Journal: Scientific Reports

Article Title: Spatial organization of Clostridium difficile S-layer biogenesis

doi: 10.1038/s41598-020-71059-x

Figure Lengend Snippet: New surface S-layer colocalizes with areas of new peptidoglycan synthesis. ( a ) Examples of timepoints from real-time widefield fluorescent HADA signal (left panels) and phase contrast (center panels) of C. difficile 630 cells chased for HADA stain. Frame time represented in minutes, scale bar indicates 6 µm. ( b ) Airyscan confocal image of a C. difficile 630 cell grown with HADA to label peptidoglycan (Blue) and chased to reveal darker areas of newly synthesized peptidoglycan in the cell wall. This chase was followed by a short expression of SlpA R20291 which was specifically immunolabeled with Cy5 (White). After merging HADA and Cy5 channels (righthand panels, duplicated for clarity), cell sides (yellow bars) were selected for signal intensity analysis. For this example cell the intensity plots for cell sides (i) and (ii) are shown in panel C. Scale bar indicates 6 µm. ( c ) Intensity plot depicting signal from HADA (Blue) and Cy5 (Grey) along the yellow bars illustrated in ( b ). Trace (i) was calculated to have a significant positive correlation and (ii) a significant negative correlation.

Article Snippet: Images were taken on a Nikon Ti eclipse widefield imaging microscope using NIS elements software or a ZEISS LSM 880 with Airyscan using ZEN imaging software.

Techniques: Staining, Synthesized, Expressing, Immunolabeling

Journal: Scientific Reports

Article Title: Spatial organization of Clostridium difficile S-layer biogenesis

doi: 10.1038/s41598-020-71059-x

Figure Lengend Snippet: SecA2-SNAP localization and new S-layer. ( a ) Widefield phase contrast (left panels) and fluorescent (right panels) images of wild type C. difficile 630 or 630 secA2-snap cells stained with TMR-Star (red). Scale bar indicates 3 µm. ( b ) Airyscan confocal image displaying SecA2-SNAP-TMR-Star signal distribution in C. difficile 630 cells. Scale bar indicates 3 µm. ( c ) Airyscan confocal image showing the localization of SecA2-SNAP-TMR-Star (red) in relation to the synthesis of peptidoglycan (dark patches lacking blue HADA stain) and newly synthesized S-layer (Cy5, white). Scale bar indicates 3 µm. ( d ) Graph displaying the proportion of cell area with internal SecA2-SNAP-TMR-star signal or outline with R20291-LMW-SLP-Cy5 signal. Mean and SD of results from n = 47 cells is shown. One-way ANOVA analysis identified a significant difference in mean values *p < 0.0001.

Article Snippet: Images were taken on a Nikon Ti eclipse widefield imaging microscope using NIS elements software or a ZEISS LSM 880 with Airyscan using ZEN imaging software.

Techniques: Staining, Synthesized

Journal: Scientific Reports

Article Title: Spatial organization of Clostridium difficile S-layer biogenesis

doi: 10.1038/s41598-020-71059-x

Figure Lengend Snippet: Sites of S-layer secretion. ( a ) Schematic diagram illustrating the position of stained SNAP tagged SlpA constructs expressed in C. difficile 630 cells: SNAP-Cell TMR-Star stained SlpA-SNAP (left panel) or SlpA-DHFR-SNAP (center panel) and SNAP-Surface 549 stained SlpA-SNAP (right panel). Colored as in Fig. b with SNAP tags represented as an orange coil. SlpA-SNAP is exported and cleaved into LMW-SLP and HMW-SLP-SNAP. The DHFR domain (dark gray oval) of SlpA-DHFR-SNAP blocks the translocon channel during export, leaving the TMR-Star bound SNAP tag in the cytosol. SNAP-Surface 549 stains extracellular HMW-SLP-SNAP only. ( b ) Widefield phase contrast (left panels) and fluorescent SNAP-Cell TMR-Star signal (right panels) of C. difficile 630 cells stained with SNAP-Cell TMR-Star imaged with and without induction of SlpA-SNAP expression. Scale bar indicates 3 µm. ( c ) Overlay of fluorescent signal in the induced sample (from ( b )) with areas taken for the plot profiles labelled (yellow lines, i–iii). Scale bar indicates 3 µm. ( d ) SlpA-SNAP-Cell TMR-Star profile plots of i-iii (from ( c )) of phase contrast signal (black) and SNAP-Cell TMR-Star signal (red). ( e ) SlpA-DHFR-SNAP in C. difficile 630 cells (labelled as in ( b )). ( f ) Overlay of signal in the induced sample (from ( e ), labelled as in ( c )). ( g ) SlpA-DHFR-SNAP-Cell TMR-Star profile plots of i–iii (from ( f )) (labelled as in ( d )). ( h ): Widefield phase contrast (left panels) and fluorescent SNAP-Surface 549 signal (right panels) of C. difficile 630 cells stained with SNAP-Surface 549 imaged with and without induction of SlpA-SNAP expression. ( i ) Overlay of signal in the induced sample (from ( h ), labelled as in ( c )). ( j ) HMW-SLP-SNAP-Surface 549 profile plots of i-iii (from ( i )) of phase contrast signal (black) and SNAP-Surface 549 (red).

Article Snippet: Images were taken on a Nikon Ti eclipse widefield imaging microscope using NIS elements software or a ZEISS LSM 880 with Airyscan using ZEN imaging software.

Techniques: Staining, Construct, Expressing

Journal: iScience

Article Title: MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels

doi: 10.1016/j.isci.2022.104459

Figure Lengend Snippet: High MASTL levels correlate with OCT1 and mammosphere formation (A) Western blotting of β3-integrin (ITGB3), OCT1, MASTL, and GAPDH in MDA-MB-231 cells grown as a monolayer (2D) or as mammospheres. (B) Relative protein expression of MASTL to GAPDH, experimental setup shown in (A). (n = 3 biologically independent experiments, unpaired t-test, mean ± SD). (C) Western blotting of OCT1, MASTL, and GAPDH in MDA-MB-231 cells silenced with siControl or siOCT1 for 48 h. (D) Relative protein expression of MASTL to GAPDH, experimental setup shown in (C), two datapoints collected after 96 h and three after 48 h (n = 5 biologically independent experiments, one sample t-test, mean ± SD). (E) Western blotting of OCT1, MASTL, and tubulin in MDA-MB-231 cells overexpressing EGFP-control (enhanced green fluorescent protein) or hemagglutinin (HA)-tagged OCT1. (F) Relative protein expression of MASTL to tubulin, experimental setup shown in (E). (n = 3 biologically independent experiments, one sample t-test, mean ± SD). (G) Representative flow cytometry histograms of MDA-MB-231 cells treated with DMSO control or DCF-DA (2ʹ,7ʹ-Dichlorofluorescin Diacetate) to measure ROS activity after silencing with siControl (gray), siMASTL#6 (red), or siMASTL#7 (orange) for 48 h. (H) Geometric Mean of the DCF-DA signal (ROS activity) in the experimental setup described in (G). (n = 3 biologically independent experiments, unpaired t-test, mean ± SD). (I) Representative images of tetracycline-induced shControl and shMASTL#3 MDA-MB-231 cells grown in mammosphere culture conditions (7 days) and stained with Calcein (Nikon Eclipse Ti-E widefield microscope, Hamamatsu Orca C13440 Flash 4.0 ERG [b/w] sCMOS camera and Plan Apo lambda 20×/0.80, WD 1,000-μm objective). (J) Average mammosphere size (average ferret diameter of spheres in μm) and spheres/1,000 cells plated, experimental setup shown in (I). (n = 3 biologically independent experiments, eight replicate wells/experiment, unpaired t-test, mean ± SEM). (K) Western blotting of OCT1, MASTL, and GAPDH in tetracycline-induced (4 days) shControl and shMASTL#3 MDA-MB-231 cells. (L) Relative protein expression of MASTL to GAPDH, experimental setup shown in (K). (n = 3 biologically independent experiments, one sample t-test, mean ± SD). (M) Representative flow cytometry histograms of CD44 expression in tetracycline-induced (4 days) shControl and shMASTL#3 MDA-MB-231 cells. (N) Geometric mean of CD44 expression in the experimental setup described in (M). (n = 5 biologically independent experiments, unpaired t-test, mean ± SD). See also Figure S2 .

Article Snippet: Images were captured with (Nikon Eclipse Ti-E widefield microscope, Hamamatsu Orca C13440 Flash 4.0 ERG [b/w] sCMOS camera and Plan Apo lambda 20×/0.80, WD 1,000-μm objective).

Techniques: Western Blot, Expressing, Flow Cytometry, Activity Assay, Staining, Microscopy