Journal: Communications Biology

Article Title: NLS-binding deficient Kapβ2 reduces neurotoxicity via selective interaction with C9orf72-ALS/FTD dipeptide repeats

doi: 10.1038/s42003-024-07412-x

Figure Lengend Snippet: A Representative images of primary cortical neurons co-transfected with (GR) 100 -mCherry and GFP or GFP-Kapβ2 W460A:W730A . GFP signal is shown in green, mCherry is shown in red, Tuj1 is shown in magenta, and nuclei are shown in blue. Scale bar = 10 μm. B Quantification of the number of (GR) 100 aggregates per neuron in primary cortical neurons co-transfected with (GR) 100 -mCherry and GFP or GFP-Kapβ2 W460A:W730A . The number of neurons quantified (n) in each condition is indicated in the figure. Data shown are mean ± SEM. Unpaired Student’s t -tests were used to compare different conditions; *** p ≤ 0.001. C Quantification of the size of (GR) 100 aggregates formed in primary cortical neurons co-transfected with (GR) 100 -mCherry and GFP or GFP-Kapβ2 W460A:W730A . The number of aggregates quantified ( n ) in each condition is indicated in the figure. Data shown are mean ± SEM. Unpaired Student’s t -tests were used to compare different conditions; ns: p > 0.05. D Quantification of the mean mCherry fluorescence intensity in each (GR) 100 aggregates formed in primary cortical neurons co-transfected with (GR) 100 -mCherry and GFP or GFP-Kapβ2 W460A:W730A . The number of aggregates quantified (n) in each condition is indicated in the figure. Data shown are mean ± SEM. Unpaired Student’s t -tests were used to compare different conditions; *** p ≤ 0.001. E Analysis of percentage of survival neurons 5 days after co-transfected with (GR) 100 -mCherry and GFP or GFP-Kapβ2 W460A:W730A . N = 3. Unpaired Student’s t-test was used to compare different conditions. F Viability assay of primary cortical neurons treated with indicated R-DPR alone or with Kapβ2 WT or Kapβ2 W460A:W730A . 5μM of DRAQ7, 2.5μM of the indicated R-DPRs and proteins were added into the culture media of treated cortical neurons. Cells positive for DRAQ7 fluorescence (observed in Cy5 channel) were counted 18 hours after treatment to assess the viability of the neurons. Data are represented as median ± S.E.M., n = 3, m (fields of view) = 5. Nested Student’s t -test was used to compare different conditions, **** p ≤ 0.0001. G The indicated R-DPRs (2.5μM) and Kapβ2 WT or Kapβ2 W460A:W730A (2.5 μM) were added into the culture media of treated primary cortical neurons. DIC images of the treated primary cortical neurons were taken 18 hours after treatment to assess the morphology of the neurons. Scale bar = 20 μm.

Article Snippet: DRAQ7 fluorescence was then semi-automatically quantified in each cell and graphed using Prism GraphPad 10.0.3.

Techniques: Transfection, Fluorescence, Viability Assay

Journal: PLoS ONE

Article Title: Time resolved 3D live-cell imaging on implants

doi: 10.1371/journal.pone.0205411

Figure Lengend Snippet: CYTO-ID Red labeled human gingival fibroblasts were treated with or without chlorhexidine. CYTO-ID Red labeled gingival fibroblasts, fixed with 4% w/v PFA in PBS and subsequently stained with DRAQ7 served as a positive control for the LIVE/DEAD staining: (A) CYTO-ID Red, (B) DRAQ7, and (C) their overlay. Labeled gingival fibroblasts without addition of chlorhexidine served as a negative control after DRAQ7 staining: (D) CYTO-ID Red, (E) DRAQ7, and (F) their overlay. CYTO-ID Red labeled gingival fibroblasts were treated with 132 mM chlorhexidine for 2 hours prior DRAQ7 staining: (G) CYTO-ID Red, (H) DRAQ7, and (I) their overlay. Arrows show live cells without DRAQ7 nucleus staining. The samples were examined under the CLSM (Leica TCS SP2). Scale bars: 100 μm.

Article Snippet: The non-toxic DRAQ7 DNA dye was able to pass cell membrane of compromised cells, since they were fixed with 4% w/v PFA in PBS (335.2, Carl Roth GmbH, Germany) to generate a positive control of cell damage Consequently, the fibroblasts had damaged cell membranes and emitted both dyes, the CYTO-ID Red was located at the cell membranes and the DRAQ7 at the nuclei ( ).

Techniques: Labeling, Staining, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: Time resolved 3D live-cell imaging on implants

doi: 10.1371/journal.pone.0205411

Figure Lengend Snippet: CYTO-ID Red labeled human gingival fibroblasts were treated with different concentrations of chlorhexidine for 8 hours prior DRAQ7 staining. LIVE/DEAD stained gingival fibroblasts after treatment with 18 μM chlorhexidine: (A) CYTO-ID Red, (B) DRAQ7, and (C) their overlay. LIVE/DEAD stained gingival fibroblasts after treatment with 36 μM chlorhexidine: (D) CYTO-ID Red, (E) DRAQ7, and (F) their overlay. LIVE/DEAD stained gingival fibroblasts after treatment with 72 μM chlorhexidine: (G) CYTO-ID Red, (H) DRAQ7, and (I) their overlay. LIVE/DEAD stained gingival fibroblasts after treatment with 181 μM chlorhexidine: (J) CYTO-ID Red, (K) DRAQ7, and (L) their overlay. The samples were examined under the CLSM (Leica TCS SP2). Scale bars: 200 μm.

Article Snippet: The non-toxic DRAQ7 DNA dye was able to pass cell membrane of compromised cells, since they were fixed with 4% w/v PFA in PBS (335.2, Carl Roth GmbH, Germany) to generate a positive control of cell damage Consequently, the fibroblasts had damaged cell membranes and emitted both dyes, the CYTO-ID Red was located at the cell membranes and the DRAQ7 at the nuclei ( ).

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Time resolved 3D live-cell imaging on implants

doi: 10.1371/journal.pone.0205411

Figure Lengend Snippet: (A) MIP of the live cell stain with CYTO-ID Red (green) and dead cell stain with DRAQ7 (red) at the different time points. Rectangles indicate area of zoomed in versions of each MIP. (B) Fluorescence intensity profile of the MIPs (see corresponding image above in A). The profile was measured top-down and averaged for the full width of the titanium implant. (C) The difference spectrum for consecutive profiles in B.

Article Snippet: The non-toxic DRAQ7 DNA dye was able to pass cell membrane of compromised cells, since they were fixed with 4% w/v PFA in PBS (335.2, Carl Roth GmbH, Germany) to generate a positive control of cell damage Consequently, the fibroblasts had damaged cell membranes and emitted both dyes, the CYTO-ID Red was located at the cell membranes and the DRAQ7 at the nuclei ( ).

Techniques: Staining, Fluorescence