Journal: Nature Communications

Article Title: Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes

doi: 10.1038/s41467-022-32579-4

Figure Lengend Snippet: a Live-cell fluorescence imaging of stage II-V gametocytes transfected with the nuclear localisation signal (NLS)-mCherry reporter. NLS-mCherry (NLS-mCh, red) labels the elongated nucleus, with pointed extensions (yellow arrowheads). Subpellicular (green arrowheads) and nuclear (cyan arrowheads) populations of microtubules are labelled with Tubulin Tracker (TT, cyan). The nucleus contracts to a more spherical morphology and the nuclear and sub-pellicular microtubule populations are disassembled in stage V gametocytes. The chromatin (Hoechst, grayscale) is present as punctate features in the nucleus (white arrowheads). b Live-cell fluorescence imaging of stage II/III gametocytes in the Pf EB1-GFP/NLS-mCherry co-transfectant parasite line. NLS-mCherry (NLS-mCh, red) delineates the nucleus; and Pf EB1-GFP (EB1-GFP, green) is in the same compartment. Chromatin (Hoechst, grayscale) is distributed along the nuclear microtubules. (See Tubulin Tracker (TT, cyan) overlap with Pf EB1-GFP, cyan arrows). Additional images are presented in Supplementary Fig. . c Quantitative analysis of the contour lengths for the Pf EB1-GFP (EB1) and nuclear microtubules (MT) labelled with Tubulin Tracker (mean ± SD is shown; n = 20 cells per stage). Differences in the length of nuclear microtubule features were evaluated by two-way ANOVA Tukey’s test, **** p < 0.0001. The blue dashed line indicates the limit of resolution (250 nm) of the microscope. d Images showing that the nuclear microtubules (as marked by anti-GFP ( Pf EB1-GFP), green) are depolymerised and the subpellicular microtubules (white arrowheads) are modified with polyglutamate (polyE, red) in stage IV gametocytes. Anti-β-tubulin (anti-β-tub, magenta) labels both nuclear and subpellicular microtubules. The chromatin was labelled with DAPI (grayscale). e Quantitative analysis of anti-polyE fluorescence intensity at different stages of development (mean ± SD is shown; n = 15 cells). Differences in polyE fluorescence signal were determined using a paired one-way ANOVA Tukey’s, I vs. II and II vs. IIIa: NS > 0.9999; IIIa vs. IIIb, IIIb vs. IV and IV vs. V: **** p < 0.0001. Differential interference contrast (DIC) images are shown. Scale bars: 2 μm. Additional images are presented in Supplementary Fig. . Source data for Fig. c, e is provided in the Source Data file.

Article Snippet: Parasite chromatin was labelled with DAPI (2 μg/mL in PBS, Thermo Scientific) for 15 minutes at room temperature, followed by washes with PBS buffer.

Techniques: Fluorescence, Imaging, Transfection, Cotransfection, Microscopy, Modification

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A & B : HEK-293 cells were transiently transfected with D 1 R::RFP and D 5 R::GFP and then grown on a cover slip or in a polarized state in a Transwell ™ insert; the colocalization of these receptors was evaluated via laser scanning confocal microscopy. Wheat germ agglutinin (WGA) tagged with Alexa Fluor□ 647 was used to visualized the plasma membrane, while DAPI (#H-1500, Vector Laboratories, Burlingame, CA) was used to visualized the nuclei. Colocalization was observed as discrete areas of white (red+green+blue) or yellow (red+green). Serial images along the X, Y, and Z axes were obtained in cells grown in Transwell ® and stitched together to show 3D colocalization of the receptors. 630x magnification, scale bar = 10 μm, n=3 independent experiments. C : hRPTCs were double-transfected with D 1 R and D 5 R tagged with the C and N termini of the fluorescent protein EYFP, respectively. The cells were grown on cover slips for 48 hours post-transfection, serum-starved for 2 hrs, and then prepared for confocal microscopy. The interaction of the tagged receptors results in the reconstitution and fluorescence of EYFP (pseudocolored green). An overlay of the BiFC signal and the nucleus (pseudocolored blue) is shown to indicate the distribution of the D 1 R-D 5 R complexes, 630x magnification, scale bar =10 μm, n=3 independent experiments. Transfection of D 1 R-EYFP alone was used as negative control.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Transfection, Confocal Microscopy, Plasmid Preparation, Fluorescence, Negative Control

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A section of the human kidney was double immunostained for endogenous D 1 R (red) and D 5 R (green) ( A ). WGA (magenta) was used to visualize the plasma membrane, e.g., apical brush border of proximal tubules, while DAPI was used to visualize the nuclei. Colocalization in yellow is indicated by the arrows. DIC = differential interference microscopy. Sections of a mouse kidney infused with either vehicle (Basal) or fenoldopam was double immunostained for endogenous D 1 R (green) and D 5 R (red) ( B ). The RPT marker CD15 and DAPI were used to visualize the brush border and nuclei, respectively. Colocalization is indicated by the yellow or white areas in merged images. 630x magnification, scale bar=10 μm, n=3 independent experiments.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Microscopy, Marker

Journal: bioRxiv

Article Title: An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

doi: 10.1101/2020.06.15.151456

Figure Lengend Snippet: (A) CRMP2 is cleaved at sites near T509 in its C-terminal tail. Left inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. Right inset: the abundance ratios of the identified phosphosites in the C-terminal tails of CRMP2 at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. P in red sphere: phosphorylation. (B) A model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our proteomic findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyses processive phosphorylation of CRMP2 at three other sites in the order of S518, T514 and T509. In excitotoxic neurons, cleavage of CRMP2 generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514 and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514 and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. (C) Structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). Dotted line shows the disordered C-terminal tail region. (D) Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2, anti-pT509 CRMP2 and tubulin antibodies. Asterisks: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. (E) Fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows indicate dendritic blebs. The close-up views of the images in the rectangles marked by white dotted lines are shown. Inset: The number of dendritic blebs per mm 2 in control and the glutamate treated neurons in three biological replicates. **: p < 0,01; ***: p <0.001.

Article Snippet: For all experiments, DNA counterstain DAPI (Molecular Probes, Thermo Fisher Scientific) was applied before coverslipping with ProLong gold anti-fade reagent (Invitrogen).

Techniques: Phospho-proteomics, Control, Mutagenesis, Western Blot, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: cGAS-activating lupus autoantibody for cancer immunotherapy

doi: 10.1101/2023.02.11.527649

Figure Lengend Snippet: Human U87 and murine PPQ glioma cells treated with IgG control, DX1, or 4H2 were immunostained to detect antibody penetration and counterstained with DAPI. No significant uptake of IgG control was observed in either cell line. DX1 and 4H2 penetrated and localized into the nuclei and cytoplasm of cells, respectively. Representative merged images are shown. Bar = 10 µm.

Article Snippet: After a final series of washes, cells were treated with Prolong Gold Antifade Reagent with DAPI (Cell Signaling) and imaged using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) under light, DAPI, GFP, or RFP filters.

Techniques: Control

Journal: bioRxiv

Article Title: cGAS-activating lupus autoantibody for cancer immunotherapy

doi: 10.1101/2023.02.11.527649

Figure Lengend Snippet: ( a-c ) The effect of supplemental nucleoside on cellular penetration by DX1 and 4H2 into PPQ cells was determined by ImageJ quantification of fluorescence signal. ADE supplementation increased the efficiency of cellular penetration by DX1 by a factor of 1.70±0.01 (****P<0.0001) ( a ) but did not enhance 4H2 penetration ( b ). In contrast, GUO supplementation increased 4H2 cellular penetration by factor of 3.20±0.30 (****P<0.0001) ( c ), correlating with the known affinity of 4H2 for GUO. ( d ) 4H2 penetrates lung adenocarcinoma cells and localizes into cytoplasm. Representative images of control and 4H2-treated Cal12T cells immunostained for antibody penetration and counterstained with DAPI are shown. ( e ) Lysates of Cal12T cells treated with 0-1 mg/mL 4H2 for 24 hours were analyzed by western blot probed with primary actin antibody for loading control and an anti-mouse secondary antibody to detect the actin primary and 4H2 (both murine). 4H2 HC and LC ran at their expected MWs, showing the antibody is not significantly degraded 24 hours after cellular penetration. ( f ) Visualization of live A549 lung adenocarcinoma cells treated with FITC-labeled 4H2 and MitoTracker Red FM and Hoechst counterstains show 4H2 penetration into cytoplasm. Images of Cal12T cells from this experiment are shown in Supp. Fig. 1b . ( g ) Representative images of Cal12T cells treated with 4H2 immunostained for 4H2 (green) and markers of specific organelles (red), including EEA1 for endosomes, LAMP1 for lysosomes, COX IV for mitochondria, and RCAS1 for Golgi apparatus), and DAPI nuclear counterstained (blue). 4H2 signal did not specifically co-localize with any of the organelles, including endosomes and lysosomes. Error bars = SEM. Scale bars = 10 µm.

Article Snippet: After a final series of washes, cells were treated with Prolong Gold Antifade Reagent with DAPI (Cell Signaling) and imaged using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) under light, DAPI, GFP, or RFP filters.

Techniques: Fluorescence, Control, Western Blot, Labeling

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 1. NPC characterization. A) Phase contrast microscopy of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Microscopy, Control, Knockdown, Derivative Assay, Fluorescence, Marker, Expressing, Immunocytochemistry

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 2. NPC viability. A) Cell cycle analysis by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean 6 SD, *p = 0.047). B) Survival under nutritional and toxicant stress. NPCs propagated in medium without glucose (NG) untreated or treated with 20 mM rotenone (R) or 20 mM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean 6 SEM). C) Cell viability assayed by plate reader based high throughput screen (HTS) of NPCs untreated (HG), treated with 20 mM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 mM of the RedOx indicator C12-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean 6 SEM, *p#0.05). D) Cell viability assayed by flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C12-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin- V Alexa-660 nm conjugated antibody) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *p = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **p = 0.004). doi:10.1371/journal.pone.0112413.g002

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Cell Counting, High Throughput Screening Assay, Fluorescence, Membrane

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 3. Mitochondrial membrane potential (MMP) and energy balance. A) Fluorescence microscopy of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 mM Rotenone (HG+R) or with 1 mM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 mm). B) Plate reader based high throughput screen (HTS) of MMP in live NPCs loaded with 20 mM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean 6 SEM, Ctrl/SNCA-

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Membrane, Fluorescence, Microscopy, Control, Negative Control, High Throughput Screening Assay

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 4. Protein biosynthesis and proteasome function. A) Mitochondrial protein biosynthesis and protein import. Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 mm, 5 mm). B) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy of adherent NPCs cultured with 20 mM rotenone alone or with 10 mM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 mM of the aggresome/ proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 mm). D) Proteasome activity measured by flow cytometry evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 1006[MRFU MG132 treated2MRFU untreated]/MRFU MG132 treated (n = 3, mean 6 SD, APF Ctrl/SNCA-Tri: 51/120, *p = 0.041). doi:10.1371/journal.pone.0112413.g004

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Expressing, Transduction, Activity Assay, Fluorescence, Microscopy, Cell Culture, Staining, Flow Cytometry

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 5. Reactive oxygen species (ROS) production. A) Fluorescence microscopy of live adherent NPCs untreated (HG) or treated with 100 mM TBHP (HG+TBHP), loaded with CM-H2DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 mm). B) Plate reader based HTS of ROS levels in adherent NPC in 96- well plates and treated as under A). Relative CM-H2DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *p#0.046, **p#0.009, ***#0.001). C) ROS production rates by HTS plate reader analysis of CM-H2DCFDA fluorescence development over time (D RFU CM-H2DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/ SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *p#0.010, **p#0.007, ***p#0.001). D) Mitochondrial superoxide production rates assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (D RFU MitoSOX/min + H33342) (n = 4, mean 6 SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/ 5.5/3.7, NG: 2.3/5.2/0.8,*p#0.038, **p#0.007). doi:10.1371/journal.pone.0112413.g005

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Fluorescence, Microscopy, Staining

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 6. Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 mM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean 6 SD, Ctrl/SNCA-Tri: 3.4/4.9, *p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl2 were assayed 1 hr. after treatment with 4 mM staurosporine under NG conditions. MPT opening results in entry of CoCl2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 mm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 mm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 mM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean 6 SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *p#0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Negative Control, Fluorescence, Microscopy, Staining, Software

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 7. Apoptosis sensitivity and caspase activation. A) Caspase 3 activity in cell lysates from adherent NPCs either left untreated or treated with 20 mM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p#0.050, **p#0.0035; from three independent experiments). B) Kinetics of caspase 3/7 activity in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as DmM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean 6 SEM). doi:10.1371/journal.pone.0112413.g007

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Activation Assay, Activity Assay, Fluorescence, Bradford Protein Assay