 kilobase (kb) major domain. Shown are the putative subdomains of centromere protein (CENP)-A, with higher densities indicated by darker shading. The polymerase chain reaction (PCR) microarray fragments are shown below (see Figure 2). The fragments examined using the oligo array are shown in gray. (b) DNA obtained from chromatin immunoprecipitation (ChIP) using CENP-A from cell line BBB was hybridized to a 70 mer oligonucleotide microarray containing two distinct subdomains of the major neocentromere domain, a 1.6 kb region at the 5' end (PCR fragments 3 and 4; Table 1) and a 2 kb region within the domain (PCR fragments 20 and 21). Three independent biological replicates were performed, and the mean log 2 Cy-5:Cy-3 intensity ratio (CENP-A ChIP to input) from each biologic replicate was scale normalized (SN). The result for each 70 mer oligomer is shown plotted on the y-axis with the standard error.

Article Snippet: Each qRT-PRC was performed in a 10 μl volume using equal amounts of input and ChIP DNA (2 to 5 ng), 20 mmol/l Tris HCl (pH 8.4), 5 mmol/l MgCl 2 , 0.2 mmol/l dNTPs, 0.2 μmol/l of each primer, and 0.025U of Platinum Taq DNA polymerase (Invitrogen).

Techniques: Polymerase Chain Reaction, Microarray, Chromatin Immunoprecipitation

Journal: PLoS ONE

Article Title: Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

doi: 10.1371/journal.pone.0033788

Figure Lengend Snippet: A. Gene expression heat maps of C T measurements of 15 genes by microfluidic qRT-PCR assays performed on single MCF7 cells before and after labeling and capture by the MagSweeper. Each gene is measured in triplicate for each single cell. Some single cell expression variation is inherent among individual cells, but the overall pattern showed no marked effect by our isolation protocol. B. Average plating efficiency (percent of single cells that formed colonies after seven days) of MCF7 cells; either control, labeled with beads, or labeled and captured by the MagSweeper, performed in triplicate. This demonstrates that cell viability was not affected by our purification protocol.

Article Snippet: Target genes were preamplified using TaqMan gene expression assays (20x) (Applied Biosystems) and CellsDirect qRT-PCR kit (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Labeling, Isolation, Purification

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 modulates pancreatic tumor growth. A) Immortalized human pancreatic duct cells (hTERT-HPNE and HPDE6c7), human and murine PDA cell lines, and human patient derived-organoids (PDOs) demonstrate nSMase activity. Values shown are technical replicates of one lysate per cell line. B) SMPD3 expression was analyzed in an existing human pancreas organoid RNA-Seq dataset and is depicted in normal (n=11), primary (n=35), and metastatic (n=9) human organoids. Log of normalized counts of SMPD3 expression are plotted. C) Immunocytochemistry demonstrates nSMase2 expression in two pancreatic metastatic PDOs. Scale bar is 100uM. D) Nanoparticle tracking analysis of CD63 + sEVs upon GW4869 and vehicle treatment is depicted. E) IVIS imaging results at Day 7, 14, and 19 are graphed. Eight animals were injected with each cell line. F) Images depict bioluminescence signal during IVIS imaging at day 19. G) Pancreas weight/body weight at day 19 is graphed. Average values for the KPC scrambled shRNA are 0.04540 ± 0.001635 (n=8) and KPC Smpd3 shRNA 1 are 0.02817 ± 0.0008858 (n=7). H) Rag 1 KO mice injected orthotopically with KPC Smpd3 shRNA1 and KPC Smpd3 shRNA2 cell lines lived significantly longer than Rag1 KO mice injected orthotopically with KPC scrambled shRNA cells ( P =0.0013 using log-rank test and P =0.0001 using log-rank test, respectively). The median survival of Rag 1 KO mice injected orthotopically with KPC scrambled shRNA cells (n=12) is 19 days, KPC Smpd3 shRNA 1 cells (n=6) is 31.50 days, and KPC Smpd3 shRNA 2 cells (n=6) is 36.50 days.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Derivative Assay, Activity Assay, Expressing, RNA Sequencing, Immunocytochemistry, Imaging, Injection, shRNA

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: S m pd3 regulates proliferation of PDA cells in vivo . A) SMPD3 is expressed in a panel of human pancreatic duct cells (n=2 cell lines), human PDA cells (n=12 cell lines), and mouse PDA cells (n=3 cell lines). B) qPCR demonstrates a reduction in Smpd3 expression in KPC Smpd3 shRNA 1 LucFlag (n=3) and KPC Smpd3 shRNA 2 LucFlag (n=3) cell lines when compared to the KPC scrambled shRNA LucFlag (n=3) control cell line. C) Mean ± standard deviation for tumor volume of cell lines injected subcutaneously are graphed. Results of an unpaired t test comparing KPC scrambled shRNA and KPC Smpd3 shRNA 2 cell lines at day 17 are depicted on the graph. For all cell lines, N=6 animals. D) Mean ± standard error of the mean for tumor weight at Day 17 of cell lines injected subcutaneously are graphed. For all cell lines, N=6 animals. E) Immunofluorescence images and quantification of epithelial (CK19 + , Vimentin - , Dapi + ), proliferative (Ki67 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag line (n=4) are 39.33% ± 2.046%, KPC Smpd3 shRNA 1 LucFlag line (n=5) are 28.88% ± 4.096%, and KPC Smpd3 shRNA 2 LucFlag line (n=5) are 15.83% ± 1.960. Scale bar is 50uM. F) Immunofluorescence images and quantification of epithelial (E-cadherin + , Vimentin - , Dapi + ), apoptotic (Cleaved caspase 3 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.299% ± 0.2075%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.082% ± 0.2126%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=5) are 0.9832% ± 0.1307. Scale bar is 50uM. G) CTG assay or colony formation assay results for the indicated cell lines are depicted. H) Reduction of Smpd3 in epithelial pancreatic cancer cells modestly reduces fibrosis. IHC demonstrates loss of nSMase2 in epithelial pancreatic cancer cells in tumors generated using the KPC Smpd3 shRNA 1 LucFlag and KPC Smpd3 shRNA 2 LucFlag cell lines. One scale bar (1000uM) on the picrosirius red image is representative for all picrosirius red and corresponding brightfield images. One scale bar (200uM) on the nSMase2 IHC image is representative for all nSMase2 IHC images. I) Quantification of polarized light using the picrosirius red stained images is depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.734% ± 0.3760%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.319% ± 0.1328%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=4) are 0.9188% ± 0.1124.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: In Vivo, Expressing, shRNA, Control, Standard Deviation, Injection, Immunofluorescence, Generated, CTG Assay, Colony Assay, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 expression is upregulated in PanIN. A) Schematic details the construct design for the Smpd3 floxed mouse. B-C) For examination of nSMase2 expression in the indicated normal murine pancreatic cell types, nSMase2 IHC sections from 3 C57BL/6J mice were scored. Preneoplasia and neoplasia lesions were scored from KPC mice (n=10 animals scored for acinar-to-ductal metaplasia (ADM), n=12 animals scored for PanIN, n=10 animals scored for primary tumor, and n=4 animals scored for metastasis). Scale bars for IHC images are 40 uM. nSMase2 expression was scored on a scale of 0-3 and is depicted in (C). The scoring system used is 0=absent, 1=low, 2=medium, 3=high. Values depicted are representative for individual animals. D) Subcellular localization of nSMase2 was scored using the same slides and animals used for expression scoring in . Results from Chi-squared tests using a 2 x 3 table are shown. E-F) H&E and IHC for nSMase2 images are displayed for the indicated genotypes.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 ablation reduces formation of neoplasia and prolongs survival of KPC mice. A) KPC; Smpd3 f/f mice lived significantly longer than KPC; Smpd3 wt/wt mice ( P =0.0039 using log-rank (Mantel-Cox) test). The median survival of KPC; Smpd3 f/f mice (n=41) is 23.71 weeks, KPC; Smpd3 f/wt mice is 21.57 weeks (n=45), and KPC; Smpd3 wt/wt mice (n=47) is 20.00 weeks. B) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt mice (n=30), KPC; Smpd3 f/wt mice (n=27), and KPC; Smpd3 f/f mice (n=24). (C) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt (n=29), KPC; Smpd3 f/wt (n=25), and KPC; Smpd3 f/f (n=22) mice. Using Fisher’s exact tests, no significant differences were observed in the number or site of macrometastatic lesions present at end-stage in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , or KPC; Smpd3 f/f mice. If macrometastasis was present, only one site was observed per animal in these cohorts. D-E) Quantification of alcian blue staining demonstrates significantly reduced PanIN lesions in KPC; Smpd3 f/f mice (n=5) when compared to KPC; Smpd3 wt/wt mice (n=9) ( P =0.0154). H&Es used to score edema are depicted. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PanIN-bearing KPC; Smpd3 f/f mice. F) Edema scoring for KPC; Smpd3 wt/wt (n=9), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=7) mice is depicted. The scoring system used is 1=low, 2=medium, and 3=high. G-H) The percentage of pancreatic area occupied by PDA is significantly higher in KPC; Smpd3 wt/wt mice (74.16 ± 8.705) when compared to KPC; Smpd3 f/f mice (30.51 ± 17.72) ( P =0.0497) at 19-21 weeks of age. Average percent area is graphed in H (n=4 KPC; Smpd3 f/f mice, n=4 KPC; Smpd3 f/wt mice, n=5 KPC; Smpd3 wt/wt mice). Dotted lines show the PDA area in representative images. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PDA-bearing KPC; Smpd3 f/f mice at 19-21 weeks of age.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of the KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. A-F) Western blot depicts nSMase2 expression levels in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. G) Pictures of cell lines taken immediately before RNA isolation for RNA sequencing are shown. Scale bars are 100uM.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: RNA sequencing demonstrates a role for Smpd3 in modulation of cellular pathways and regulation of PDA subtype. A) Average luminescence values from 3-5 independent experiments per cell line are graphed. Average values for primary PDA cell lines generated from KPC; Smpd3 wt/wt mice (n=17 cell lines) are 98,614 ± 5,684, KPC; Smpd3 f/wt mice (n=14 cell lines) are 106,061 ± 6,699, and KPC; Smpd3 f/f mice (n=15 cell lines) are 88,460 ± 6,672. B) Average number of colonies from at least 3 independent colony formation assays per cell line are depicted. Average values from KPC; Smpd3 wt/wt mice (n=14 cell lines) are 26.02 ± 3.652, KPC; Smpd3 f/wt mice (n=14 cell lines) are 24.16 ± 2.519, and KPC; Smpd3 f/f mice (n=13 cell lines) are 21.34 ± 4.872. C) Heat maps depict expression of significantly deregulated molecules in Tumor Microenvironment Pathway and Hepatic Fibrosis Signaling Pathway in all three PPT cell lines. Molecules shown in heat maps include those molecules within the Ingenuity pathway analysis (IPA) pathway that were significantly deregulated in any comparison between PPT groups. D) IPA results show a bubble category chart of significantly deregulated pathways when comparing KPC; Smpd3 f/f PPT and KPC; Smpd3 wt/wt PPT cell lines. A positive z-score represents upregulation, and a negative z-score indicates downregulation of a pathway in KPC; Smpd3 f/f PPT when compared to KPC; Smpd3 wt/wt PPT cell lines. A gray circle depicts significant overrepresentation of a pathway, the direction of which cannot yet be determined. Select pathways are annotated. E) Picrosirius red stained images are shown for KPC; Smpd3 wt/wt (n=6), KPC; Smpd3 f/wt (n=5), and KPC; Smpd3 f/f (n=6) mice at 10-11 weeks of age. The small piece of intestine in the lower left corner of the KPC; Smpd3 f/wt image was excluded from the quantification. Scale bar is 1000uM. Inset shows brightfield image. F) Transcriptional subtyping of end-stage pancreatic tumor cell lines generated from KPC; Smpd3 wt/wt (PPT and MPT), KPC; Smpd3 f/wt (PPT), KPC; Smpd3 f/f (PPT and MPT) as well as KPC Mock OE and KPC Smpd3 OE lines is depicted. Genes used to identify subtypes according to the Moffitt classification are shown. G) Pie charts depict the number of cell lines with the classical and basal-like Moffitt subtypes. When comparing KPC; Smpd3 wt/wt PPT vs KPC; Smpd3 f/f PPT using Moffitt classification and Chi square test, p-value=0.0339.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Generated, Expressing, Comparison, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of RNA sequencing data from murine polyclonal pancreatic cancer cell lines. A) Heat map showing Integrin Signaling in all three PPT cell lines. B) Estimated counts for select genes identified from Sleuth comparison of polyclonal cell lines from end-stage PPTs of KPC; Smpd3 wt/wt (n=8), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=10) mice are graphed. Each box plot represents the distribution of estimated counts quantified by kallisto with 100 bootstrap samples. For each gene, the most significant protein-coding isoforms are shown. C) The top twenty pathways from IPA comparing KPC; Smpd3 f/f MPT vs KPC; Smpd3 wt/wt MPT are shown. D) A heat map including significantly differentially regulated molecules in the Pentose Phosphate Pathway are shown for KPC; Smpd3 wt/wt MPT and KPC; Smpd3 f/f MPT groups. E) The six differentially regulated pathways when comparing KPC Smpd3 OE vs KPC Mock OE are shown. F) Log of transcript per million (TPM) values for Smpd3 gene are graphed for KPC Mock OE and KPC Smpd3 OE sequenced cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: K P C ; Smpd3 f/f pancreata display significantly fewer activated stellate cells and fibroblasts when compared to both KPC; Smpd3 f/wt and KPC; Smpd3 wt/wt pancreata. A) Immunofluorescence images depict co-labeling with alpha SMA, Gfap, Desmin, and Dapi in the pancreas of 10–11-week-old mice. For KPC; Smpd3 wt/wt , n=6 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=5 animals. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel C. Scale bar is 20uM. B) Immunofluorescence images depict co-labeling with alpha SMA, Fap, Vimentin, and Dapi in the pancreas of 10–11-week-old mice. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel D. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, KPC; Smpd3 f/f , n=6 animals. Scale bar is 50uM. C) Quantification of alpha SMA, Gfap, Desmin, and Dapi quadruple positive cells is depicted. D) Quantification of alpha SMA, Fap, Vimentin, and Dapi quadruple positive cells is depicted. E) Pie charts depict Moffitt classification PDA subtypes for KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of lipidomics data from murine polyclonal pancreatic cancer cell lines. A) Heat map shows IPA Ceramide Signaling Pathway in all three PPT cell lines. B) Heat map shows differentially expressed lipids in comparisons amongst our murine polyclonal pancreatic cancer cell lines. Groups compared are listed as A-B where depicted upregulation or downregulation of a lipid would be in A when compared to B. C) Principal component analysis of our polyclonal murine pancreatic cancer cell lines based on lipid composition is depicted. D) Box plots showing expression of selected ceramide species in our KPC; Smpd3 wt/wt PPT, KPC; Smpd3 f/wt PPT, KPC; Smpd3 f/f PPT, KPC; Smpd3 wt/wt MPT, and KPC; Smpd3 f/f MPT cell lines. E) Box plots showing expression of selected phosphatidylcholine species in our KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of exosomes isolated from KPC; Smpd3 wt/wt PPT and KPC; Smpd3 f/f PPT cell lines. A) CD63eGFP particles per cell are graphed for KPC; Smpd3 wt/wt CD63eGFP cell lines (n=6) and KPC; Smpd3 f/f CD63eGFP cell lines (n=6). B) Fractions were subjected to WB for sEV markers to determine the fractions that exosomes are contained within using the C-DGUC exosome isolation protocol. Expected band sizes are listed for each marker. C) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt uninjected mice (n=30), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=15), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=15), KPC; Smpd3 f/f uninjected mice (n=24), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=9), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . D) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt uninjected mice (n=29), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=14), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=12), KPC; Smpd3 f/f uninjected mice (n=22), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=8), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . E) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 wt/wt are 0.04396 ± 0.006112 (n=28), KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.05477 ± 0.007678 (n=15), and KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 f/f are 0.06809 ± 0.009409 (n=16). F) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 f/f are 0.05085 ± 0.005912, KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.06149 ± 0.01434, and KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 f/f are 0.04654 ± 0.008279. G) Pancreas images at dissection for the indicated groups are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Isolation, Marker, Injection, Dissection

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: PDA cell exosomes generated through nSMase2 accelerate PDA progression. A) Schematic of exosome injection study. B) Probability of survival for the indicated groups is graphed. Log-rank test was performed between the indicated groups. C-D) Volcano plot (C) and heat map (D) depict differentially expressed exosomal miRNAs isolated from KPC; Smpd3 f/f and KPC; Smpd3 wt/wt polyclonal PDA PPT cell lines. E) Exosomal protein abundance results for KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines are depicted. The x-axis shows the log2 fold change for KPC; Smpd3 f/f over KPC; Smpd3 wt/wt and the y-axis shows −log10(q-value). Significantly upregulated proteins (q-value < 0.05) are shown in green and significantly downregulated proteins are shown in red. F) Heatmap of top 40 differentially abundant exosomal proteins comparing KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt , displaying log2 normalized expression. Red indicates lower abundance of a given protein in each sample and green indicates higher abundance. Rows and columns are arranged based on hierarchical clustering dendrograms (not displayed). The color bar at the top indicates the genotype. G) IPA results depict significantly differentially regulated pathways when comparing exosomal proteins from KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Injection, Isolation, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: sEVs generated independently of nSMase2 promote a proinflammatory macrophage phenotype. A) Heat map depicts scaled marker expression in KPC; Smpd3 wt/wt and KPC Smpd3 f/f pancreata using our 17-marker myeloid spectral flow cytometry panel. B) t-SNE of 200,683 cells stained with our spectral flow panel depict 12 clusters identified for KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata through FlowSOM clustering. C) Heat map of scaled marker expression and number of cells for clusters shown in B is displayed. D) Bar graphs depict percent cellular subpopulations from KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata for the indicated markers after gating in FlowJo. E) Immunofluorescence images depict co-labeling with F4/80, iNOS, and Dapi at 10-11 weeks of age. Scale bar is 20uM. F) Quantification of intralobular F4/80 + and iNOS + copositive cells in KPC; Smpd3 wt/wt (n=5 animals), KPC; Smpd3 f/wt (n=6 animals), and KPC; Smpd3 f/f (n=5 animals) pancreata at 10-11 weeks of age is depicted. G) Schematic of experiment to assess how nSMase2-mediated exosome biogenesis affects polarization of macrophages. H-J) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Marker, Expressing, Flow Cytometry, Staining, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Functional effects of PDA cell sEVs on macrophages. A) Immunofluorescence images depict co-labeling with F4/80, Ly6G, and Dapi at 10-11 weeks of age. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=6 animals. Scale bar is 20uM. B) Quantification of interlobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. C) Quantification of intralobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. D-F) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Functional Assay, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: SMPD3 is an independent prognostic factor for pancreatic cancer patient survival. A) Log2 of the normalized counts are plotted for normal human pancreatic SMPD3 expression (n=39 samples) and primary PDA SMPD3 expression (n=39 samples). B) Log2 of the normalized counts are plotted for PPT SMPD3 expression (n=231 samples) and MPT SMPD3 expression (n=158 samples) in PDA patients. C) Kaplan-Meier survival curve of the whole cohort (N = 94) with the patients dichotomized into SMPD3 high and low expression based on the median mRNA expression value. SMPD3 high group had significantly better survival (median survival of 26.5 vs 15.0 months; P = 0.0364). A (0) value indicates a censored event and a (1) value indicates a death event. D) Kaplan-Meier survival curve of the subset of the whole cohort of patients who underwent adjuvant chemotherapy (> 95% single agent gemcitabine) after surgical resection (N = 58). SMPD3 expression was significantly associated with better survival (median survival of 34.3 vs 15.9 months; P = 0.0289) indicating SMPD3 is a biomarker of adjuvant gemcitabine response. E) Kaplan-Meier survival curve of the subset of the whole cohort of patients who did not undergo adjuvant chemotherapy after surgical resection (N = 36). SMPD3 expression was not associated with survival (P = 0.8001). F-G) Kaplan-Meier curves depict patient survival based on SMPD3 expression in locally advanced primaries or metastatic PDA from the COMPASS trial for patients who received chemotherapy with modified Folfirinox or Gemcitabine plus Abraxane. SMPD3 expression was stratified into high and low with low expression defined as the maximal chi-squared statistic. H) Log2 of SMPD3 expression by hypoxia status defined by . I) Log2 of SMPD3 expression in defined Waddell structural variation subtypes in COMPASS cases is depicted. J) Kaplan-Meier curves depict patient survival based on cytoplasmic and membranous nSMase2 expression in treatment naïve primary resected PDA. Tumor, and not stromal, cell nSMase2 expression was scored in 143 patients. When comparing survival of PDA patients with low cytoplasmic nSMase2 expression (n=89, mean survival=30.1 months) and high cytoplasmic nSMase2 expression (n=54, mean survival=29.1 months), p=0.627 using the log-rank test. When comparing survival of PDA patients with low membrane nSMase2 expression (n=65, mean survival=25.6 months) and high membrane nSMase2 expression (n=78, mean survival=32.9 months), p=0.052 using the log-rank test.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Biomarker Discovery, Modification, Membrane

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Pathways analysis of pancreatic nSMase2 expression in PDA patients based on treatment with chemotherapy in addition to the effect of nSMase2 expression on chemosensitivity and vasculature integrity. A) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs receiving adjuvant chemotherapy. The threshold line represents statistical significance. B) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs not receiving adjuvant chemotherapy. C) Representative images used to score the TMA for nSMase2 low and nSMase2 high membrane and cytoplasmic labeling. D) Images depict dextran and tomato lectin, a marker of blood vessels in mice, immunofluorescence at 10-11 weeks of age in KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreases. E) Percent pancreatic neoplastic area per field occupied by dextran is graphed for KPC; Smpd3 wt/wt (n=8) and KPC; Smpd3 f/f (n=6) mice. F) Negative log of gemcitabine IC50 for KPC; Smpd3 wt/wt , KPC; Smpd3 f/f , KPC Mock OE, and KPC Smpd3 OE cell lines is graphed. G) Quantification of total HIF1α in nSMase2 high and nSMase2 low primary pancreatic tumors of PDA patients is depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Membrane, Labeling, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 regulates PDA vasculature development. A-B) Heat maps showing VEGF Signaling and HIF1α Signaling pathways in all three PPT cell lines. C) Log of transcript per million (TPM) values for Vegfa gene are graphed for sequenced PPT cell lines. Wilcoxon test was performed. D) Pancreatic CD31 and CK19 immunolabeling at 19-21 weeks of age in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is depicted. Scale bar is 20uM. E) Quantification of pancreatic CD31 immunofluorescence in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is shown. F) Pancreatic CD31 and CK19 immunolabeling at 21 days post orthotopic injection of KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines into Rag1 KO mice 21 days post injection is depicted. Scale bar is 20uM. G) Quantification of pancreatic CD31 immunofluorescence in Rag1 KO mice injected with KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines 21 days post injection is depicted. H) Immunohistochemistry of CD31 in primary tumors from PDA patients is depicted with low and high nSMase2 expression. I) Percent CD31 positive area in primary nSMase2 low (n=19) and nSMase2 high (n=34) pancreatic tumors from PDA patients is shown. J) Immunohistochemistry of HIF1α is depicted in primary tumors from PDA patients with low and high nSMase2 expression. K) Percent nuclear HIF1α epithelial tumor cells in primary nSMase2 low (n=5) and nSMase2 high (n=6) PDA patient pancreatic tumors is shown. L) Ratio of pancreas weight to body weight for gemcitabine and vehicle treated Rag1 KO mice 21 days after pancreatic orthotopic injection of the depicted cell lines is shown. For KPC scrambled shRNA vehicle treated mice, n=4, for KPC scrambled shRNA gemcitabine treated mice, n=9, for KPC Smpd3 shRNA 1 vehicle treated mice, n=10, for KPC Smpd3 shRNA 1 gemcitabine treated mice, n=10, for KPC Smpd3 shRNA 2 vehicle treated mice, n=10, and for KPC Smpd3 shRNA 2 gemcitabine treated mice, n=10. M) Kaplan-Meier survival curves show probability of survival for vehicle-injected KPC; Smpd3 wt/wt mice (n=12, median survival=20.29 weeks), gemcitabine-injected KPC; Smpd3 wt/wt mice (n=19, median survival=23.43 weeks), vehicle-injected KPC; Smpd3 f/f mice (n=15, median survival=23.29 weeks), gemcitabine-injected KPC; Smpd3 f/f mice (n=16, median survival=19.86 weeks). A black line indicates a censored animal.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Protein-Protein interactions, Immunolabeling, Immunofluorescence, Injection, shRNA, Immunohistochemistry, Expressing

Journal: Cellular microbiology

Article Title: Incoming human papillomavirus 16 genome is lost in PML protein-deficient HaCaT keratinocytes

doi: 10.1111/cmi.12708

Figure Lengend Snippet: (A) Cells were grown on glass coverslips and infected with EdU-labeled HPV16 PsV. At 24 hpi cells were fixed and processed for the detection of EdU-labeled DNA (red), PML (green), NUP153 (blue) and DAPI (gray). (B) PML NBs and (C) EdU-labeled viral pseudogenome number was counted manually in z-stacks spanning the whole nucleus for each cell. 15 AGS cells and 13 EBV-harboring AGS cells were included in the count. P value was determined using Student's t-test. (D) RT-qPCR for viral transcription at 48 hpi with HPV16 quasivirions. cDNA samples were analyzed for HPV16E7 and the data shown are fold changes relative to AGS cells. Error bars represent SEM of five independent experiments.

Article Snippet: Antibodies and reagents Antibodies used for the study were as follows: PML (BETHYL; catalog number (#) A301-167A), lamin A/C mouse (Sigma; #SAB4200263), Nup153 (Covance; #MMS-102P), pStat1 and Stat1 (Cell Signaling; #9167S and 9176S, respectively), and β-actin (Santa Cruz; #sc-47778).

Techniques: Infection, Labeling, Quantitative RT-PCR