hela-mz cells  (Microsynth ag)

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: Thioperamide treatment of tissue culture cells. BHK, CHO and HeLa cells were treated with thioperamide and processed as in Fig C. Low‐magnification views are shown after staining with anti‐LBPA antibodies (green) and DAPI (blue). n = 3 independent experiments with > 200 images acquired and analysed automatically. Cholesterol quantification by mass spectrometry. A431 cells were treated with DMSO alone, pitolisant 10 μM (Pito), thioperamide 10 μM (thio) or U18666A 10 μM (U18) for 18 h at 37°C. After extraction, free cholesterol, cholesteryl esters and total cholesterol were quantified by mass spectrometry and normalized to the DMSO controls ( n = 3 independent experiments, error bars = SD, one‐way ANOVA, **** P < 0.0001). Screen data plot of the cholesterol content of LBPA endosomes. Automated unbiased quantification of the filipin integrated fluorescence signal (cholesterol content) in LBPA‐containing endosomes, after treatment with each compound of the Prestwick library. As in Fig D, each dot is the average of replicates in the duplicate plates, and ≥ 600 cells were analysed per compound—only compounds that showed < 20% toxicity are plotted. The fluorescence signal of LBPA endosomes was used to segment the imaged and generate a mask, which was then applied on the micrographs to quantify the integrated intensity of filipin staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated.

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Staining, Mass Spectrometry, Extraction, Fluorescence

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: A, B HeLa MZ cells were treated or not with thioperamide, embedded in Epon and processed for electron microscopy (A). Arrows point at structures with the characteristic appearance of multivesicular endosomes. Alternatively, cryosections were prepared and labelled with anti‐LBPA antibodies followed by 5 nm protein A‐gold (B); micrographs are same as in Fig A, but without pseudo‐colouring. Scale bars: (A) 1 μm; (B) 100 nm.

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Electron Microscopy

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: A, B Immunogold labelling with anti‐LBPA antibodies. HeLa MZ cells were treated or not with thioperamide for 18 h and processed for electron microscopy. Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5 nm protein A‐gold. Multivesicular endosomes are pseudo‐coloured (see Fig for uncoloured images). Scale bars, 200 nm. The data in (A) were quantified in a double‐blind analysis of two sets of 16 micrographs for each condition; the number of gold particles per endosome is shown in a scatter plot (B) for each endosome identified without any bias in each micrograph of the two control (DMSO) and thioperamide‐treated (THIO) samples. C Distribution of LBPA‐containing endosomes in the perinuclear region. Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells, after treatment with each compound of the Prestwick library as in Fig C. As in Fig D, each dot is the average of replicates in the duplicate plates, and ≥ 600 cells were analysed per compound—only compounds that showed < 20% toxicity are plotted. The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell, calculated from the nuclei DAPI staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated. D Total number of LBPA endosomes. The number of individual LBPA‐positive structures was measured in the whole cell. The z ‐factors are shown to evaluate the distribution of endosomes containing LBPA; colour code as in (C).

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Electron Microscopy, Control, Fluorescence, Staining

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: Analysis of endosomal compartments by immunofluorescence. HeLa MZ cells were treated or not with thioperamide or U18666A for 18 h and processed for immunofluorescence microscopy after labelling with DAPI (nuclei) and antibodies against the indicated proteins. Lipid droplets were labelled with bodipy . The bar graph shows the quantification by automated microscopy of the intensity of the fluorescence signals; TfR: transferrin receptor ( n = 3 independent experiments, 200 images analysed per experiments, error bars = SD). Infection with vesicular stomatitis virus (VSV). HeLa cells treated or not with thioperamide for 15 h were further incubated for 3 h in the presence of the drug and recombinant VSV expressing a GFP‐tagged version of the viral phosphoprotein (P‐eGFP) , at low physiologically relevant MOI (1.0). Cells were labelled with DAPI (nuclei) and analysed by fluorescence microscopy. The bar graph shows the percentage of infected cells after quantification by automated microscopy ( n = 3 independent experiments, 100 images analysed per experiments, error bars = SD, two‐way ANOVA, **** P < 0.0001). Acidic pH of endosomes and lysosomes. HeLa cells were treated or not with thioperamide as in (A) and incubated with LysoTracker to label acidic compartments. For comparison, cells were also treated for a short (2 h) period with the V‐ATPase inhibitor bafilomycin A1. Cells were then labelled with DAPI (nuclei) and processed for fluorescence microscopy. The bar graphs show the integrated intensity (left) and number (right) of labelled structures after quantification by automated microscopy ( n = 3 independent experiments, 64 images analysed per experiments, error bars = SD). EGF receptor degradation. HeLa MZ cells treated or not with thioperamide as in (A) were challenged with 100 nM EGF for 60 min and further incubated at 37°C for the indicated time periods with or without the drug, up to a total time period of 18 h. Cells were labelled with DAPI (nuclei) and antibodies against the EGF receptor and processed for fluorescence microscopy. The panels show examples of the EGF receptor distribution after 60 min. The bar graph shows the integrated intensity of the labelled structures after quantification by automated microscopy ( n = 3 independent experiments, 100 images analysed per experiments, error bars = SD).

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Immunofluorescence, Microscopy, Fluorescence, Infection, Virus, Incubation, Recombinant, Expressing, Comparison

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: A Effects of compounds that target histamine receptor family members. The outline shows the compounds (listed in ) that target histamine receptor family members as antagonists (HRH1, HRH2, HRH3 and HRH4) or agonists. These include the compounds originally present in the Prestwick library as well as additional compounds selected from the literature. Cells treated with this small library of compounds were analysed by automated microscopy after labelling with DAPI and antibodies against LBPA. Compounds that increased LBPA staining intensity, such as thioperamide (Fig ) or pitolisant (Fig B), are green, while compounds without effects are red ( n = 2 independent experiments with 96 images acquired and analysed automatically). B Effect of pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10 μM for 18 h at 37°C were labelled with DAPI and anti‐LBPA antibodies and analysed by fluorescence microscopy (quantification in Fig A). C, D LBPA intensity in a mixed population of cells expressing HRH3‐GFP. After transfection with HRH3‐GFP, uncloned stably expressing cells (C) were labelled with anti‐LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3‐GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red) ( n = 2 independent experiments with 500,000 cells analysed automatically, error bars = SD). E–G LBPA intensity in cells expressing or not HRH3‐GFP. HeLa MZ cells stably expressing HRH3‐GFP grown in 96‐well plates were separately treated, with four different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; and siHRH3#4) or with control non‐target (siNT) siRNA (E). HRH3‐GFP was analysed by fluorescence microscopy in four columns of cells per condition; micrographs are stitched together in the montage. Panel (F) shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F) were analysed by automated microscopy, and the integrated intensities of LBPA (left panel) and HRH3‐GFP (right panel) signals are compared (G) ( n = 2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Microscopy, Staining, Fluorescence, Expressing, Transfection, Stable Transfection, Control

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: HeLa MZ or A431 cells were treated with DMSO (0 control, black bars), thioperamide or pitolisant at the indicated concentrations for 18 h. Cells were labelled with anti‐LBPA antibodies and analysed by automated microscopy. The intensity of LBPA staining was quantified and is normalized to the DMSO controls ( n = 3 independent experiments with 288 images analysed per condition, error bars = SD). HeLa MZ cells were treated with DMSO, with 10 μM thioperamide or with 10 μM pitolisant for the indicated time periods. Cells were labelled with anti‐LBPA antibodies and analysed by automated microscopy. The intensity of LBPA staining was quantified and is normalized to the DMSO controls ( n = 3 independent experiments with 288 images analysed per condition). As in (B), nuclei were stained with DAPI and the number of nuclei per micrograph was quantified ( n = 3 independent experiments with 288 images analysed per condition).

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Control, Microscopy, Staining

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: Effects of thioperamide on cholesterol‐dependent transcriptional regulation. The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18 h. Total mRNA was extracted, and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT–PCR ( n = 3 independent experiments, error bars = SD, one‐way ANOVA, * P < 0.05; ** P < 0.005). Quantification of cholesterol in NPC null mice by enzymatic analysis. WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed. The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue. The total content of LBPA in the same liver extracts was quantified by mass spectrometry (see ) and is therefore expressed as a percentage of total phospholipids. The total LBPA content of WT mice was only marginally increased by the drug, either because the doses were low, or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level (despite much effort, we were unable to visualize LBPA in liver samples by immunocytochemistry). The relative ratio of LBPA versus cholesterol values is shown.

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Enzymatic Assay, Mass Spectrometry, Immunocytochemistry

Journal: EMBO Reports

Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice

doi: 10.15252/embr.201847055

Figure Lengend Snippet: The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide for 18 h. Total mRNA was extracted, and the indicated mRNAs were quantified by RT–PCR ( n = 3 independent experiments, error bars = SD).

Article Snippet: Our HeLa MZ and A431 cells were authenticated by Microsynth (Balgach, Switzerland) and are mycoplasma‐negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction