Journal: Autophagy

Article Title: Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity

doi: 10.1080/15548627.2021.1961067

Figure Lengend Snippet: Mycolactone induces an increase in autophagic flux in primary dermal microvascular endothelial cells and fibroblasts. Cells were exposed to mycolactone (Myco) at the minimal inhibitory concentration alongside a solvent control (0.05% DMSO, equivalent dilution to the mycolactone) for the longest timepoint tested. where included, bafilomycin A 1 (BAF) was used for 4 h at cell-specific saturating concentrations, and at the longest timepoint tested when in combination with mycolactone. PP242 was used at 1 μM for 1 h. All immunofluorescence images were obtained using a Nikon A1 confocal laser scanning microscope. For all panels, unless otherwise indicated, statistical comparisons are to the DMSO-treated cells of that particular cell type; ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. (A) HDMEC exposed to 10 ng/ml mycolactone with and without 100 nM BAF for 8 h were fixed with methanol for LC3B staining (scale bar: 50 µm). values represent the number of puncta detected per cell for at least 36–49 cells. Data are representative of 2 independent experiments (scale bar: 50 µm). (B) HFFF exposed to 31.25 ng/ml mycolactone with and without 200 nM BAF for 8 h were fixed with methanol for LC3B staining (scale bar: 50 µm). Values represent the number of puncta detected per cell for at least 63–90 cells. Data are representative of 2 independent experiments. (scale bar: 50 µm) experiments. (C) HFFF cells exposed to 31.25 ng/ml mycolactone and/or 200 nM BAF were lysed directly in gel sample buffer prior to immunoblotting. The migration relative to known molecular mass markers is shown. Data are representative of 2 independent experiments. (D) Expression of tandem-tagged LC3B in HeLa cells. Cells transiently transfected with pDEST-mCherry-eGFP-LC3B were exposed to mycolactone for 4 h before the addition of 100 nM (BAF) and incubation for a further 4 h before fixation. Values represent the number of puncta detected per cell for at least 8–10 transfected cells. Data are representative of 4 independent experiments (scale bar: 10 μm). (E) HDMEC and HFFF were exposed to mycolactone for various times or PP242, then fixed for WIPI2 staining (scale bar: 50 µm). Values represent the puncta detected in each cell for 25–50 cells. Data are representative of 2 independent experiments. (F) HDMEC were exposed to 10 ng/ml mycolactone for 8 h, or PP242 then fixed for RB1CC1 or ATG16L1 staining (scale bar: 50 µm). Values represent the puncta detected in each cell for 25–50 cells. Data are representative of 2 independent experiments. (G) HDMEC were exposed to 10 ng/ml mycolactone for 8 h then fixed for co-staining with WIPI (green) and RB1CC1 or ATG16L1 (red) (scale bar: 10 μm).

Article Snippet: Slides were imaged on a Zeiss Axiovert S100 TV inverted fluorescence microscope or Nikon A1 confocal laser scanning microscope and NIS Elements software.

Techniques: Concentration Assay, Solvent, Control, Immunofluorescence, Laser-Scanning Microscopy, Staining, Western Blot, Migration, Expressing, Transfection, Incubation

Journal: Autophagy

Article Title: Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity

doi: 10.1080/15548627.2021.1961067

Figure Lengend Snippet: Accumulation of SQSTM1 and ubiquitinated proteins in mycolactone-exposed cells. Cells were exposed to mycolactone (Myco) at the minimal inhibitory concentration, alongside a solvent control (0.05% DMSO, equivalent dilution to the mycolactone) for the longest timepoint tested. Where included, bafilomycin A 1 (BAF) was used at cell-specific saturating concentrations. All immunofluorescence images were obtained using a Nikon A1 confocal laser scanning microscope. For all immunoblots, the migration relative to known molecular mass markers is shown. For all panels, unless otherwise indicated, statistical comparisons are to the DMSO-treated cells of that particular cell type, ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. (A) HDMEC exposed to 10 ng/ml mycolactone with and without 100 nM BAF for 8 h were fixed with PFA for SQSTM1 staining with a mouse anti-SQSTM1 antibody (scale bar: 50 µm). Values represent the number of puncta detected per cell for 45–100 cells. Data are representative of 2 independent experiments. (B) HFFF exposed to 31.25 ng/ml mycolactone with and without 200 nM BAF for 8 h were fixed with PFA for SQSTM1 staining with a mouse anti-SQSTM1 antibody (scale bar: 50 µm). Values represent the number of puncta detected per cell for 45–100 cells. Data is representative of 2 independent experiments. (C) HeLa cells were exposed to 31.25 ng/ml mycolactone for 24 h, in the presence or absence of 100 nM BAF for the final 12 h, then fixed with methanol for SQSTM1 with a mouse anti-SQSTM1 antibody (green) and LC3B (red) staining. Nuclei were counterstained with DAPI (blue) (scale bar: 10 µm). Values are means of 3 independent experiments ± SEM. (E) HeLa cells were left untreated, exposed to mycolactone for different times (as indicated) or 50 µM chloroquine (CQ) for 12 h. The 48-h timepoint was performed without and with 50 µM CQ (for the final 12 h). Cells were harvested in RIPA lysis buffer, then soluble and insoluble fractions were prepared as described in Methods prior to immunoblotting. Data are representative of 3 independent experiments. (F) HeLa cells exposed to 31.25 ng/ml mycolactone for various times or 10 µM MG132 for 8 h were lysed directly in gel sample buffer prior to immunoblotting. Data are representative of 2 independent experiments. (G) HeLa cells were exposed to 31.25 ng/ml mycolactone for the times indicated, or 10 µM MG132 for 8 h then fixed with PFA for total ubiquitin staining with FK2 (scale bar: 10 µm). Data are representative of 2 independent experiments. (H) HeLa cells were exposed to 31.25 ng/ml mycolactone for 16 h then fixed with PFA for total ubiquitin with FK2 (green) and SQSTM1 with a rabbit anti-SQSTM1 antibody (red) staining (scale bar: 10 µm).

Article Snippet: Slides were imaged on a Zeiss Axiovert S100 TV inverted fluorescence microscope or Nikon A1 confocal laser scanning microscope and NIS Elements software.

Techniques: Concentration Assay, Solvent, Control, Immunofluorescence, Laser-Scanning Microscopy, Western Blot, Migration, Staining, Lysis, Ubiquitin Proteomics

Journal: Autophagy

Article Title: Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity

doi: 10.1080/15548627.2021.1961067

Figure Lengend Snippet: Increased expression of SQSTM1 enhances cell survival in the presence of mycolactone but is not required for initiation of autophagy. Cells were exposed to 31.25 ng/ml mycolactone (Myco), alongside a solvent control (0.05% DMSO, equivalent dilution to the mycolactone) for the longest timepoint tested. All immunofluorescence images were obtained using a Nikon A1 confocal laser scanning microscope (scale bar: 10 µm). For all immunoblots, the migration relative to known molecular mass markers is shown. For all panels, unless otherwise indicated, statistical comparisons are to the DMSO-treated cells of that particular cell type, ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.(A-C) Matched Sqstm1 WT and sqstm1 −/- MEFs were exposed to mycolactone for the indicated times. As controls, either 100 nM bafilomycin A 1 (BAF) for 12 h or 1 µM PP242 for 1 h were used, as indicated. (A) Cells were harvested in RIPA lysis buffer, then soluble and insoluble fractions were prepared as described in Methods prior to immunoblotting. Migration relative to known molecular mass markers is shown. Twelve-hour mycolactone exposure. Data are representative of 3 independent experiments. (B) Cells were fixed and stained for WIPI2 after 8 h mycolactone exposure. Values represent the means of 3 independent experiments ± SEM. (C) Cell viability in Sqstm1 WT and sqstm1 −/- MEF as measured by CellEvent assay after 24 h mycolactone exposure. Values represent the mean of 3 independent experiments ± SEM. (D) WT HeLa cells exposed to mycolactone for 16 h before fixing with PFA for immunofluorescent staining with mouse anti-SQSTM1, anti-TAX1BP1, anti-CALCOCO2, anti-NBR1 or anti-OPTN antibodies. Data are representative of duplicate experiments. (E) Cell viability for parental HeLa (WT), PentaKO cells (5KO) and PentaKO cells with reconstituted SQSTM1 expression (5KO/ SQSTM1 ) as measured by CellEvent assay after 48 h mycolactone exposure. Values represent the mean of 3 independent experiments ± SEM. (F) Skin sections from samples from healthy donor (left hand panel) and Buruli ulcer patient lesions (right hand panels), stained with anti-SQSTM1 antibody or isotype-matched control. Boxes show locations of magnified SQSTM1-stained sections. Comparable results were found for all 8 patient biopsies analyzed regardless of whether the lesion was an ulcer or a plaque.

Article Snippet: Slides were imaged on a Zeiss Axiovert S100 TV inverted fluorescence microscope or Nikon A1 confocal laser scanning microscope and NIS Elements software.

Techniques: Expressing, Solvent, Control, Immunofluorescence, Laser-Scanning Microscopy, Western Blot, Migration, Lysis, Staining

Journal: Journal of Virology

Article Title: The Conserved YAGL Motif in Human Metapneumovirus Is Required for Higher-Order Cellular Assemblies of the Matrix Protein and for Virion Production ^▿

doi: 10.1128/JVI.02694-10

Figure Lengend Snippet: Immunofluorescence analysis of the intracellular distributions of wild-type and mutant M proteins. 293T cells harboring stable replicons of the indicated hMPV-GFP-Puro clones (A and B) or Vero cells infected with the hMPV-GFP-Puro-wt or FAGL clone (C) were grown on glass coverslips and processed for immunofluorescence analysis using anti-M antibodies (red). The nuclei were stained with DAPI (blue). In panel A, the top row shows low-magnification images of the cells, whereas the bottom row presents higher-magnification images of the areas defined by the insets in the upper panels. “293T” and “Vero” represent naïve cells. Images were acquired using a motorized spinning-disc confocal microscope (Yokogawa CSU-22 confocal head and Zeiss Axiovert 200 M microscope). Maximum-intensity projections of confocal z stacks are depicted in panels A, B, and C. (D) 3D renditions (generated with the 3D volume view function of SlideBook software) of cells grown and processed as in panels A and B. Bars and grids, 10 μm.

Article Snippet: Images were acquired using a motorized spinning-disc confocal microscope (Yokogawa CSU-22 confocal head and Zeiss Axiovert 200 M microscope) and were analyzed with SlideBook software (version 4.2.0; Intelligent Imaging Innovations).

Techniques: Immunofluorescence, Mutagenesis, Clone Assay, Infection, Staining, Microscopy, Generated, Software

Journal: Journal of Virology

Article Title: The Conserved YAGL Motif in Human Metapneumovirus Is Required for Higher-Order Cellular Assemblies of the Matrix Protein and for Virion Production ^▿

doi: 10.1128/JVI.02694-10

Figure Lengend Snippet: Confocal fluorescence microscopy of cellular wild-type and mutant M proteins fused to GFP. 293T cells were transfected with plasmids expressing the wild-type (wt) M protein or the indicated M mutants, all fused to GFP. The cells were fixed, and their nuclei were stained with Hoechst dye (blue). (A) Low-magnification images of the cultures. (B) Higher-magnification images of the areas defined by the insets in panel A. (C) Confocal images of single cells of the indicated transfected cultures. Orthogonal images are presented above and to the left of each regular imaging plane (each central panel is an x-y image, the top panel is an x-z image, and the left panel is a z-y image). Images were acquired using a motorized spinning-disc confocal microscope (Yokogawa CSU-22 confocal head and Zeiss Axiovert 200 M microscope). Bars, 10 μm. GFP, free GFP that is not fused to the M protein.

Article Snippet: Images were acquired using a motorized spinning-disc confocal microscope (Yokogawa CSU-22 confocal head and Zeiss Axiovert 200 M microscope) and were analyzed with SlideBook software (version 4.2.0; Intelligent Imaging Innovations).

Techniques: Fluorescence, Microscopy, Mutagenesis, Transfection, Expressing, Staining, Imaging