Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: SOX9 protein and SOX9 mRNA expression in AsPC-1, BxPC-3, Colo357, Capan-2, MiaPaCa-2, and Panc1 cells. ( A ) Western blot analysis of the expression of E-cadherin (CDH1), cytokeratin-19 (KRT19), vimentin (VIM), SOX9, and GAPDH in the investigated cell lines. Pancreatic cancer cells were seeded in 6-well plates (0.5 × 10 6 cells per well). After 48 h, cell lysates were prepared by adding SDS sample buffer (200 µL per well). ( B ) Densitometric quantitation of Western blots from ( A ). The levels of the SOX9 protein were normalized to the levels of the GAPDH protein. ( C ) Real-time qPCR analysis of the mRNA expression of SOX9 in pancreatic cancer lines. The results are presented as the mean ± SEM of three independent experiments and normalized against the HPRT expression. ( D ) Immunofluorescence imaging of the SOX9 expression in Panc1 cells. Cells were stained for total cytokeratin (green) and SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 100 µm.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Quantitation Assay, Immunofluorescence, Imaging, Staining

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: The effect of SOX9 downregulation on the expression levels of protein markers of differentiation and developmental transcription factors in pancreatic cancer cells. ( A ) Western blot analysis of the expression of SOX9 in AsPC-1, BxPC-3, Colo357, Capan-2, MiaPaCa-2, and Panc1 cells transfected with control siNeg and siSOX9 ( n = 3). GAPDH and beta-tubulin (TUBB) were used as loading and normalization controls. ( B ) Immunofluorescence imaging of the SOX9 expression in Panc1 cells transfected with control siNeg and siSOX9. Cells were stained for total cytokeratin (green) and SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 50 µm. ( C ) Western blot analysis of the expression of epithelial cell protein markers in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( D ) Heatmap of differentially expressed epithelial cell proteins from ( C ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( E ) Western blot analysis of the expression of mesenchymal cell protein markers in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( F ) Heatmap of differentially expressed mesenchymal cell proteins from ( E ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( G ) Western blot analysis of the expression of developmental transcription factors in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( H ) Heatmap of differentially expressed developmental transcription factors from ( G ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Transfection, Control, Immunofluorescence, Imaging, Staining

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: The effect of SOX9 downregulation on the expression levels of cell cycle protein regulators. ( A ) Western blot analysis of the expression of cell cycle protein regulators in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( B ) Heatmap of differentially expressed cell cycle protein regulators from ( B ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( C ) Immunofluorescence imaging of p21 Waf1/Cip1 (CDKN1A) expression in Colo357 and Panc1 cells transfected with control siNeg and siSOX9. Cells were stained for SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. ( D , E ) Relative Western blot, RT-qPCR, and RNA-Seq data quantification of the levels of SOX9, SNAI1, SNAI2, FOXA2, GATA4, CDKN1A, TP53, and PTEN in control siNeg and siSOX9 transfected Colo357 ( D ) and Panc1 ( E ) cells. RT-qPCR and RNA-Seq data are means ± SEM from three technical replicates and representative of at least three experiments. * p ≤ 0.05; ** p ≤ 0.01 compared with siNeg control.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Transfection, Control, Immunofluorescence, Imaging, Staining, Quantitative RT-PCR, RNA Sequencing

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: Effect of SOX9 expression on cell proliferation, cellular senescence, apoptosis, and cell migration. ( A ) Effect of SOX9 downregulation on the cell proliferation of BxPC-3, Colo357, MiaPaCa-2, and Panc1. Pancreatic cancer cells seeded in 6-well plates were transfected with siSOX9 and control siNeg. After 72 h, the cell number was measured by counting cells using the TC20 automated cell counter. Data were expressed as the percentage from control cell growth. Data are means ± SEM from three technical replicates and representative of at least three experiments. * p ≤ 0.05; ** p ≤ 0.01 compared with siNeg control. ( B ) Effect of SOX9 downregulation on the senescence-associated β-galactosidase (SA-β-Gal) activity of BxPC-3, Colo357, MiaPaCa-2, and Panc1 cells. SA-β-Gal activity data were expressed relative to SA-β-Gal activity levels in siNeg and siSOX9 transfected cells. ( C ) Effect of SOX9 downregulation on caspase 3/7, caspase 8, and caspase 9 activities of BxPC-3, Colo357, MiaPaCa-2, and Panc1 cells. Caspase activity data were expressed relative to caspase activity levels in siNeg and siSOX9 transfected cells, respectively. ( D ) Panc1 cells were transfected with siSOX9 and siNeg for 72 h and then analyzed by annexin V–AF488/DAPI staining with flow cytometry analysis. The lower right area (Q3) shows early apoptotic cells, and the upper right area (Q2) shows late apoptotic cells. ( E ) Summary graphs of the flow cytometry results. ( F ) Western blot analysis of SOX9 and CDKN1A expression in Panc1 cells transfected with pCMV6 empty plasmid and pCMV6-SOX9-FLAG after 72 h post-transfection. GAPDH was used as control. ( G ) Effect of SOX9 upregulation on cell proliferation of BxPC-3 and Colo357. Pancreatic cancer cells seeded in 6-well plates were transfected with empty pCMV6 vector and pCMV6-SOX9-FLAG. After 72 h, the cell number was measured by counting cells using the TC20 automated cell counter. ( H ) Western blot analysis of the expression of SOX9, TP53, CDKN1A, and CDKN1B in Panc1 cells transfected with control siNeg and siSOX9. Cell lysates were prepared at 72, 96, and 120 h after first siRNA transfection. GAPDH was used as loading control. ( I ) The representative images displayed the GFP distributions of transplanted Panc1-EGFP control siNeg transfected cells in Danio rerio embryo. The microinjection site Danio rerio embryo is indicated with a red circle. The bioimaging of injected embryos was performed 2 days after cell transplantation. PH, phase contrast; FL, fluorescence. ( J ) The representative image displayed the GFP distributions of transplanted Panc1-EGFP siSOX9 transfected cells in Danio rerio embryo. The microinjection site Danio rerio embryo is indicated with a red circle. The bioimaging of injected embryos was performed 2 days after cell transplantation. PH, phase contrast; FL, fluorescence.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Migration, Transfection, Control, Activity Assay, Staining, Flow Cytometry, Western Blot, Plasmid Preparation, Microinjection, Injection, Transplantation Assay, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Schematic illustration of the PSMA and esterase dual responsive CPT nanoparticles (CPT-WT-H NPs) with negatively charged surface, prolonged blood circulation, excellent PSMA-targeted delivery, enhanced tumor accumulation, potent antitumor efficacy and low side effects.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Characterization of CPT-WT-H NPs. ( A ) A TEM image of CPT-WT-H NPs dispersed in PBS (pH 7.4); inserts are the photographs of CPT-WT-H NPs in PBS in bright field (left) and under UV light at 365 nm with a red laser passing through (right). ( B ) DLS histograms of CPT-WT-H NPs in PBS. ( C ) Change of hydrodynamic diameter (Dh) of CPT-WT-H NPs in PBS for 120 h. ( D ) Schematic of sequential PSMA hydrolysis of CPT-WT-H (CPT12A-βAsp-GluγGluγGluγGlu) to yield the PSMA hydrolysate CPT12A-βAsp. ( E ) Characterization of in vitro PSMA response of CPT-WT-H NPs by LC-MS.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vitro, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: In vitro cytotoxicity of CPT-WT-H NPs and CPT against PSMA-expressing (PSMA + ) cancer cell LNCaP-FGC ( A ) and non-PSMA-expressing (PSMA − ) cancer cells HepG2 ( B ), MCF-7 ( C ), HeLa ( D ), DU145 ( E ), PC-3 ( F ) and normal liver cells LO2 ( G ), the IC 50 values of CPT and CPT-WT-H NPs on different cell lines ( H ). *** p < 0.001, vs LNCaP-FGC cells.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vitro, Expressing

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Cellular uptake of CPT-WT-H NPs in vitro. ( A ) CLSM images of LNCaP-FGC (PSMA + ) cells incubated with CPT-WT-H NPs (10 μM) for 1 and 4 h. ( B ) CLSM images of HepG2 (PSMA − ) cells incubated with CPT-WT-H NPs (10 μM) for 1 and 4 h. Nuclei were stained by PI (red), the blue color was indicative of CPT. ( C ) Mean fluorescence intensity of CPT-WT-H NPs internalized by LNCaP-FGC and HepG2 cells after incubation for 1, and 4 h. *** p < 0.001. ( D ) Flow cytometry analysis for apoptosis of LNCaP-FGC (PSMA + ) (1) and HepG2 (PSMA − ) (2) cells induced by CPT-WT-H NPs for 72 h. Q1, necrotic cells; Q2, late apoptotic cells; Q3, living cells; Q4, early apoptotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vitro, Incubation, Staining, Fluorescence, Flow Cytometry

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: In vivo NIRF imaging of DiR-doped CPT-WT-H NPs. ( A ) In vivo NIRF images of the MCF-7 tumor-bearing nude mice (PSMA-producing) after intravenous injection of free DiR (0.25 mg·mL −1 , 100 µL) or DiR-doped CPT-WT-H NPs (0.25 mg·mL −1 , 100 µL). ( B ) Integrated fluorescence intensity of tumor obtained after intravenous injection of DiR-doped CPT-WT-H NPs (0.25 mg·mL −1 , 100 µL). ( C ) Ex vivo NIRF images and ( D ) Integrated fluorescence intensity of the major organs and tumors excised at 48 h post intravenous injection of DiR-doped CPT-WT-H NPs, *** p < 0.001, vs tumor. ( E ) In vivo pharmacokinetics profiles of CPT-WT-H NPs s at the equivalent dose (30 mg·kg −1 ) in BALB/c mice. Error bars indicate SD (n = 6).

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vivo, Imaging, Injection, Fluorescence, Ex Vivo

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: CPT-WT-H NPs significantly inhibited tumor progression and dramatically reduced side effects in an MCF-7 human breast cancer model. ( A ) The growth curves of MCF-7 tumor after treatment, which was initiated on day 13 post tumor inoculation, and drugs were intravenously injected every 2 days for 8 times (n = 6/group). ( B ) Changes of body weight in each group. ( C ) The tumor pictures in each group. ( D ) Excised tumor weight in each group. ( E ) Excised tumor volume in each group. * p < 0.05; *** p < 0.001. ( F ) Histological analysis of tumor sections stained with H&E.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: Injection, Staining

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Representative H&E-stained histological images obtained from liver ( A ) and kidneys ( B ) after intravenous administration of irinotecan (10 mg·kg −1 ) and CPT-WT-H NPs (10, 30, 60 mg·kg −1 ). The PBS-treated group and the untreated group were used as the control. (I: PBS, II: CPT-WT-H NPs (10mg·kg −1 ), III: CPT-WT-H NPs (30mg·kg −1 ), IV: CPT-WT-H NPs (60mg·kg −1 ), V: Irinotecan (10 mg·kg −1 ), VI: Normal group). ( C ) Blood biochemical analysis of after the antitumor efficacy evaluation. The PBS-treated group and the untreated group were used as the control. The main parameters of hepatic and renal function are alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE). Error bars indicate SD (n = 6). * p < 0.05; *** p < 0.001.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: Staining

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: Characterization and in vitro DC-activation of neoantigen nanovaccines. a 1 H NMR of DSPE-PEG 2000 -NHS (vehicle), DSPE-PEG 2000 -peptide and peptide (peptide: M27). b MALDI-TOF–MS of DSPE-PEG 2000 -peptide (peptide: M27). c Size, PDI, encapsulation efficiency, drug loading content and zeta-potential of nanovaccines. d Size of nanovaccines. e The transmission electron microscopy (TEM) image of nanovaccines. f Curves of peptide (M27) release from nanovaccines in different solutions. g Proportion of mature DC (CD11c + CD80 + CD86 + ) after incubation with normal saline (NS), peptide (Tyrp1, M20 or M27) or nanovaccines (Tyrp1-NP, M20-NP or M27-NP) for 48 h. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0026 (M20 vs M20-NP), ** P = 0.0029 (NS vs M27), * P = 0.0429 (M27 vs M27-NP), *** P < 0.001

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: In Vitro, Activation Assay, Encapsulation, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Incubation, Saline

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: Lymph node-targeting characteristic of neoantigen nanovaccines. a Equivalent peptide-NIR797 (Free vaccine group) and DSPE-PEG 2000 -peptide-NIR797 (Nanovaccine group) were separately mixed with Montanide™ ISA 51 and injected subcutaneously at the tail base of mice. The fluorescence distribution in mice at different time points was photographed by small animal in vivo imaging (n = 3). b Fluorescence image of inguinal lymph nodes 48 h after injection. c The average radiant efficiency of inguinal lymph nodes, spleens and kidneys 48 h after injection. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0062. d – e A FITC labeled model antigen ovalbumin (OVA) was used to evaluate the distribution of nanovaccines. d Localization of nanovaccines and CD3 + T cells in inguinal lymph nodes 48 h after subcutaneous injection of DSPE-PEG 2000 -OVA-FITC, was shown by immunofluorescence staining. Nanovaccine: green (FITC); T cells (CD3): gray (Cy5); Scale: 500 μm. e Localization of nanovaccines and DCs in lymph nodes 48 h after subcutaneous injection of DSPE-PEG 2000 -OVA-FITC. Nanovaccine: green (FITC); DCs (CD11c): red (Cy3); Scale: 25 μm

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Injection, Fluorescence, In Vivo Imaging, Labeling, Immunofluorescence, Staining

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: T cell responses activated by neoantigen nanovaccines. One week after last treatment, Proportions of mature DCs (CD11c + CD80 + CD86 + ) in lymph nodes ( a ), proportions of neoantigen specific T cells (CD3 + CD8 + M27-H 2 K b+ ) in spleens ( b ) and tumors ( c ), and proportions of effector memory T cells (CD3 + CD8 + CD44 + CD62L − ) in spleens ( d ) were analyzed by flow cytometry. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0034 ( a ), ** P = 0.0026 (d, NS vs Nanovaccine), ** P = 0.0056 (d, Free vaccine vs Nanovaccine), *** P < 0.001. e Lymphocytes in spleens were incubated with CFSE labeled B16F10 melanoma cells and MFC forestomach cancer cells at effector-to-target ratio (E: T) of 10:1. PI was added 4 h after incubation and the percentage of dead tumor cells (CFSE + PI + / CFSE + ) was analyzed by flow cytometry. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P = 0.0114 (NS vs Free vaccine), ** P = 0.0046 (Free vaccine vs Nanovaccine). f Cytokines in the supernatant after co-incubation of lymphocytes and tumor cells. P -values were determined by two-way ANOVA with Tukey’s HSD multiple comparison post hoc test. *** P < 0.001. The level of TNF-α ( g ) and IL-6 ( h ) in the tumor microenvironment. i Proportions of regulatory T cells (CD3 + CD4 + Foxp3 + ) in the tumor microenvironment. j The expression of PD-L1 in tumors. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0014 ( g ), *** P < 0.001

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Flow Cytometry, Incubation, Labeling, Comparison, Expressing

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A Hoechst staining of RPMI8226 cells in the presence of an EGF neutralizing antibody (10 μg/ml) for 24 h and the result quantification (right panel). B Hoechst staining of CTR-KD or AL-KD RPMI8226 cells in the presence of recombinant EGF protein (10 ng/ml) for 24 h and the result quantification (right panel). C Hoechst staining of AL-KD1 and AL-KD2 RPMI8226 cells, cocultured with BMSC in the presence of ALCAM-Fc chimera fusion protein (AL-Fc, 0.5 μg/ml) for 24 h and the result quantification (right panel). D Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC in the presence of EGFR inhibitor (gefitinib, 200 nΜ) for 24 h and the result quantification (right panel). E Hoechst staining of RPMI8226 cells in the presence of CD6 antibody (10 μg/ml) and the result quantification (right panel). Data are the mean of three independent experiments in three replicates. * p < 0.05.

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: Staining, Recombinant

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A MM cells RPMI8226, either CTR-KD or AL-KD, were treated with melphalan (Mel, 15 μM) or bortezomib (BTZ, 5 nM) for 24 h. The SP cell ratio was examined by Hoechst staining. B The RPMI8226 cells were treated by melphalan as described above. The cell cycle was analyzed after Hoechst staining. C Cell-cycle quantification. D After Hoechst staining, the apoptotic cells were analyzed by annexin V staining. E ALCAM and EGFR expression on MM cells after Mel or BTZ treatment were detected by flow cytometry. MFI mean fluorescence index. F Examination of SP cells after EGFR inhibitor (gefitinib, 200 nM) and melphalan treatment. Data are the mean of three independent experiments in three replicates. * p < 0.05.

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: Staining, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A Scheme graph showing the animal study to evaluate the efficacy of the combination therapy (Mel and EGFR inhibitor) in vivo. The mice were treated by intraperitoneal injection of melphalan (60 μg/mouse per time, 4 times within 10 days) or gefitinib (500 μg/mouse per time, 4 times within 10 days), or a combination of both. Each group contained 8 mice. B Tumor-bearing mice were subjected to in vivo bioluminescent imaging (IVIS) before and after treatment. Five out of seven representative results are shown. C The relative luciferase activity of IVIS was calculated. D The tumor-bearing mice were treated twice as described above. Then, mice BM cells were analyzed by Hoechst staining for MM SP in vivo. Two out of three representative results are shown in the left panel, and result quantification is shown in the right panel. E Tumor burden was evaluated by circulating IgG2b. F Treatment efficacy was evaluated by mouse survival. G MM tumor-bearing mice were treated by intraperitoneal injection of PBS, bortezomib (15 μg/mouse per time, 4 times within 10 days), or a combination of bortezomib and gefitinib (500 μg/mouse per time, 4 times within 10 days). The PBS group contained 4 mice; the other treated group each contained 6 mice. Tumor burden was evaluated by circulating IgG2b. H Treatment efficacy was evaluated by mouse survival (* p < 0.05).

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: In Vivo, Injection, Imaging, Luciferase, Activity Assay, Staining

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A ALDEFLUOR analysis of BM cells from non-tumorous donors ( n = 5) and MM patients ( n = 7). Two representative results are shown. B BM cells from 42 newly diagnosed MM patients were analyzed by flow cytometry for ALCAM expression in CD138 + MM cells and ALDEFLUOR staining. Results show 2 representatives of ALCAM-high MM versus ALCAM-low MM, and corresponding ALDEFLUOR staining results. C Graphic model of EGFR-targeting therapy in MM treatment (* p < 0.05).

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: Flow Cytometry, Expressing, Staining

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer, pSi-shUSP22-1 or pSi-shUSP22-2 plasmid. ( A ) Western blot analysis of USP22 expression; β-actin was used as endogenous control. ( B ) Cell viability was measured using MTT assay at 1, 2, 3, and 4 d after transfection. ( C ) EdU staining used to examine the proliferation. Scale bar: 5 μm. ( D ) Percentage of EdU-positive staining in (C). ( E ) Effect of USP22 knockdown on cell proliferation evaluated by colony formation assay. After 21 d of transfection, cells were stained with Giemsa solution. ( F ) Histograms representing the colony formation number in (E). ( G ) Western blot conducted to analyze the expression of USP22 and proliferation-related proteins including Rb, p-Rb, Akt, p-Akt, and cyclin D2. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B, D, and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, MTT Assay, Staining, Knockdown, Colony Assay

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A and B ) After 48 h of transfection, the scratch wound-healing assay was performed to evaluate the effect of USP22 on cell migration. The representative images of cell migration were shown in (A), and the width of wounds at the indicated times was shown in (B). ( C ) Invasion of CAL-62 and 8505C cells analyzed by transwell assay. The invaded cells were stained with DAPI. Scale bar: 5 μm. ( D ) Number of DAPI-positive cells per field counted under a fluorescent microscopy. ( E ) Immunofluorescence staining was performed to analyze the effect of USP22 on the expression of E-cadherin and vimentin in 8505C cells. Scale bar: 5 μm. ( F and G ) Expression of invasion-related proteins containing BMI-1, E-cadherin, vimentin, and snail in ATC cells was measured by qPCR (F) and western blot (G). GAPDH and β-actin were used as endogenous controls, respectively. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with the group at 0 h after transfection (B). ## P < 0.01, as compared with mock or pSilencer group at 24 h after transfection (B). * P < 0.05, as compared with mock or pSilencer group (D and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Migration, Transwell Assay, Staining, Microscopy, Immunofluorescence, Expressing, Western Blot

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A ) Apoptosis of 8505C cells analyzed by flow cytometry. ( B ) Apoptosis rate in (A) was calculated. ( C ) Fluorescent TUNEL assay conducted to determine the apoptosis of 8505C cells. Scale: 10 μm. ( D ) Percentage of TUNEL-positive cells in (C). ( E ) CAL-62 and 8505C cell apoptosis evaluated by nucleosomal fragmentation assay. ( F ) Quantification of caspase-3 activity in CAL-62 and 8505C cells. ( G ) Western blot analyses of apoptosis-related protein (Bid, Bax, cl-caspase-3, caspase-3, and Bcl-2) expressions in CAL-62 and 8505C cells. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B and D–F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, TUNEL Assay, Activity Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: Female six-week-old SCID mice were inoculated subcutaneously into right hind flanks or injected via tail vein with stably expressed pLKO.1 or pLKO.1-shUSP22 8505C-luc cells. Mock-treatment was used as control. ( A ) Tumor growth progression was measured by in vivo luciferase imaging of the xenografts at days 1, 5, 9, and 12 d after inoculation. ( B ) Representative gross photos of tumors 22 d after subcutaneous xenografting ( n = 6). ( C ) Tumor volumes of subcutaneous implantation models of ATC were monitored and calculated after 0, 7, 11, 13, 17, 20, and 22 d of inoculation. D–F. After 28 d of injection through tail vein, the lungs were removed and photographed ( D ), the number of metastatic nodules in lungs was counted ( E ), and the weight of lungs was measured ( F ). ( G ) Fluorescence TUNEL assay was carried out to determine cell apoptosis in the same tumor tissues as indicated above. The rate of TUNEL-positive cells was calculated. Scale bar: 10 μm. ( H ) qPCR assay was performed to detect the mRNA expression of USP22 in tumor tissues from (B). ( I ) Protein expression of USP22 in tumor tissues was analyzed by western blot. ( J ) Representative results of western blot analyses of cyclin D2, Akt, p-Akt, E-cadherin, vimentin, Bax, Bcl-2, cl-caspase-3, and caspase-3 in tumor tissues. (I and J) β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pLKO.1 group (C and E–H).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Injection, Stable Transfection, Control, In Vivo, Luciferase, Imaging, Fluorescence, TUNEL Assay, Expressing, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Identification of cancer stem cells and a strategy for their elimination

doi: 10.4161/cbt.29854

Figure Lengend Snippet: Figure 1. Internalization of exogenous dsDNA by ascites cells of mouse Krebs-2 tumor. ( A ) Fluorescence analysis of TAMRA-labeled Alu DNA and TAMRA-dUTP precursor by ascites form of Krebs-2 tumor cells. Notably, ascites cells fail to incorporate TAMRA-dUTP. Bar corresponds to 50 µm. ( B ) Same as above, zoom-in, bars correspond to 10 µm. Top and side views of a TAMRA-positive cell are shown on the bottom right panel. ( C ) Fluorescence analysis of total human TAMRA-labeled DNA fragments (0.2–6 kb) internalized by ascites Krebs-2 tumor cells. Bars correspond to 10 µm. ( D ) Molecular analysis of dsDNA internalization by Krebs-2 ascites cells. Upper panel: αP 32 -labeled PCR-amplified GFP fragment was used to directly monitor DNA internalization by ascites cells (probe size is shown on the *GFP lane), M, DNA weight marker; CP+DNA, Krebs-2 ascites cells collected 18 h post CP injection and incubated with labeled DNA (for 1, 2, 4 and 8 h); DNA, Krebs-2 ascites cells from CP-untreated mice and incubated with labeled DNA (for 1, 2, 4 and 8 h); ВMC, bone marrow cells from an intact mouse incubated with labeled DNA (for 1, 2, 4 and 8 h). Lower panel: Non-labeled linearized pEGFP-N1/HindIII DNA was used to detect internalization of exogenous DNA by cells. Internalization was visualized by Southern blot using 32 P-labeled GFP DNA as a probe. Treatments and collection timepoints are the same as on the upper panel. Southern blot and gel images are shown. Bone marrow cells were used as a positive control, where DNA internalization is well-documented. ( E ) FACS profiles showing dsDNA internalization upon increasing concentration of the labeled substrate in the medium. Graph summarizing FACS data are shown below. ( F ) Flow cytometry analysis of dynamics of Alu -TAMRA dsDNA internalization in course of incubation with Krebs-2 tumor cells. Data shown on the FACS plots are converted into a graph format (bottom right).

Article Snippet: DNA concentration and incorporation of dUTP-TAMRA were measured using Nanodrop (Eppendorf) and calculated by comparing the signal before and after PCR re-precipitation.

Techniques: Fluorescence, Labeling, Amplification, Marker, Injection, Incubation, Southern Blot, Positive Control, Concentration Assay, Flow Cytometry

Journal: Cancer Biology & Therapy

Article Title: Identification of cancer stem cells and a strategy for their elimination

doi: 10.4161/cbt.29854

Figure Lengend Snippet: Figure 2. Analysis of tumorigenic properties of TAMRA-positive cells. ( A ) Overlap between CD34 + and TAMRA + ascites cell populations: 1, cell cycle profile for Krebs-2 ascites cells; 2, FACS analysis confirming the presence of CD34 + and TAMRA + cells in the same sample of ascites; 3 and 4, FACS analysis of TAMRA and/or FITC positive cells. Vast majority of CD34 + cells are also TAMRA + (3), whereas about half of TAMRA + cells are also CD34 + (4); 5, cytofluorescence analysis showing co-staining of ascites Krebs-2 cells in a sample used for FACS profiling. Bars correspond to 10 µm. ( B ) Analysis of engraftment potential of TAMRA + , TAMRA − cells. 1, The plots illustrate the dynamics of tumor formation (percentage of mice with detectable tumors); 2, FACS profile of Krebs-2 ascites cells following incubation with TAMRA-DNA; 3, FACS profile of an enriched sample of TAMRA positive cells to be used for engraftment. ( C ) Analysis of engraftment potential of CD34 + and CD34 − cells. 1, The plots illustrate the dynamics of tumor formation (percentage of mice with detectable tumors); 2, FACS analysis of Krebs-2 ascites cell samples after incubation with isotype control (left) and CD34 specific antibodies (right). ( D ) 1–6 spatial organization of TAMRA-positive cells in solid form of Krebs-2 tumor. Bars correspond to 20 µm (1–4) and 10 µm (5). Well-defined foci of cells internalizing TAMRA-DNA. Each focus includes tens to hundreds of cells. 7, Histology analysis of a solid form of Krebs-2 tumor. Tumor tissue displays pronounced cellular atypia and aggressive growth pattern. Stretches of tumor cells (arrows) are interlaced with the fragments of muscle fibers. 8 and 9, FACS quantification of TAMRA positive cells in a solid form of Krebs-2 tumor, bar corresponds to 20 µm. ( E ) Spatial organization of TAMRA positive cells in spleen and liver. 1 and 2, upon co-incubation of tissue samples with TAMRA-labeled DNA, followed by cryosectioning and confocal imaging; 3 and 4, upon physical homogenization of the tumor down to individual cells, co-incubation with TAMRA-labeled DNA and FACS analysis. Spleen samples lack any cells showing TAMRA-DNA internalization. Single TAMRA positive cells were detected in the liver. Bars correspond to 50 µm. ( F ) 3D reconstruction of a solid form of Krebs-2 tumor showing localization of TAMRA-DNA internalizing cell clusters.

Article Snippet: DNA concentration and incorporation of dUTP-TAMRA were measured using Nanodrop (Eppendorf) and calculated by comparing the signal before and after PCR re-precipitation.

Techniques: Staining, Incubation, Control, Labeling, Imaging, Homogenization

Journal: Cancer Biology & Therapy

Article Title: Identification of cancer stem cells and a strategy for their elimination

doi: 10.4161/cbt.29854

Figure Lengend Snippet: Figure 3. Internalization of Alu fragment DNA by human glioma cells. ( A ) Phase-contrast image of adherent cell fraction and neurospheres from human glioblastoma cell culture. ( B ) Fluorescence microscopy analysis of TAMRA-labeled DNA internalization by glioblastoma cells. Background images show the magnitude of non-specific autofluorescence. Notably, labeled DNA probe displays specific nuclear localization in the cells from neurosphere fraction, and only a single specific positive signal was observed across all adherent cells analyzed. ( C ) Cytofluorescence analysis of TAMRA-labeled DNA in freely-floating neurosphere cell cultures. Several neurospheres are shown. Bottom image represents cytospinned neurospheres stained with DAPI. ( D ) 3D reconstituted image of a neurosphere with cells internalizing TAMRA-labeled dsDNA (arrowheads). ( E ) Visualization of GFP expression in neurospheres that have internalized pEGFP-N1 plasmid. As a control, we provide the fluorescence image of a neurosphere (1) and dying glioma cells (2) neither of which were incubated with plasmid DNA. Unless specified otherwise, bars correspond to 50 µm.

Article Snippet: DNA concentration and incorporation of dUTP-TAMRA were measured using Nanodrop (Eppendorf) and calculated by comparing the signal before and after PCR re-precipitation.

Techniques: Cell Culture, Fluorescence, Microscopy, Labeling, Staining, Expressing, Plasmid Preparation, Control, Incubation

Journal: Cancer Biology & Therapy

Article Title: Identification of cancer stem cells and a strategy for their elimination

doi: 10.4161/cbt.29854

Figure Lengend Snippet: Figure 4. Pathomorphology, histofluorescence and PCR analysis of glioblastoma xenotransplants from brains of NOD/SCID mice. ( A ) Brain MRI of experimental animals. Upper panel: Serial mouse brain MRI sections on day 28 after glioma adherent cell fraction engraftment. Middle panel: Serial mouse brain MRI sections on day 28 after glioma neurosphere cell injection. Lower panel: Serial mouse brain manganese-enhanced MRI sections (same animal as above). Arrows indicate localization of the growing transplant. ( B ) Histology analysis of paraffin sections of glioma transplants and original glioblastoma biopsy. Black arrows on mouse brain sections indicate graft cells, and white arrows show normal brain tissue (mouse 1). Tumor displays surface localization and proliferation in interlobar gyri (central sulcus in this case). HE staining, 200× magnification. Mouse 2: large tumor lining the brain ventricle wall. Central part of the tumor is infiltrated with inflammatory cells (mostly granulocytes). Black arrows point to the cell infiltrate; white arrows show normal brain tissue. HE staining, 400× magnification. Original human tumor: 1, endothelial hyperplasia, vessel proliferation, 200× magnification; 2, cell and nuclear polymorphisms, 400× magnification; 3, endothelial hyperplasy, focal necrosis in the tumor, 200× magnification. AE staining. ( C ) PCR analysis of DNA isolated from the brain tissue carrying glioma graft and from the liver of the same animal (negative control). Human Alu -specific primers were used in amplification reaction. ( D ) Histofluorescence analysis of cryosections of mouse brains with human glioma graft. 1 and 2, overview of a tumor focus cross-section. Arrow indicates a demarcation line between the brain and the tumor. Zoom-in insets are shown in 3 and 4. Red signals correspond to graft cells that internalized TAMRA- Alu DNA. Bars correspond to 10 µm (1 and 2) and 50 µm (3–5); 6, 3D reconstruction of the tumor fragment. Red dots correspond to individual cells or clusters of cells.

Article Snippet: DNA concentration and incorporation of dUTP-TAMRA were measured using Nanodrop (Eppendorf) and calculated by comparing the signal before and after PCR re-precipitation.

Techniques: Injection, Staining, Isolation, Negative Control, Amplification

Journal: Cancer Biology & Therapy

Article Title: Identification of cancer stem cells and a strategy for their elimination

doi: 10.4161/cbt.29854

Figure Lengend Snippet: Figure 7. Cytofluorescence and flow cytometry analyses of TAMRA- Alu DNA internalization by various cellular communities. Field view is shown for microscopy imaging. DNA is stained with DAPI, FITC denotes CD34 + staining, and TAMRA corresponds to TAMRA- Alu DNA. ( A ) FACS profiles showing localization of TAMRA-labeled DNA in mouse lung tumor ascites. ( B ) FACS profiles showing localization of TAMRA-labeled DNA in mouse hepatoma ascites. ( C ) Internalization of TAMRA-labeled DNA by CD34 + human mesenchymal stem cells (short-term culture). ( D ) 3D reconstitution image of TAMRA-DNA internalization by human mesenchymal stem cells showing cytoplasmic localization of the probe. ( E ) FACS profiles showing localization of TAMRA-labeled DNA in CD34 + human bone marrow cells (short-term culture). ( F ) Cytofluorescence analysis of human bone marrow stem cells showing TAMRA-DNA labeling in CD34 + cells. ( G and H ) Non-tumorigenic human HEP-G 2 cells grown in immunocompromised mice display no TAMRA-DNA signal in cellular compartments on either FACS profiles ( G ) or upon cytofluorescence analysis ( H ).

Article Snippet: DNA concentration and incorporation of dUTP-TAMRA were measured using Nanodrop (Eppendorf) and calculated by comparing the signal before and after PCR re-precipitation.

Techniques: Flow Cytometry, Microscopy, Imaging, Staining, Labeling, DNA Labeling

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-C: Bacterially purified GST-Bub2 or GST-Bub2-Q132L, MBP-Bfa1 and 6xHis-Tem1 proteins were used to measure the kinetics of hydrolysis+dissociation (γ[ 32 P]GTP) or dissociation only (γ[ 35 S]GTP) using a filter binding assay (see ). Graphs show average values and standard deviations from three independent experiments. D: Exponentially growing cultures of the indicated strains were shifted to nocodazole containing medium at t = 0. Cell samples were withdrawn at the indicated time for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a checkpoint defect (indicated by re-budding in the absence of proper chromosome segregation) was scored in cycling cultures of the indicated strains shifted either to 14°C for 16h (left graph) or to 37°C for 3h (right graph). F-G: Exponentially growing cells with the indicated genotypes were arrested in G1 by α-factor and released into fresh medium at time 0. At 70’ after release α-factor was re-added to prevent cells from entering a second cell cycle. Cell samples were collected for FACS analysis of DNA contents (F) and for tubulin staining by indirect immunofluorescence (G). H: Cells were treated as in (F-G). TCA extracts were prepared from cell samples at the indicated time points to monitor kinetics of Bfa1-HA6 phosphorylation and Clb2 accumulation and degradation by western blot analysis. Pgk1 was used as loading control. I: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. J-K: Localization of eGFP- tagged Bub2/Bub2-Q132L, Tem1, Bfa1 (J) and Cdc15-GFP (K) was analysed by fluorescence microscopy after formaldehyde fixation.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Filter-binding Assay, Staining, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Cycling cells co-expressing Spc72-Bfa1-eGFPand Spc42-mCherry to mark the SPB (upper panel) or co-expressing Tem1-eGFP and Tub1-GFP (to mark microtubules, lower panel) were analysed to study the distribution of Spc72-Bfa1-eGFP (A) and Tem1-eGFP (B) at SPBs in SPC72-BFA1 bfa1Δ cells. C-D: Cycling cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). Cell samples were withdrawn at the indicated times for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a SPOC defect was scored after propidium iodide staining of cycling cultures of cells with the indicated genotypes after shift to 14°C for 16h. The histograms on the right side represent the DNA contents of the same cells as measured by FACS analysis. F: Percentage of metaphase cells with Cdc15-GFP at 0, 1 or 2 SPBs was scored in the indicated strains after formaldehyde fixation. Metaphases were identified by means of the Tub1-mCherry co-expressed marker. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD and incubated at the indicated temperature. H: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YP medium containing either glucose or galactose and incubated at 25°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Staining, Marker, Incubation

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A: Bacterially purified 6XHis-Tem1 and 6XHis-Tem1-Q79L were loaded with γ[ 32 P]GTP either in the absence or in the presence of recombinant MBP-Bfa1 and incubated at 30°C for 10 minutes. The mixture was then added to GST-Bub2 or buffer alone and kinetics of GTP hydrolysis and dissociation was followed by a filter-binding assay (see details in ). Graphs show average values and standard deviations from three independent experiments. B: Wild type and TEM1-Q79L cells were arrested in G1 by α-factor and then released into fresh medium at 25°C (t = 0). Cell samples were withdrawn every 10’ to measure kinetics of budding and spindle formation/elongation after in situ immunostaining of tubulin. C: Actomyosin ring contraction has been visualized by live cell imaging of wild type and TEM1-Q79L expressing Myo1-GFP (n = 30). D: Logarithmically growing cultures of cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. E: The percentage of cells with binucleate cell bodies accompanied or not by SPOC defect was scored after DAPI staining of cycling cells of the indicated strains shifted to 14°C for 16h. F: Logarithmically growing cultures of strains with the indicated genotypes were shifted to nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD or YP galactose plates and incubated at 30°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Recombinant, Incubation, Filter-binding Assay, In Situ, Immunostaining, Live Cell Imaging, Expressing, Flow Cytometry, Staining

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: List of non-essential genes implicated in microtubules dynamics or spindle positioning identified in the SGA screen with TEM1-Q79L .

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Migration

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-PK, anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. C-F: Localization of eGFP- tagged Tem1 and Tem1-Q79L (C-D) or Bfa1-eGFP (E-F) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative cells of each strain in anaphase. G: Fluorescence intensity ratios were calculated between the two SPBs in anaphase cells of the indicated strains (see details in ).

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Immunoprecipitation, Western Blot, Fluorescence, Microscopy, Marker

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Distribution of Cdc15-GFP (A) or Mob1-GFP (B) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative wild type and TEM1-Q79L cells expressing Cdc15-GFP in metaphase. C: Serial dilutions of stationary phase cells with the indicated genotypes were spotted on YPD and incubated at the indicated temperatures for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Fluorescence, Microscopy, Marker, Expressing, Incubation

Journal: Journal of Translational Medicine

Article Title: pSTAT3: a target biomarker to study the pharmacology of the anti-IL-21R antibody ATR-107 in human whole blood

doi: 10.1186/1479-5876-11-65

Figure Lengend Snippet: ATR-107 concentration-dependently inhibited IL-21 induced STAT3 phosphorylation. A : Purified T cells from healthy donor peripheral blood were pre-incubated with ATR-107 at concentrations from 0.03 ng/ml to 100 ng/ml or control antibody for 30 minutes, followed by stimulation with IL-21 (10 ng/ml) for an additional 15 minutes. Total and phospho–STAT3 was detected by Western blot. B : Healthy donor whole blood was pretreated with ATR-107 or control Ab for 30 minutes prior to stimulation with 10 ng/ml IL-21 for 15 minutes. Cells were fixed, permeabilized, stained with A647-labeled anti–phospho–STAT-3 antibody, and analyzed using flow cytometry gated on CD4 + T cells. Representative histogram showing the baseline (gray area), IL-21 + control Ab (red line) and IL-21 with various amount of ATR-107. C : Inhibition of ATR-107 on pSTAT3 mean fluorescence intensity (MFI) induced by 10 ng/ml IL-21(n = 6). D : Representative image of 6 individual cells detected by ImageStream cytometer (ISC) after treatment with 10 ng/ml IL-21 for 15 minutes. In addition to the bright field image (left column), cells were stained with CD4 (blue), DAPI (green) and pSTAT3 (red). Merged imaging shows pSTAT3 staining overlap with the nuclear (DAPI) staining (far right column). E : Translocation of pSTAT3 was completely blocked by pre-treatment of the samples with ATR-107 (4.3 ug/mL) for 30 minutes. F : Overlay of similarity of nuclear pixel images of DAPI and AF647 dyes in CD4 + T cells. Autoflurorescence (AutoFl) was a measurement of the sample treated with neither ATR-107 nor IL-21. The data were analyzed by the IDEAs software (Amnis Corporation).

Article Snippet: Then the membrane was rinsed three times with PBS + 0.1% Tween-20, and incubated with the primary antibodies at 4°C overnight in PBS + 5% BSA. pSTAT3 (Tyr705, Cat#: 9145 s) and STAT3 (Cat#: 9139 s) antibodies (Cell Signaling Technology) were used at 1:1000 dilution.. HRP-conjugated secondary antibody was used at 1:10,000 dilution and incubated with the membrane at room temperature for one hour.

Techniques: Concentration Assay, Phospho-proteomics, Purification, Incubation, Control, Western Blot, Staining, Labeling, Flow Cytometry, Inhibition, Fluorescence, Cytometry, Imaging, Translocation Assay, Software

Journal: Journal of Translational Medicine

Article Title: pSTAT3: a target biomarker to study the pharmacology of the anti-IL-21R antibody ATR-107 in human whole blood

doi: 10.1186/1479-5876-11-65

Figure Lengend Snippet: ATR-107 behaves like a non-competitive antibody. Summarized results of ATR-107 inhibition on STAT3 phosphorylation in peripheral T ( A ) and B ( B ) cells induced by different amount of IL-21. Whole blood was pre-incubated with various amounts of ATR-107, as indicated on the graphs, for 30 minutes, followed by 1–1,000 ng/ml of IL-21 stimulation for 15 minutes. The reaction was stopped, and the RBC removed by adding the BD Lyse/fix buffer. The remaining cells were then washed, permeabilized and stained for pSTAT3. Shown are the means ± SE from 2–3 separate experiments. % of Max was defined as: (MFI – background) / (Max response – background) X 100. Same data was re-plotted with IL-21 at the x-axis ( C , D ).

Article Snippet: Then the membrane was rinsed three times with PBS + 0.1% Tween-20, and incubated with the primary antibodies at 4°C overnight in PBS + 5% BSA. pSTAT3 (Tyr705, Cat#: 9145 s) and STAT3 (Cat#: 9139 s) antibodies (Cell Signaling Technology) were used at 1:1000 dilution.. HRP-conjugated secondary antibody was used at 1:10,000 dilution and incubated with the membrane at room temperature for one hour.

Techniques: Inhibition, Phospho-proteomics, Incubation, Staining

Journal: Journal of Translational Medicine

Article Title: pSTAT3: a target biomarker to study the pharmacology of the anti-IL-21R antibody ATR-107 in human whole blood

doi: 10.1186/1479-5876-11-65

Figure Lengend Snippet: ATR-107 inhibition on IL-21 induced STAT3 phosphorylation correlates inversely with IL-21R occupancy, ex vivo (A, B). Receptor occupancy assays and pSTAT3 assays were performed side by side in T cells ( A ) and B cells ( B ). Briefly, whole blood samples were pre-incubated with ATR-107 for 15 minutes at 37°C, and stained with cell surface markers (CD4, CD19) for 15 minutes. Then either ATR-107-A647 or 10 ng/ml of IL-21 were added to the wells, and incubated at 37°C for another 15 minutes (for pSTAT3 assay) or 20 minutes (for receptor occupancy assay), followed by the addition of the Lyse/fix buffer to stop the reaction and to remove RBC. After centrifugation, the cell pellets were washed twice with PBS + 2% FBS. Samples were then collected with (for pSTAT3 assay) or without (for receptor occupancy assay) permeabilization and stained with pSTAT3 A647. Data shown are the means ± SE from 8 donors.

Article Snippet: Then the membrane was rinsed three times with PBS + 0.1% Tween-20, and incubated with the primary antibodies at 4°C overnight in PBS + 5% BSA. pSTAT3 (Tyr705, Cat#: 9145 s) and STAT3 (Cat#: 9139 s) antibodies (Cell Signaling Technology) were used at 1:1000 dilution.. HRP-conjugated secondary antibody was used at 1:10,000 dilution and incubated with the membrane at room temperature for one hour.

Techniques: Inhibition, Phospho-proteomics, Ex Vivo, Incubation, Staining, Centrifugation