Journal: Cell Reports Medicine

Article Title: Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer

doi: 10.1016/j.xcrm.2023.101335

Figure Lengend Snippet:

Article Snippet: PDTOs were generated in low-viscosity matrix (LVM) suspension culture medium as described previously., Tumor samples obtained at resection or biopsy were washed 3–5 times with PBS, minced with scissors into pieces that can pass through 1-mL tips, digested at 37°C for 30–60 min with 0.1 mg/mL Dispase II (Life Technologies, 17105-041) and 200 U/ml Collagenase IV (Life Technologies, 17104019) in DMEM/F12 (Life Technologies, 11320082), and then washed with PBS for another 5–10 times at 200 g for 3 min before being plated into 6-well plates (Interpath, 657185) in PDTO LVM culture medium consisting of advanced DMEM/F12 (Thermo Fisher Scientific, 12634028), 1x GlutaMax supplement (Gibco, 35050061), 10 mM HEPES (Sigma-Aldrich, H3375-1KG), 1x B-27 supplement (Life Technologies, 17504001), 1x N2 supplement (Life Technologies, catalog number: 17502001), 10 mM nicotinamide (Sigma-Aldrich, 72340-1KG), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich, A7250-1KG), 20 ng/mL recombinant human basic FGF (Life Technologies, PHG0263), 50 ng/mL recombinant human EGF (Life Technologies, PHG0313) and 500 nM A83-01 (Tocris Bioscience, 2939), with the addition of 100 μg/mL primocin, 10 μM Y27632 (Stemcell Technologies, 72308) and 5% Matrigel (Corning, 356234).

Techniques: Recombinant, Sequencing, Software, Inverted Microscopy

Journal: PLoS ONE

Article Title: NPM1 Silencing Reduces Tumour Growth and MAPK Signalling in Prostate Cancer Cells

doi: 10.1371/journal.pone.0096293

Figure Lengend Snippet: ( a ) NPM1 knockdown does not alter LNCaP cells morphology. LNCaP cells were stably transfected with control shRNA (shScr) or specific NPM1 shRNA (shNPM1). NPM1 mRNA and protein levels were analysed by RT-qPCR and western blotting respectively. Morphology of cells was observed by inverted microscopy and photographed. ( b ) NPM1 knockdown inhibits LNCaP cells clonogenicity. Cells were seeded at low confluence over one week, then fixed with methanol and stained with 5% Giemsa blue before microscopic observation. Pictures are representative of three independent experiments with consistent results. The graph represents the number of cell clones (>50 cells) in the shNPM1 condition, calculated as the mean ± SD of the number of clones counted per field, on 5 random fields, using the ImageJ free software and expressed relatively to the number of clones counted in the control condition. ( c, d ) NPM1 controls proliferation of prostate cancer cells. Five thousand cells were seeded per well in a 96-wells plate and cultured for 48 hours. ( c ) Cells were then incubated with a BrdU labeling solution for 2.5 hours and BrdU incorporation was measured by densitometric analysis at 655 nm. ( d ) Proliferation was also analysed by RT-qPCR assay by evaluating PCNA relative mRNA level accumulation normalized using β-actin mRNA level. All data are representative of at least three independent triplicate experiments and BrdU incorporation as mean of triplicate experiments of 96 points each. Data are expressed as the mean ± SD.

Article Snippet: Total cellular RNA was extracted using TRIzol reagent (Life Technologies) and cDNA was synthesized with 200 U of Moloney murine leukemia virus-reverse transcriptase (Promega), 5 pmol of random primers (C1181, Promega), 40 U RNAsin (Promega), and 2.5 mM deoxynucleotide triphosphate. mRNA levels were quantitated on a Mastercycler ep Realplex (MasterCycler2, Eppendorf, Le Pecq, France) with Mesagreen QPCR Mastermix Plus for SYBR (Promega).

Techniques: Knockdown, Stable Transfection, Transfection, Control, shRNA, Quantitative RT-PCR, Western Blot, Inverted Microscopy, Staining, Clone Assay, Software, Cell Culture, Incubation, Labeling, BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: NPM1 Silencing Reduces Tumour Growth and MAPK Signalling in Prostate Cancer Cells

doi: 10.1371/journal.pone.0096293

Figure Lengend Snippet: ( a ) NPM1 controls EGF expression. Relative EGF mRNA levels compared to β-actin were analyzed by RT-qPCR in LNCaP cells expressing control (shScr) or NPM1 specific shRNA (shNPM1). ( b ) NPM1 control EGF promoter activity. shScr and shNPM1 LNCaP cells were transfected with the phEGF-luciferase reporter plasmid. EGF promoter activity was evaluated by measuring the luciferase activity 24 hours later. Results of the assay were standardized using the CMV promoter as control and expressed as fold-induction over control cells (shScr). ( c ) NPM1 controls activation of the EGF/EGFR pathway downstream effectors. Proteins, extracted from shScr and shNPM1 LNCaP cells cultured in RPMI 1640 10%FBS, were electrophoresed by SDS-PAGE. Transferred membranes were immunoblotted with indicated antibodies. Histograms show the band quantification reported to the β-actin level. Blots are representative of three independent experiments with consistent results. Data are representative of at least three independent experiments and are expressed as the mean ± SD.

Article Snippet: Total cellular RNA was extracted using TRIzol reagent (Life Technologies) and cDNA was synthesized with 200 U of Moloney murine leukemia virus-reverse transcriptase (Promega), 5 pmol of random primers (C1181, Promega), 40 U RNAsin (Promega), and 2.5 mM deoxynucleotide triphosphate. mRNA levels were quantitated on a Mastercycler ep Realplex (MasterCycler2, Eppendorf, Le Pecq, France) with Mesagreen QPCR Mastermix Plus for SYBR (Promega).

Techniques: Expressing, Quantitative RT-PCR, Control, shRNA, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Activation Assay, Cell Culture, SDS Page

Journal: Current Protocols in Toxicology

Article Title: Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction

doi: 10.1002/cptx.99

Figure Lengend Snippet: Preparation of human primary foreskin fibroblasts (FSK) for transmission electron microscopy (TEM) analysis (Basic Protocol ). ( A ) Flowchart depicting our strategy for embedding FSK cells in EPON resin and ( B ) trimming for ultramicrotome ultra‐thin sectioning. After 48 hr at 55°C‐60°C, the resin‐embedded sample is removed from the microcentrifuge tube with a razor blade. The excess resin is cut with a hacksaw and mounted on a wooden stick with epoxy adhesive. The mounted sample can be placed on the ultramicrotome holder and trimmed. The tip of the trapezoid can be a point, giving square sections, or a ridge, resulting in rectangular sections. At this point, the sample is ready for the preparation of the sections for TEM.

Article Snippet: Fresh human circumcision specimen obtained from a young healthy donor: size 2 × 2 cm 2 (see Strategic Planning) 70% ethanol for cleaning laminar flow hood (see Strategic Planning) Dulbecco's phosphate‐buffered saline (DPBS; Millipore, cat. no. D8537) 200 U/ml collagenase A (Sigma‐Aldrich, cat. no. C0130) Complete fibroblast cell culture medium (FBM; see ) 0.05% (w/v) trypsin‐EDTA (Gibco, cat. no. T4049) Laminar flow biosafety cabinet 37°C water bath 100‐ml beaker (Corning, cat. no. 1000‐100) 35‐ and 100‐mm plastic petri dishes (Corning, cat. no. CLS3294; Corning, cat. no. CLS3296), gelatin coated as described in Strategic Planning Sterile forceps Sterile tweezers 1.5‐ml sterile microcentrifuge tubes (Eppendorf or similar) 40‐μm sterile cell strainer (Corning, cat. no. 431750) 37°C humidified 5% carbon dioxide incubator Eppendorf tabletop microcentrifuge Inverted microscope T‐75 cell culture flasks, vented cap (Sigma, cat. no. CLS3290), gelatin coated as described in Strategic Planning Additional reagents and equipment for counting viable cells with a hemocytometer and trypan blue (see Current Protocols article: Phelan, ) NOTE : All reagents should be purchased sterile or be filter sterilized using a 0.2‐μm filter.

Techniques: Transmission Assay, Electron Microscopy, Thin Sectioning, Adhesive

Journal: Cell Reports Medicine

Article Title: Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer

doi: 10.1016/j.xcrm.2023.101335

Figure Lengend Snippet:

Article Snippet: PDTOs were generated in low-viscosity matrix (LVM) suspension culture medium as described previously., Tumor samples obtained at resection or biopsy were washed 3–5 times with PBS, minced with scissors into pieces that can pass through 1-mL tips, digested at 37°C for 30–60 min with 0.1 mg/mL Dispase II (Life Technologies, 17105-041) and 200 U/ml Collagenase IV (Life Technologies, 17104019) in DMEM/F12 (Life Technologies, 11320082), and then washed with PBS for another 5–10 times at 200 g for 3 min before being plated into 6-well plates (Interpath, 657185) in PDTO LVM culture medium consisting of advanced DMEM/F12 (Thermo Fisher Scientific, 12634028), 1x GlutaMax supplement (Gibco, 35050061), 10 mM HEPES (Sigma-Aldrich, H3375-1KG), 1x B-27 supplement (Life Technologies, 17504001), 1x N2 supplement (Life Technologies, catalog number: 17502001), 10 mM nicotinamide (Sigma-Aldrich, 72340-1KG), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich, A7250-1KG), 20 ng/mL recombinant human basic FGF (Life Technologies, PHG0263), 50 ng/mL recombinant human EGF (Life Technologies, PHG0313) and 500 nM A83-01 (Tocris Bioscience, 2939), with the addition of 100 μg/mL primocin, 10 μM Y27632 (Stemcell Technologies, 72308) and 5% Matrigel (Corning, 356234).

Techniques: Recombinant, Sequencing, Software, Inverted Microscopy