Journal: Scientific Reports

Article Title: High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria

doi: 10.1038/s41598-017-17878-x

Figure Lengend Snippet: High content image analysis reveals the impact of miR-124 on primary motor neuron morphology. ( a ) Values for nine individual miRNAs, all transfected at 0.5 ng/µl, to mouse primary motor neurons. miRNA expression levels displayed on the Y-axis as 40 minus qPCR cycle threshold (40-Ct), on a Log scale. All miRNAs were significantly overexpressed. ( b ) A diagram describing the method: Spinal motor neurons were isolated from E13.5 mouse embryos and seeded on a 384 multiwell plate. Culture was transfected with different miRNA mimics using Bravo automated liquid handling robot. 72 hrs later, cells were fixed, stained with anti Tuj1 antibody and DAPI. Two fluorescent micrographs were captured per well (ImageXpress Micro and MetaXpress2 software, Molecular Devices). ( c ) Cell numbers (Cell), neurite outgrowth per cell (outgrowth) and number of branches per cell (branches), were quantified with serial doses of the stress-inducing agent, Sodium Arsenite (15, 30 and 60 µM, for 60 minutes). See Methods and Sup. Figure . ( d ) None of the nine miRNAs tested influenced cell numbers. miR-124 was the only miRNA to reduce mean axonal outgrowth per cell and mean number of branches per cell. 500 Tuj1+ neurons quantified per field, 2 fields/well and 6 wells per treatment in five independent experimental repeats. Data collected from >30,000 Tuj1+ neurons per treatment. Averages \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm $$\end{document} ± SEM, Student’s t-test. *P-value < 0.05.

Article Snippet: For high content image analysis, eight/two micrographs taken in 24 /384 well plate setups, respectively, using automated fluorescence microscope (ImageXpress Micro and MetaXpress software, Molecular Devices).

Techniques: Transfection, Expressing, Isolation, Staining, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Anti-Inflammatory and Antioxidant Properties of Physalis alkekengi L. Extracts In Vitro and In Vivo : Potential Application for Skin Care

doi: 10.1155/2022/7579572

Figure Lengend Snippet: Antioxidant effect of PEs in vivo . Thirty zebrafish embryos were incubated either with PEs at different concentrations or Tripeptide-1 as the positive control (PC) for 24 h. After, toxicity in zebrafish embryos was determined through bright-field microscopy while the ROS levels were detected through a fluorescent dye H2DCFDA. Briefly, 20 μ g/mL of H2DCFDA replaced the PEs to incubate with embryos for 1 h in the dark. After washing three times, the fluorescence was observed via fluorescence microscopy (c) and the fluorescence intensity was obtained using a microplate reader (b). ∗ ∗ p < 0.01, ∗ ∗∗ p < 0.001 compared to the nontreatment group.

Article Snippet: The average fluorescent intensity of the individual group was quantified using SpectraMax i3x Multimode microplate reader and calculated through Image J.

Techniques: In Vivo, Incubation, Positive Control, Microscopy, Fluorescence

Journal: Nature Communications

Article Title: GAK and PRKCD are positive regulators of PRKN-independent mitophagy

doi: 10.1038/s41467-021-26331-7

Figure Lengend Snippet: a U2OS cells stably expressing an internal MLS-EGFP-mCherry (IMLS) reporter that is pH-responsive (yellow at neutral, red at acidic pH) were incubated for 24 h ± 1 mM DFP followed by PFA fixation, antibody staining for TIM23 (Alexa Fluor-647) and widefield microscopy. Scale bar = 10 µm, inset = 0.5 µm. b U2OS IMLS cells were transfected with 7.5 nM siRNA non-targeting control (siNT) or siULK1 for 48 h prior to 24 h treatment ± 1 mM DFP in the presence or absence of 50 nM BafA1 for the final 2 h. Western blot from cell lysates shows representative ULK1 knockdown level. The graph represents the mean red-only area per cell from fluorescence images normalised to control DMSO siNT cells ± SEM from n = 4 independent experiments. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. c U2OS IMLS cells treated with 1 mM DFP as in ( a ) and fixed for CLEM analysis. Inset of the cell area in the white box is shown by confocal analysis and EM section along with EM overlay. Scale bar = 5 µm, inset = 1 µm. d Citrate synthase activity from U2OS cells treated for 24 h ± 1 mM DFP with final 16 h in the presence of 50 nM BafA1 or DMSO, values are normalised to DMSO control from n = 6 (DMSO) or n = 3 (+BafA1) independent experiments ± SEM. Significance was determined by two-way ANOVA followed by Sidak’s multiple comparisons test. e U2OS whole-cell protein abundance was determined by mass spectrometry following treatment ±1 mM DFP 24 h. Mitochondrial proteins identified by GO analysis (term = mitochondrion) are highlighted in blue. f Mean T test difference between control and DFP samples for peptides identified in e matching GO terms related to cellular organelles. Bars represent Log2 fold change (control vs DFP) ± SEM from n = 4 independent experiments, number on bars indicate how many protein targets are included in GO analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001 and n.s. = not significant in all relevant panels. For precise P values, see the source data file.

Article Snippet: The tertiary siRNA screen was carried out utilising an ImageXpress Micro Confocal (Molecular Devices) using a ×20 objective (NA 0.45).

Techniques: Stable Transfection, Expressing, Incubation, Staining, Microscopy, Transfection, Western Blot, Fluorescence, Activity Assay, Mass Spectrometry

Journal: Nature Communications

Article Title: GAK and PRKCD are positive regulators of PRKN-independent mitophagy

doi: 10.1038/s41467-021-26331-7

Figure Lengend Snippet: a – e Primary siRNA screen data. U2OS cells were transfected with a pool of three sequence variable siRNA oligonucleotides per gene target (2.5 nM per oligo) for 48 h before the addition of 1 mM DFP for 24 h. Cells were PFA fixed and imaged using a ×20 objective (35 fields of view per well). The red area per cell was normalised to the average of siNT controls and adjusted so that the DFP siNT control was 0 from n = 14 plates ( a , b ) or n = 6 plates ( c – e ) ±SEM. siULK1 and BafA1 (red bars) are positive controls. siRNA targets containing similar lipid-binding domains were assayed and plotted together as shown for a PX domains, b FYVE domains, c C1 domains, d C2 domains, e GRAM, ENTH, PROPPIN domains. f Summary of the significance of different lipid-binding domains relative to the total tested from ( a – e ) proteins containing more than one type of domain are represented in each category. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test to the siNT control where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and n.s. = not significant in all relevant panels. For precise P values, see the source data file.

Article Snippet: The tertiary siRNA screen was carried out utilising an ImageXpress Micro Confocal (Molecular Devices) using a ×20 objective (NA 0.45).

Techniques: Transfection, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: GAK and PRKCD are positive regulators of PRKN-independent mitophagy

doi: 10.1038/s41467-021-26331-7

Figure Lengend Snippet: a Summary of significant targets identified across primary, secondary (7.5 nM individual siRNA oligos) and tertiary screens (15 nM each oligo) siRNA screens. Cells were transfected for 48 h prior to 24 h of 1 mM DFP treatment. Bars represent mean fold change in mitophagy relative to the siNT controls ± SEM from n = 12 (siNT, primary + secondary), n = 13 (SNX10, primary), n = 14 (SYTL5 + ULK1, primary), n = 17 (siNT, secondary) or n = 6 (all others) independent plates. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test to the siNT control. b Protein–protein interaction networks for candidate proteins (see “Methods”) were plotted by % of interacting proteins belonging to each highlighted compartment, value in brackets represents total number of interacting proteins. Dashed lines indicate average values from all screened proteins for mitochondria (red) or autophagosome (orange). c siRNA treatment with indicated oligos for 48 h prior to 24 h treatment ± 1 mM DFP and subsequent analysis of citrate synthase activity levels. Values were normalised to the siNT control and plotted ± SEM for n = 4 independent experiments. Significance was determined by two-way ANOVA followed by Sidak’s multiple comparison test. d U2OS cells treated ± 1 mM DFP for 24 h were enriched from a post-nuclear supernatant (PNS) for mitochondria followed by western blotting for the indicated proteins. e U2OS IMLS cells treated ± 1 mM DFP for 24 h followed by PFA fixation and staining for endogenous PRKCD (Alexa Fluor-647). Scale bar = 20 µm. f U2OS cells treated ± 1 mM DFP for 24 h ± 50 nM BafA1 for the final 16 h and blotted for the indicated proteins. g Quantitation of PRKCD and TIM23 levels to β-actin in ( f ) from n = 3 independent experiments ± SEM. Significance was determined by two-way ANOVA followed by Dunnett’s multiple comparison test to the control. h U2OS cells (−) or those stably expressing PRKCD wild type (WT) or PRKCD ∆C1 ∆C2 (∆lipid-binding domains—LBD) were enriched from a post-nuclear supernatant (PNS) for cytosol and mitochondrial fractions followed by western blotting for the indicated proteins. Representative from n = 2 experiments * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and n.s. = not significant in all relevant panels. For precise P values, see the source data file.

Article Snippet: The tertiary siRNA screen was carried out utilising an ImageXpress Micro Confocal (Molecular Devices) using a ×20 objective (NA 0.45).

Techniques: Transfection, Activity Assay, Western Blot, Staining, Quantitation Assay, Stable Transfection, Expressing, Binding Assay