Journal: Angewandte Chemie (International ed. in English)

Article Title: Ultrafluorogenic Coumarin-Tetrazine Probes for Real-Time Biological Imaging **

doi: 10.1002/anie.201403890

Figure Lengend Snippet: a) Mitochondrial imaging: OVCA-429 cells with RFP-tagged mitochondria were incubated with an anti-mitochondria-TCO antibody, rinsed briefly, and then imaged after addition of 100nM HELIOS-388H in PBS. Colocalization analysis in ImageJ (Costes auto-threshold) was used to generate the merged image in the right-hand panel. b) Actin imaging: COS-1 cells were incubated with phalloidin-TCO (1µg/mL) and DRAQ5 nuclear counterstain (1µM, BioStatus), rinsed briefly, and then imaged upon addition of the indicated HELIOS probe at 100nM. Control images were collected at matched dye concentrations in the absence of phalloidin-TCO.

Article Snippet: Colocalization analysis in ImageJ (Costes auto-threshold) was used to generate the merged image in the right-hand panel. b) Actin imaging: COS-1 cells were incubated with phalloidin-TCO (1µg/mL) and DRAQ5 nuclear counterstain (1µM, BioStatus), rinsed briefly, and then imaged upon addition of the indicated HELIOS probe at 100nM.

Techniques: Imaging, Incubation

Journal: Stem cell research

Article Title: Derivation and characterization of a UCP1 reporter human ES cell line

doi: 10.1016/j.scr.2018.04.007

Figure Lengend Snippet: Differentiation protocol and characterization of mCherry positive cells. (A) Scheme of the stepwise differentiation strategy to differentiate UCP1: mCherry H1 hES cells to brown adipocytes. (B) Fluorescence imaging of the differentiation of UCP1: mCherry H1 hES cells at days 0, 6, 14 and 20 of differentiation. Scale bar = 50 μm. (C) qRT-PCR analysis of differentiating reporter line cultures. Expression was normalized to GAPDH (n = 3 biological replicates, Student’s t-test, *p < 0.05, data represented as mean ± S.E.M). (D) Immunostaining of UCP1: mCherry H1 hES cell-derived cells using antibodies against mCherry (red) and UCP1 (green). Differentiated mCherry positive cells show co-expression of UCP1. Arrow refers to cell in inset image. Scale bar = 50 μm. (E) Mitotracker staining (green) of UCP1: mCherry H1 hES cell-derived cells. Scale bar = 50 μm. (F) Immunostaining of UCP1: mCherry H1 hES cell-derived cells using antibody against mCherry (red) and BODIPY against lipid droplets (green). Arrow refers to cell in inset image. Scale bar = 100 μm. (G) Oxygen consumption assay on reporter line derived brown adipocytes. (H) Glycerol release assay of UCP1: mCherry H1 hES cell-derived cells. Cells were incubated with either 20 pm isoproterenol or 10 pm forskolin for 1 h (n = 3 biological replicates, Student’s t-test, *p < 0.05, data represented as mean ± S.E.M).

Article Snippet: White adipocyte cells were incubated with 10 μΜ forskolin and imaged using a Nikon Biostation CT live imaging system for 36 h. Fluorescence intensity was quantified using ImageJ.

Techniques: Fluorescence, Imaging, Quantitative RT-PCR, Expressing, Immunostaining, Derivative Assay, Staining, Glucose Assay, Incubation

Journal: Stem cell research

Article Title: Derivation and characterization of a UCP1 reporter human ES cell line

doi: 10.1016/j.scr.2018.04.007

Figure Lengend Snippet: Real-time monitoring of UCP1 upregulation in UCP1: mCherry H1 hES cell-derived white adipocytes. (A) Representative phase contrast and fluorescence images of UCP1: mCherry H1 hES cell-derived white adipocytes after stimulation with 10 μΜ forskolin. Time lapse photos at 0, 18, and 36 h after forskolin addition. Arrow refers to cell in inset image. (B) Quantification of fluorescence intensity of mCherry positive cells after 10 μm forskolin stimulation over 36 h.

Article Snippet: White adipocyte cells were incubated with 10 μΜ forskolin and imaged using a Nikon Biostation CT live imaging system for 36 h. Fluorescence intensity was quantified using ImageJ.

Techniques: Derivative Assay, Fluorescence

Journal: ACS Central Science

Article Title: Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry

doi: 10.1021/acscentsci.8b00933

Figure Lengend Snippet: Airway inflammation features increased CG activity. (a) Representative confocal images of sputum-derived human neutrophils (DNA panel, orange arrowheads) incubated with mSAM (D/A panel). (b) Quantification of cathepsin G (CG) activity on 14 CF and 10 healthy donor neutrophils by confocal microscopy (mSAM). (c) Donor/acceptor increase after 30 min of incubation of sSAM with sputum supernatants (7 healthy donors and 27 CF patients) and 12 COPD bronchial lavage (BL) fluids. (d) Mean donor/acceptor values of 5 patients after 1 and 20 min of incubation with mSAM in the presence or absence of ACT [3 μM]. (e) Fluorimetric quantification of CG activity in the mucus supernatants of CF patients in the presence of a small-molecule CG inhibitor I (CGI) [25 μM], 1 and 30 min after sSAM addition. Scale bars: 10 μm. Images are representative of the 14 and 10 subjects shown in part b. For each condition and patient, 50–100 cells were analyzed by microscopy. Each human sample was measured in duplicate. Statistics were calculated by Wilcoxon rank sum test.

Article Snippet: In 50 μL of PBS, 30 000 human CF or healthy donor sputum cells were incubated with mSAM [2 μM], Draq5 DNA stain (BioStatus Limited, Shepshed, U.K.) (1:1000 dilution), for 20 min at room temperature in 1.5 mL Eppendorf tubes.

Techniques: Activity Assay, Derivative Assay, Incubation, Confocal Microscopy, Microscopy

Journal: Cancers

Article Title: Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment

doi: 10.3390/cancers10100375

Figure Lengend Snippet: Mefloquine and nelfinavir synergize to kill Tsc2 −/− Mouse embryonic fibroblasts (MEFs), ELT3-T3 and sporadic cancer cells. Dose response curves were performed in Tsc2 +/+ and Tsc2 −/− MEFs using flow cytometry to measure cell death following treatment with ( A ) nelfinavir (NFV); ( B ) mefloquine (MQ) and ( C ) combined mefloquine with a fixed concentration of 10 µM nelfinavir (MQ/NFV); ( D ) Tsc2 +/+ and Tsc2 −/− MEFs; ( E ) ELT3-T3 and ELT3-V3; ( F ) MCF7, HCT116 and NCI-H460 were treated with either DMSO, etoposide (ETO), 10 µM mefloquine (MQ), 10 µM nelfinavir (NFV) or mefloquine combined with nelfinavir (MQ/NFV) for 48 h. Cells were then tested by flow cytometry and cells were separated into viable and non-viable cell populations via DRAQ7 staining. Statistical significance is shown with combination treated Tsc2 −/− MEFs or the ELT3-V3 cells to their wild-type controls, and comparing single drug treatment of mefloquine and combination with the MCF7, HCT116 and NCI-H460.

Article Snippet: Treated cells were collected and incubated with 3 μM DRAQ7 (Biostatus, Leicestershire, UK) at room temperature for 10 min. Flow cytometry was performed using a FACS Calibur flow cytometer (Becton Dickinson (Cowley, UK)) with excitation set at 488 nm and detection of fluorescence in log mode at wavelengths greater than 695 nm.

Techniques: Flow Cytometry, Concentration Assay, Staining

Journal: Cancers

Article Title: Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment

doi: 10.3390/cancers10100375

Figure Lengend Snippet: Mefloquine and nelfinavir prevents colony formation and spheroid growth. ( A ) Colony formation was tested in Tsc2 −/− MEFs seeded on soft agar that were treated for 14 days with Dimethyl Sulfoxide (DMSO), 10 µM mefloquine (MQ), 10 µM nelfinavir (NFV) or in combination. Tumor diameters were measured using Image J; scale bar is 200 μm. Significance was observed when comparing combined nelfinavir and mefloquine treatment to DMSO vehicle control. ( B ) Tsc2 −/− MEF spheroids were treated under the same conditions as ( A ) for 96 h. DRAQ7 was supplemented for the final 36 h to monitor cell death before images were taken and DRAQ7 fluorescence quantified. ( C ) Spheroids treated in ( B ) were re-plated onto standard tissue culture plates and grown in drug-free media. Images were taken every 24 h and the area of outgrowth calculated using Image J, scale bar is 200 μm and outgrowth area is graphed.

Article Snippet: Treated cells were collected and incubated with 3 μM DRAQ7 (Biostatus, Leicestershire, UK) at room temperature for 10 min. Flow cytometry was performed using a FACS Calibur flow cytometer (Becton Dickinson (Cowley, UK)) with excitation set at 488 nm and detection of fluorescence in log mode at wavelengths greater than 695 nm.

Techniques: Control, Fluorescence

Journal: Cancers

Article Title: Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment

doi: 10.3390/cancers10100375

Figure Lengend Snippet: Mefloquine and nelfinavir drug cytotoxicity is not associated with mTORC1 hyperactivity and causes minimal autophagy inhibition. ( A ) Tsc2 −/− MEFs, NCI-H460, MCF7 and HCT116 cells were pre-treated with 50 nM rapamycin (RAP) for 1 h, where indicated, before being treated with 10 μM nelfinavir (NFV) and 10 µM mefloquine (MQ) for 48 h. Cells were stained with DRAQ7 and % cell death determined using flow cytometry. ( B ) Western blotting was carried out to determine rp-S6 phosphorylation at Ser235/236 in the cells treated in ( A ) after 48 h of treatment. ( C ) Tsc2 +/+ and Tsc2 −/− cells were treated with DMSO, 10 µM mefloquine (MQ), 20 µM chloroquine (CQ), 10 µM mefloquine or 20 µM chloroquine combined with 10 µM nelfinavir for 3 h. Accumulation of lipidated LC3-II were analyzed by Western blot. Total protein levels of β-actin were used as a loading control.

Article Snippet: Treated cells were collected and incubated with 3 μM DRAQ7 (Biostatus, Leicestershire, UK) at room temperature for 10 min. Flow cytometry was performed using a FACS Calibur flow cytometer (Becton Dickinson (Cowley, UK)) with excitation set at 488 nm and detection of fluorescence in log mode at wavelengths greater than 695 nm.

Techniques: Inhibition, Staining, Flow Cytometry, Western Blot, Phospho-proteomics, Control

Journal: Cancers

Article Title: Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment

doi: 10.3390/cancers10100375

Figure Lengend Snippet: Mefloquine and nelfinavir combined drug treatment induces cytotoxicity via energy stress in Tsc2 −/− MEFs. ( A ) The RNA sequencing data used for C−E was assessed for gene-expression of genes involved in energy homeostasis. A heatmap for a panel of energy stress-linked genes is shown. Differences of mRNA expression between Tsc2 +/+ and Tsc2 −/− MEFs treated with mefloquine and nelfinavir is shown as a volcano plot ( B ) and graphed ( C ). ( D ) Tsc2 −/− cells were treated with DMSO, 10 μM mefloquine and 10 μM nelfinavir combination (MQ/NFV) or mefloquine/nelfinavir combination with the addition of 8 mM methyl pyruvate (MQ/NFV/MP) for 48 h. Cells were then stained with DRAQ7 and % cell death determined by flow cytometry. ( E ) Tsc2 −/− were treated with either DMSO or 10 μM mefloquine and 10 μM nelfinavir combination in the presence or absence of 8 mM methyl pyruvate for 24 h and total and phosphorylated ACC and AMPK was determined by western blot. ( F ) Tsc2 +/+ and Tsc2 −/− cells were treated with either DMSO or 10 μM mefloquine and 10 μM nelfinavir combination in the presence or absence of 8 mM methyl pyruvate for 6 and 24 h, where indicated. Total protein levels of ACC, CHOP GADD34 and ATF4 as well as phosphorylated ACC were detected by Western blot.

Article Snippet: Treated cells were collected and incubated with 3 μM DRAQ7 (Biostatus, Leicestershire, UK) at room temperature for 10 min. Flow cytometry was performed using a FACS Calibur flow cytometer (Becton Dickinson (Cowley, UK)) with excitation set at 488 nm and detection of fluorescence in log mode at wavelengths greater than 695 nm.

Techniques: RNA Sequencing, Gene Expression, Expressing, Staining, Flow Cytometry, Western Blot