Journal: Heliyon

Article Title: Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations

doi: 10.1016/j.heliyon.2024.e27888

Figure Lengend Snippet: Illustration of connexin43 dynamics in HeLa cells as captured via (A) Fluorescence cryoSIM microscope and (B) Soft X-ray tomography. Green fluorescence represents Cx43-EMD-tagged HeLa cells, red represents fluorescently labelled F-Actin, and magenta represents fluorescently labelled mitochondria. Scale bars (a) 10 μm (b) 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MitoTracker Deep Red (125 nM; Fisher Scientific) for labelling mitochondria, F-Actin CellMask Orange/Red (1 × ; Fisher Scientific) for labelling filamentous actin, LysoTracker Blue (1 μM; Fisher Scientific) for labelling lysosomes and Hoechst 33342 Nuclear Blue (2 drops per mL of media; Fisher Scientific) dyes were all added to each well containing grids and incubated at 5% CO 2 and 37 °C for 30 min to allow for the cellular internalisation of the dyes.

Techniques: Fluorescence, Microscopy, Tomography

Journal: Heliyon

Article Title: Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations

doi: 10.1016/j.heliyon.2024.e27888

Figure Lengend Snippet: The impact of inflammatory insult on connexin43 channels' fate. Example 3D cryoSIM data from (A) Control Cx43-EMD transfected HeLa cells, (B) Cx43-EMD transfected HeLa cells exposed to pro-inflammatory factors for 24 h, and (C) Cx43-EMD transfected HeLa cells exposed to pro-inflammatory factors and XG19 simultaneously for 24 h. Green fluorescence represents Cx43-EMD-tagged in HeLa cells, red represents fluorescently labelled lysosomes, and magenta represents fluorescently labelled mitochondria. These example images are the bottom, middle and top slices from cryoSIM 3D data showing the distribution of Cx43 as connexons being degraded by lysosomes, hemichannels being transported to the cell membrane or GJ plaques. (D) Chances for the occurrence of connexosomes at cell interfaces. (E) Cx43 co-localisation with lysosome and (F) GJ plaque analyses in control Cx43-EMD transfected HeLa cells (blue bar, n = 18), Cx43-EMD transfected HeLa cells exposed to pro-inflammatory factors for 24 h (pink bar, n = 12) and Cx43-EMD transfected HeLa cells exposed to pro-inflammatory factors and XG19 simultaneously for 24 h (green bar, n = 18). (G) Degradation of connexosomes (green) by lysosomal contents (red). Scale bars = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MitoTracker Deep Red (125 nM; Fisher Scientific) for labelling mitochondria, F-Actin CellMask Orange/Red (1 × ; Fisher Scientific) for labelling filamentous actin, LysoTracker Blue (1 μM; Fisher Scientific) for labelling lysosomes and Hoechst 33342 Nuclear Blue (2 drops per mL of media; Fisher Scientific) dyes were all added to each well containing grids and incubated at 5% CO 2 and 37 °C for 30 min to allow for the cellular internalisation of the dyes.

Techniques: Control, Transfection, Fluorescence, Membrane

Journal: Heliyon

Article Title: Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations

doi: 10.1016/j.heliyon.2024.e27888

Figure Lengend Snippet: Soft X-ray tomography-guided comparison of mitochondria morphology. Example 5 μm × 5 μm cropped out section of a slice from a 3D tomogram showing morphological variations of mitochondria in: (A) Cx43-EMD transfected control HeLa cells (B) Cx43-EMD transfected HeLa cells at 24 h of pro-inflammatory factors (C) Cx43-EMD transfected HeLa cells at 24 h under pro-inflammatory condition and XG19 simultaneously. (D&E) Ultrastructural interactions at different 3D spaces in the cell showing the degradation of mitochondria by lysosomes. Graphs showing significant differences in (F) mitochondria surface area and (G) mitochondria volume in non-transfected control HeLa cells (blue bar, n = 29), non-transfected HeLa cells at 24 h of pro-inflammatory factor (pink bar, n = 45) and non-transfected HeLa cells at 24 h of pro-inflammatory conditions and XG19 simultaneously (green bar, n = 48). Graphs showing significant differences in (H) mitochondria surface area and (I) mitochondria volume in Cx43-EMD transfected control HeLa cells (blue bar, n = 34), Cx43-EMD transfected HeLa cells at 24 h of inflammatory stress (pink bar, n = 18) and Cx43-EMD transfected HeLa cells at 24 h of inflammatory stress and XG19 simultaneously (green bar, n = 14) cells. Green fluorescence in images represents connexin43-EMD tagged connexons, connexosomes or GJ plaques, red represents fluorescently labelled lysosomes, and magenta represents fluorescently labelled mitochondria. Orange arrows are pointing to mitochondria. Scale bars: (A–C) = 1 μm; inset = 0.25 μm; (D&E) = 0.25 μm *p < 0.05 and **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MitoTracker Deep Red (125 nM; Fisher Scientific) for labelling mitochondria, F-Actin CellMask Orange/Red (1 × ; Fisher Scientific) for labelling filamentous actin, LysoTracker Blue (1 μM; Fisher Scientific) for labelling lysosomes and Hoechst 33342 Nuclear Blue (2 drops per mL of media; Fisher Scientific) dyes were all added to each well containing grids and incubated at 5% CO 2 and 37 °C for 30 min to allow for the cellular internalisation of the dyes.

Techniques: Tomography, Comparison, Transfection, Control, Fluorescence

Journal: Heliyon

Article Title: Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations

doi: 10.1016/j.heliyon.2024.e27888

Figure Lengend Snippet: Example slices from cryoSIM fluorescence 3D data showing changes in Cx43 channels' states in (A) control, (B) 1-h inflammatory stress and (C) 2 h inflammatory stress plus 1 h of XG19 co-treatment. (D–F) Insets of different areas in panels A–C highlight the distribution of connexons, GJ plaques or connexosomes. (G) GJ plaque coverage in Cx43-EMD transfected control HeLa cells (blue bar, n = 22), Cx43-EMD transfected HeLa cells vitrified 1 h after inflammatory insult (pink bar, n = 5), and Cx43-EMD transfected HeLa cells vitrified 2-h after inflammatory trigger and 1-h of XG19 co-treatment in the second hour of inflammatory insult (green bar, n = 18). (H) GJ plaque length in Cx43-EMD transfected control HeLa cells (blue bar, n = 28), Cx43-EMD transfected HeLa cells vitrified 1-h after inflammatory insult (pink bar, n = 5), and Cx43-EMD transfected HeLa cells vitrified 2-h after inflammatory trigger and 1-h of XG19 co-treatment in the second hour of inflammatory insult (green bar, n = 39). (I) Cell length of Cx43-EMD transfected control HeLa cells (blue bar, n = 20), Cx43-EMD transfected HeLa cells vitrified 1-h after inflammatory insult (pink bar, n = 8), and Cx43-EMD transfected HeLa cells vitrified 2-h after inflammatory trigger and 1-h of XG19 co-treatment in the second hour of inflammatory insult (green bar, n = 22). (J) Chances for the occurrence of connexosomes at cell interfaces in Cx43_EMD transfected control HeLa cells (blue portion, n = 6), Cx43-EMD transfected HeLa cells vitrified 1-h after inflammatory insult (pink portion, n = 4) and Cx43-EMD transfected HeLa cells vitrified 2-h after inflammatory trigger and 1-h of XG19 co-treatment in the second hour of inflammatory insult (green portion, n = 5). Green fluorescence represents connexin43-EMD tagged connexons, connexosomes or GJ plaques, cyan represents fluorescently labelled lysosomes, red represents fluorescently labelled F-Actin and magenta represents fluorescently labelled mitochondria. Scale bars: (A–C) 10 μm; (insets) 2.5 μm *P < 0.05 and **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MitoTracker Deep Red (125 nM; Fisher Scientific) for labelling mitochondria, F-Actin CellMask Orange/Red (1 × ; Fisher Scientific) for labelling filamentous actin, LysoTracker Blue (1 μM; Fisher Scientific) for labelling lysosomes and Hoechst 33342 Nuclear Blue (2 drops per mL of media; Fisher Scientific) dyes were all added to each well containing grids and incubated at 5% CO 2 and 37 °C for 30 min to allow for the cellular internalisation of the dyes.

Techniques: Fluorescence, Control, Transfection

Journal: Heliyon

Article Title: Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations

doi: 10.1016/j.heliyon.2024.e27888

Figure Lengend Snippet: Cellular state and gross morphology. (A) 2D X-ray mosaic showing HeLa Cx43-EMD transfected control cells, (B) 2D X-ray mosaic showing HeLa Cx43-EMD transfected cells at 1 h of inflammatory insult, (C) Slices from 3D X-ray tomograms showing HeLa Cx43-EMD transfected cells at 2 h of inflammatory insult and 1 h of XG19 co-treatment in the second hour of inflammatory injury, (D) Slices from of a 3D X-ray tomogram showing branched mitochondria morphology in cells where pro-inflammatory factors were discontinued for 24 h in HeLa Cx43-EMD transfected cells, (E) Slice of a 3D X-ray tomogram showing elongated mitochondria morphology from cells where pro-inflammatory factors were discontinued for 24 h and treated with XG19 for an additional 24 h in HeLa Cx43-EMD transfected cells. (F) Analyses of gross cell morphology showing cell width in control Cx43-EMD transfected HeLa cells (blue bar, n = 28), Cx43-EMD transfected HeLa cells at 1-h pro-inflammatory trigger (pink bar, n = 8), Cx43-EMD transfected HeLa cells at 2-h of pro-inflammatory factors plus 1 h of XG19 co-treatment in the second hour of inflammatory insult (green bar, n = 23), Cx43-EMD transfected HeLa cells at 24 h of inflammatory condition + pro-inflammatory cytokines discontinued for an additional 24 h (pink dotted bar, n = 8), Cx43-EMD transfected HeLa cells at 24 h of inflammatory insult + pro-inflammatory factors discontinued for 24 h with 24 h of XG19 treatment immediately upon discontinuation of pro-inflammatory factors (green dotted bar, n = 26), Cx43-EMD transfected HeLa cells at 48 h of inflammatory condition (pink striped bar, n = 9), Cx43-EMD transfected HeLa cells at 48 h of pro-inflammatory factors & 24 h of XG19 co-treatment in the last 24 h of exposure to pro-inflammatory factors (green striped bar, n = 42). Nu = nucleus, Cyt = cytoplasm, filopodia (green arrows), nuclear membrane (purple arrows), nucleoli (yellow arrows), vacuoles/vesicles (red arrows), lipid droplets (blue arrows), mitochondria (orange arrows) 200 nm Au nanoparticles (white arrows). Red fluorescence represents F-Actin labelling and green fluorescence denote connexin43-EMD tagged connexons, connexosomes or GJ plaques. Scale bars (A&B) 10 μm, (C–E) 2 μm *p < 0.05, ***p < 0.001 and ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MitoTracker Deep Red (125 nM; Fisher Scientific) for labelling mitochondria, F-Actin CellMask Orange/Red (1 × ; Fisher Scientific) for labelling filamentous actin, LysoTracker Blue (1 μM; Fisher Scientific) for labelling lysosomes and Hoechst 33342 Nuclear Blue (2 drops per mL of media; Fisher Scientific) dyes were all added to each well containing grids and incubated at 5% CO 2 and 37 °C for 30 min to allow for the cellular internalisation of the dyes.

Techniques: Transfection, Control, Membrane, Fluorescence

Journal: Heliyon

Article Title: Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations

doi: 10.1016/j.heliyon.2024.e27888

Figure Lengend Snippet: A graphic workflow of the correlative 3D visualisation of connexons and GJs in HeLa cells using fluorescent cryoSIM and soft X-ray tomography at B24. (A) Maximum intensity projection of fluorescent cryoSIM data showing connexin43 in green and mitochondria in magenta. (B) 2D X-ray mosaic of the same area shown in panel A. (C) Slice of 3D-SIM data showing the transformation of fluorescence data to correspond to the orientation and location of the same features seen in the X-ray tomogram (D) Slice of 3D X-ray tomogram of the area in panel B. (E) Correlation of cryoSIM and X-ray dataset from the same area showing the circular loop of internalised connexin43 (connexosome shown with black arrow) and junctional connexin43 (white arrow). Green represents connexin43-EMD tagged connexons, connexosomes or GJ plaques, red represents fluorescently labelled lysosomes, cyan represents fluorescently labelled F-Actin, and magenta represents fluorescently labelled mitochondria. Scale bars (a) 10 μm (b) 10 μm (c–g) 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MitoTracker Deep Red (125 nM; Fisher Scientific) for labelling mitochondria, F-Actin CellMask Orange/Red (1 × ; Fisher Scientific) for labelling filamentous actin, LysoTracker Blue (1 μM; Fisher Scientific) for labelling lysosomes and Hoechst 33342 Nuclear Blue (2 drops per mL of media; Fisher Scientific) dyes were all added to each well containing grids and incubated at 5% CO 2 and 37 °C for 30 min to allow for the cellular internalisation of the dyes.

Techniques: Tomography, Transformation Assay, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads

doi: 10.1016/j.xpro.2024.103190

Figure Lengend Snippet:

Article Snippet: CellMask Green (1:5,000 working dilution) , Fisher Scientific , CA# H32714.

Techniques: Cell Culture, Recombinant, Blocking Assay, Saline, Software, Electron Microscopy, Staining, Microscopy, Western Blot