Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity

doi: 10.1136/jitc-2021-002408

Figure Lengend Snippet: Toxicological and immunomodulatory evaluation in vitro of intracellular TLR agonists alone or combined using primary human macrophages. Macrophages were in vitro differentiated from purified monocytes stimulated with 25 ng/mL of recombinant human macrophage colony-stimulating factor for 6 days (M0); M1 and M2 polarized macrophages were obtained by stimulation with 100 ng/mL of lipopolysaccharide+50 ng/mL of interferon-γ or 20 ng/mL of IL-4, respectively, for 24 hours. (A) Cell viability (Alamar blue) of M0 macrophages exposed 24 hours to TLR agonists alone or in different combinations. concentrations used were 5, 10, 20 and 50 µg/mL, represented by color gradient from left to right. (B–D) cytokine secretion (ELISA) of (B) CXCL10, (C) CCL5, and (D) IL-10 by macrophages exposed for 24 hours to 5 µg/mL of TLR agonists or untreated M0 macrophages. In each panel, M1 and M2 polarized macrophages from the same individuals are shown as reference populations. Each dot corresponds to macrophages from each blood donor. (E) THP-1-Lucia cells for monitoring the NF-kB signal transduction pathway were exposed for 16 hours to pIC and/or R848 at indicated concentrations. Bars represent mean±SD, n=3. (F, G) Cytotoxic activity of TLR-treated macrophages toward human Panc1 cancer cells stained with CellTrace. Each dot corresponds to macrophages from each blood donor. Bars represent mean±SEM. Statistical comparison was performed using one-way analysis of variance followed by Tukey’s multiple comparison test. Statistically significant differences are represented as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.001. IL, interleukin; NF-kB, nuclear factor-kappa B; ns, non-significant; pIC, poly(I:C); R837, imiquimod; R848, resiquimod; TLR, toll-like receptor.

Article Snippet: Human pancreatic carcinoma cell line PANC1 (ATCC), murine fibrosarcoma cell line MN/MCA1, and murine lung adenocarcinoma cell line CMT167 (ECACC) were cultured in DMEM (Lonza) with 10% FBS and 0.5% penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: In Vitro, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Transduction, Activity Assay, Staining, Comparison

Journal: British Journal of Cancer

Article Title: Inducible re-expression of p16 in an orthotopic mouse model of pancreatic cancer inhibits lymphangiogenesis and lymphatic metastasis

doi: 10.1038/sj.bjc.6604457

Figure Lengend Snippet: Induction of p16 expression inhibits lymphatic spread of MiaPaCa-2 cells to the liver hilus lymph nodes. Metastatic spread of MiaPaCa-2-TREx-p16 cells was evaluated based on inspection of lung and abdominal organs at the time of autopsy and microscopic assessment of lymph node metastasis. ( A ) Representative picture of a lymph node with cytokeratin-positive human tumour cells (stained red) surrounded by lymphocytes (images at × 10 magnification and × 20 (image inset). ( B ) Quantitative analysis of lymph node metastasis. Data represent the number of animals with presence of lymph node metastasis in the liver hilus lymph nodes (left: comparison between treatment (+Dox) and control group (−Dox). Right: subgroup analysis including only those animals with histologically confirmed expression or absence of p16 protein (−Dox/p16 vs +Dox/+p16) ( ** P <0.05) whereas in ( C ) the correlation between LVD and lymph node metastasis is shown ( * P <0.05).

Article Snippet: Briefly, 7 μ m thick cryosections were fixed in 4% paraformaldehyde/PBS, pH 7.0, for 20 min at room temperature (RT), treated with 0.3% H 2 O 2 for 10 min and incubated overnight at 4°C with anti-human p16 INK4a (NEOMarkers, CA, USA) or 1 h at RT with antibodies to mouse LYVE-1 (Relia Tech, Braunschweig, Germany), mouse CD31 (BD Pharmingen, Heidelberg, Germany) or human pan-cytokeratin-HRP (Santa Cruz Biotechnology, CA, USA) (1 : 50) followed by incubation with a biotinylated secondary antibody.

Techniques: Expressing, Staining, Comparison, Control

Journal: bioRxiv

Article Title: Spatial transcriptomic analysis of progressing oral epithelial dysplasia reveals unique differentially expressed genes and microenvironmental changes

doi: 10.64898/2026.01.07.697832

Figure Lengend Snippet: Differential gene expression in progressors (biopsy 1) and non-progressors (biopsy 1) within PanCK-positive (A-C) and PanCK-negative (D) regions of interest (ROI). (A) Heatmap depicting unsupervised clustering of samples into two groups based on differentially expressed (DE) genes. (B) Volcano plot highlighting significantly DE genes with yellow dots. (C) Violin plots illustrating the most prominently downregulated gene (CDKN2A) and upregulated gene (STOM) in progressing oral epithelial dysplasia (OED). (D) Volcano plot displaying significantly DE genes with yellow dots, alongside violin plots showing downregulated genes (S100A10 and KL4) and upregulated gene (ZFAT) in progressing OED.

Article Snippet: Slide preparation was performed according to the manufacturer’s protocol (Nanostring GeoMx) with the following digestion and heat-induced epitope retrieval (HIER) conditions: 0.1 μg/ml Proteinase K for 15 min and ER for 20 min. ROI selection (PanCK+/PanCK-) was performed in the GeoMx Digital Spatial Profiler (DSP) based on morphology and immunofluorescence staining with PanCK (Novus Biologicals, clone AE1/AE3, NBP2-33200AF488), SMA (Abcam, clone 1A4, ab202368), CD45 (CST, clone D9M8I, 13917BF), and SYTO 83 DNA staining (ThermoFisher, S11364).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Spatial transcriptomic analysis of progressing oral epithelial dysplasia reveals unique differentially expressed genes and microenvironmental changes

doi: 10.64898/2026.01.07.697832

Figure Lengend Snippet: (A) Bar graph showing the most enriched pathways in Progressors, indicating the number of enriched pathways within each category. (B) Heatmap illustrating clustering of PanCK-positive (PanCK+) regions of interest (ROI) in progressing and non-progressing oral epithelial dysplasia (OED). (C) Volcano plot displaying differentially expressed (DE) pathways, with yellow dots representing significant pathways in PanCK+ ROI of progressors and non-progressors. (D) Heatmap showing clustering of PanCK-negative (PanCK-) ROI in progressing and non-progressing OED. (E) Heatmap illustrating clustering of PanCK+ ROI in precursor lesions and subsequent oral squamous cell carcinoma (OSCC) samples. (F) Heatmap depicting clustering of PanCK- ROI in precursor lesions and subsequent OSCC samples. (G) Volcano plot with yellow dots indicating DE pathways in PanCK+ ROI of precursor lesions and subsequent OSCC.

Article Snippet: Slide preparation was performed according to the manufacturer’s protocol (Nanostring GeoMx) with the following digestion and heat-induced epitope retrieval (HIER) conditions: 0.1 μg/ml Proteinase K for 15 min and ER for 20 min. ROI selection (PanCK+/PanCK-) was performed in the GeoMx Digital Spatial Profiler (DSP) based on morphology and immunofluorescence staining with PanCK (Novus Biologicals, clone AE1/AE3, NBP2-33200AF488), SMA (Abcam, clone 1A4, ab202368), CD45 (CST, clone D9M8I, 13917BF), and SYTO 83 DNA staining (ThermoFisher, S11364).

Techniques:

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a The expression of α-SMA in tumor stroma was assayed by immunohistochemical staining, indicating that CAFs were abundant in pancreatic tumor stroma. b The morphological images of NAFs and CAFs. c Immunofluorescence staining showed the subcellular localization and the expression of α-SMA and FAP in NAFs and CAFs. Scale bar = 50 μm, magnification, ×400. d The mRNA expression levels of α-SMA, FAP, and FSP1 in NAFs and CAFs (passage 4) isolated from three patients were detected by qRT-PCR analysis. n = 3 (replicating from three patients), *** p < 0.001. e Western blot analysis shows the expression of α-SMA and FAP in NAFs and CAFs derived from four pairs of non-neoplastic pancreatic tissues and tumor tissues. f , g qRT-PCR and western blot analysis show the different expression levels of α-SMA and FAP in CAFs at different passages. Compared with the fourth-passage CAFs (CAFs-P4), the expression of α-SMA and FAP in CAFs-P8 had no significant difference, but the expression in CAFs-P12 significantly decreased. n = 3 (replicating from three patients), ns: not significantly different, * p < 0.05

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Isolation, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a , b The qRT-PCR and western blot analysis show the mRNA and protein levels of SATB-1 in SW1990 and PANC-1 cells cultured with (co-culture) or without CAFs (monoculture). n = 3, *** p < 0.001. c The qRT-PCR analysis shows the mRNA expression of SATB-1 in 32 pancreatic tumor tissues and matched non-neoplastic pancreatic tissues. Dots represent each patient, and error bars indicate standard deviation (SD). n = 3, *** p < 0.001. d Western blot analysis shows the protein levels of SATB-1 in 12 pancreatic cancer tissues (PC) and matched non-neoplastic pancreatic tissues (NP). e , f The qRT-PCR and western blot analyses show the mRNA and protein levels of SATB-1 in various pancreatic cancer cell lines. n = 3

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Co-Culture Assay, Expressing, Standard Deviation

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a Wound-healing assay shows the abilities of SW1990 and PANC-1 cells with SATB-1 silenced or co-cultured. b The transwell assay shows the fractions of migrated and invaded SW1990 and PANC-1 cells with SATB-1 silenced or co-cultured. Knockdown of SATB-1 inhibited the migration and invasion abilities of PANC-1 and SW1990 cells. Coculturing with CAFs enhanced the migration and invasion abilities of PANC-1 and SW1990 cells, but neutralization with anti-SDF-1 antibody reduced these abilities. Compared with control pancreatic cancer cells, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. c Wound-healing and transwell assays show the upregulated migration and invasion abilities of Capan-2 and BXPC-3 cells transfected with pcDNA3.1-SATB-1. Compared with control pancreatic cancer cells, n = 3, ** p < 0.01, *** p < 0.001

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Wound Healing Assay, Cell Culture, Transwell Assay, Knockdown, Migration, Neutralization, Control, Transfection

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: The relationship between SDF-1 expression and SATB-1 expression in pancreatic cancer tissues

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: Pancreatic CSCs, and cancer cell lines (AsPC-1 and PANC-1) were treated with coumarin-6 containing Mang-NPs for 2 h. Cells were incubated with Hoechst 33342 for nuclear staining. The fluorescence microscope was used to observe the uptake of Mang-NPs. Green color = Mang-NP. Blue color = Nucleus.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Incubation, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Pancreatic CSCs were treated with PLGA-NPs (NPs), Mangostin or Mang-NPs (0–10 μM) for 48 hrs, and cell proliferation was measured. Data represent mean (n = 4) ± SD. *, #, &, % and $ = significantly different from control (NPs group), and each other, P < 0.05. ( B ) and ( C ) AsPC-1 and PANC-1 cells were treated with NPs, Mangostin or Mang-NPs (0–10 μM) for 48 hrs, and cell proliferation was measured. Data represent mean (n = 4) ± SD. *, #, &, % and $ = significantly different from control (NPs group), and each other, P < 0.05. ( D ) Human pancreatic normal ductal epithelial (HPNE) cells were treated with NPs, Mangostin or Mang-NPs (0–10 μM) for 48 hrs, and cell proliferation was measured. Data represent mean (n = 4) ± SD. Mango-NPs = Mang-NPs

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Control

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Pancreatic CSCs (Pan CSCs), and cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) were treated with Mang-NPs (0–10 μM) for 21 days. Number of colonies were counted. Data represent mean ± SD. *, # and % = significantly different from control, and each other, P < 0.05. ( B ) Pan CSCs, and cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) were treated with Mang-NPs (0–10 μM) for 48 hrs. Apoptosis was measured by TUNEL assay. Data represent mean ± SD. *, # and % = significantly different from control, and each other, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Control, TUNEL Assay

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Human pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, & and # = significantly different from control, P < 0.05. ( B ) Mouse pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, & and # = significantly different from control, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Incubation, Control

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 48 h. The expression of Nanog, and c-Myc was measured by the Western blot analysis. β-actin was used as a loading control. ( B ) Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 36 h. The expression of c-Myc, Oct-4 and Nanog was measured by q-RT-PCR. Data represent mean ± SD. * and # = significantly different from control, P < 0.05. ( C ) Nanog shRNA enhances the inhibitory effects of Mang-NPs on colony formation. Pan CSCs/Scrambled and CSCs/Nanog shRNA were seeded and treated with Mang-NPs (0–10 μM) for 21 days. At the end of incubation period, number of colonies were counted. Data represent mean ± SD. *, #, %, and @ = significantly different from control, P < 0.05. ( D ) Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 48 h. The expression of Gli1, Gli2, Patched-1, and Patched-2 was measured by the Western blot analysis. β-actin was used as a loading control. ( E ), Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 36 h. The expression of Bcl-2 was measured by q-RT-PCR. Data represent mean ± SD. * = significantly different from control, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, shRNA, Incubation

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A – D ) Pancreatic CSCs, and cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) were stably transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences). Transduced CSCs and cell lines were treated with Mang-NPs (0–10 μM) for 24 h. Gli reporter activity was measured as we described . Data represent mean ± SD. *, # and % = significantly different from control, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Stable Transfection, Transduction, Luciferase, Activity Assay, Control

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Inhibition of pancreatic cancer growth and development in KPC mice. KPC mice were treated with Mang-NPs (20 mg/kg) for about 10 weeks. At the end of experiment, mice were sacrificed and pancreas weight was recorded. Data represent mean ± SD. * and # = significantly different from untreated control group, P < 0.05. ( B ) Pancreatic tissues from control and Mang-NPs-treated mice were fixed and stained with H & E. Tissue sections were visualized for the presence of PanIN (PanIN-1A, PanIN-1B, PanIN-2, and PanIN-3) lesions and PDAC. nd = not detected. ( C ) Liver metastasis. Liver tissues from control and Mang-NPs-treated mice were visualized for the presence of nodules. ( D ) Expression of stem cell marker, and pluripotency maintain factors in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of CD24, CD133, c-Myc, Nanog and Oct4 was measured. β-actin was used as a loading control. ( E ) Expression of components of Shh pathway in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of Gli1, Gli2, Patched1, Patched2, and Smoothened was measured. ( F ) Expression of Bcl2, XIAP and Cyclin D1 in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of Bcl2, XIAP and Cyclin D1 was measured. ( G ) Expression of E-cadherin, N-Cadherin, Slug, Snail, and Zeb1 in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of E-cadherin, N-Cadherin, Slug, Snail, and Zeb1 was measured.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Inhibition, Control, Staining, Expressing, Marker, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro

doi: 10.3389/fbioe.2022.872275

Figure Lengend Snippet: Immunohistochemical staining of co-cultures with HRE, co-cultures with HUVEC and tri-cultures. (A–F) : pan-cytokeratin (green) was used to detect epithelial keratins in all cultures while mucin5AC (red) showed presence of mucus and goblet cells in all cultures containing HRE with BM-MSC (A,B) and HNF (E,F) as well as in HRE-co-cultures with ASC (C) but not in tri-cultures with ASC (D) (G–L) : claudin-1 (green) indicated tight junction formation in all HRE-cultures, and α-tubulin (red) could visualize cilia in all HRE-cultures with BM-MSC (G,H,S,T) and HNF (K,L,W,X) as well as in HRE-co-cultures with ASC (L,U) but not in tri-cultures with ASC (J,V) (M,O,Q) : CD31 staining confirmed presence of endothelial cells in co-cultures of HUVEC with BM-MSC (M) , ASC (O) or HNF (Q) (N,P,R) : pan-cytokeratin stained the epithelial cell layer on top of the fibrin gel while CD31 was used to detect endothelial cells and vascular-like structures in tri-cultures containing one of the supporting cell types: BM-MSC (N) , ASC (P) or HNF (R) ; DAPI (blue) was used to counterstain cell nuclei. Representative pictures are shown. Scale bar: 50 µm.

Article Snippet: Primary antibodies included rabbit anti- pan -cytokeratin (1:200, Acris ), mouse anti-mucin-5AC (1:800, Acris ), rabbit anti-claudin-1 (1:800, Biorbyt), mouse anti-acetylated tubulin (1:800, Sigma-Aldrich) and mouse anti-CD31 (PECAM-1, 1:100; Sigma-Aldrich).

Techniques: Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: The Use of a New CellCollector to Isolate Circulating Tumor Cells from the Blood of Patients with Different Stages of Prostate Cancer and Clinical Outcomes - A Proof-of-Concept Study

doi: 10.1371/journal.pone.0158354

Figure Lengend Snippet: The CTCs were identified and enumerated via a) positive nuclear staining (Hoechst), b) positive cytokeratin staining, and c) negative CD45 staining. d) Overlay of all images showing size and morphological characteristics.

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin (BSA) (PAA) in PBS for 1 h. Primary antibodies, including anti-pan-cytokeratin-fluorescein isothiocyanate (FITC) (CK8, CK18 and CK19, Abcam) and anti-CD45- allophycocyanin (APC, Invitrogen), were added for 1 h. The wire was then rinsed 3 times with 3 ml of PBS, and the nuclei were counterstained with Hoechst 33258 (Invitrogen).

Techniques: Staining

Journal: Nature

Article Title: Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis

doi: 10.1038/nature02009

Figure Lengend Snippet: Cyclopamine treatment blocks tumour formation of human pancreatic adenocarcinoma cells after transplantation into nude mice. a, Schematic indicating sites of tumour cell and cyclopamine/vehicle injections. b, Isolated tumours derived from control or cyclopamine-treated L3.6sl and Panc 05.04 adenocarcinoma cells. Cyclopamine/vehicle injections were initiated either after palpable tumours had formed (delayed) or simultaneously with injection of tumour cells (concurrent). All pictures are shown at the same magnification. c, Weight of isolated tumours. Untreated control tumours of each cell line were adjusted to 1 to allow comparison of relative change in tumour mass. For delayed cyclopamine/vehicle injections, the values are: BxPC3, control 1 (n = 6), cyclopamine 1.1 (n = 5); Panc 05.04, control 1 (n = 5), cyclopamine 0.48 (n = 4); L3.6sl, control 1 (n = 5), cyclopamine 0.39 (n = 4). The values for concurrent cyclopamine/vehicle injections are: L3.6sl, control 1 (n = 4), cyclopamine 0.16 (n = 4). Error bars indicate standard deviation. Double asterisks, P < 0.01. d–h, Histological analysis of the effect of cyclopamine treatment on L3.6sl-derived tumours. d, e, Haematoxylin/eosin staining of sections through the peripheral tumour regions. f, g, TUNEL staining of apoptotic cells in control (f) and cyclopamine-treated (g) tumours. h, Quantification of TUNEL-positive cells in control (blue) and cyclopamine-treated (red) tumours. Error bars indicate s.e.m. Double asterisks, P < 0.01.

Article Snippet: BxPC3 and all the Panc cell lines were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), l -glutamine and penicillin/streptomycin; medium for Panc cell lines was also supplemented with insulin–transferrin–selenium (Gibco).

Techniques: Transplantation Assay, Isolation, Derivative Assay, Control, Injection, Comparison, Standard Deviation, Staining, TUNEL Assay

Journal: Cell death discovery

Article Title: Inhibition of Aurora Kinase B activity disrupts development and differentiation of salivary glands.

doi: 10.1038/s41420-020-00393-w

Figure Lengend Snippet: Fig. 8 AURKB loss disrupted acinar identity via ROS generation. A, B Mist1 (green) and pan cytokeratin (blue) expressions in E16.5+24h bud cells, scale bar = 50 µm. Remarkable loss of the acinar differentiation marker Mist1 at the protein and transcriptional levels in response to Barasertib treatment. Recovery of Mist1 levels due to ROS scavenging using aminoguanidine (number of pooled glands ≥5), analysed by ANOVA **P ≤0.01, *p ≤0.05, ****P ≤0.0001. C, D ROS production in response to AURKB inhibition in the embryonic salivary glands. ROS levels were visualised in the SMG explants after staining with DCFDA and inspection under fluorescence microscope. Analysis of corrected total fluorescence (CTF) using ImageJ revealed that Barasertib treatment for 24 h significantly increased levels of ROS in the E16.5+24h explants which were significantly reduced after co- treating the explants with aminoguanidine, analysed by ANOVA, **P ≤0.01, *p ≤0.05, ns: non-significant, p > 0.05. Scale bar = 500 μm, n = 3/group.

Article Snippet: To block endogenous peroxidase activity and nonspecific background staining sections were incubated in 3% hydrogen peroxide solution for 20–30 min. To block all epitopes on the tissue samples and prevent nonspecific antibody binding, sections were incubated with 1% BSA in 1X TBS, pH 7.6 for 5 min. Tissue sections were incubated at 4 °C overnight, with the antibody: rabbit-anti-cleaved caspase 3 (1:2500, NB100–56113, Novus Bio), rabbit–anti-Mist1 (1:200, ab187978, Abcam), mouse-anti-Cyclin D1 (1:400, NBP2–32840, Novus Bio), rabbit-anti-AURKB (1:200, ab115793, Abcam), rabbit-anti-ki67 (1:100, ab16667, Abcam), rabbit-anti-p21 (1:200, ab188224, Abcam), rabbit-anti-p.H2AX(1:200, ab81299, Abcam), rat-anti-E- Cadherin (1:200, sc-59778, Santa Cruz Biotechnology, Inc.) guinea pig-anti-pan cytokeratin (1:50, BP5069, Origene).

Techniques: Marker, Inhibition, Staining, Microscopy