Fig 2: The miR-758-3p inhibits CBX5 expression by targeting 3'-UTR. A, Predicted miR-758-3p binding site in CBX5 3'-UTR. B, Western blot to detect CBX5 expression after transfection. C, Relative luciferase activity of CBX5 in wt or mut construct. D, Correlation analysis of miR-758-3p and CBX5 in GC tissues. E, Western blot to detect CBX5 expression in GC tissues and surrounding noncancerous tissues (ns: not significant, ***P < .001). CBX5 indicates chromobox 5; GC, gastric cancer; miR-758-3p, microRNA-758-3p; mut, mutant; NC, negative control; UTR, untranslated region; wt, wild type.

Fig 3: ASFV leads to juxtaposition of host heterochromatic regions, disrupted subnuclear domains and pATR foci. (A) Heterochromatin marker HP1ß is labelled in red, while subnuclear domains (PML-NBs, nuclear speckles or Cajal bodies) and the activated ATR kinase are labelled in cyan. Cell nuclei were counterstained with DAPI (blue). (a–d) Only ASFV-infected cells (green) display the speckled pattern of enlarged SC-35 accumulations (cyan) juxtaposed to the heterochromatic regions (HP1 ß, red), as non-infected cells do not reveal the close proximity pattern (a’–d’). In contrast to non-infected cells (e’–h’), the ASFV-induced bulky heterochromatic territories (HP1ß, red) are always present within close vicinity to disrupted Cajal bodies (cyan) (e–h). Enlarged heterochromatic regions (HP1ß, red) closely neighbour reorganized PML-NBs (cyan) in infected cells (green) (i–l), rearrangements not observable in non-infected cells (i'–l’). HP1ß deposits (red) juxtapose to pATR foci (cyan), in ASFV-infected cells (green) (m–p), different chromatin/pATR appearances and distributions in non-infected cells (m’–p’). Scale bar, 10 µm. Representative microscopy images of at least three independent experiments are shown; (B–E) Relative distance between the studied subnuclear domains/pATR foci (cyan) and heterochromatic regions (red) was evaluated by radial intensity profile analysis in ASFV-infected cells (solid lines) and non-infected cells (dashed lines). Normalized fluorescence curves were obtained from 50 nuclei. Experiments were performed in triplicate and error bars represent standard errors (±SE). Radial profile analysis shows the close proximity, in ASFV-infected cells, between each subnuclear domain/pATR foci (blue solid line) and HP1ß accumulations (red solid line), crossing at a point within the given radius (1 µm). Dashed lines representing fluorescence intensities of subnuclear domains/pATR (blue) and heterochromatic regions (red) never intersect, revealing a greater distance between these nuclear domains/factors in non-infected cells.

Fig 4: Fluorescence Lifetime Imaging-Forster Resonance Energy Transfer (FLIM-FRET) analysis of potential interaction between heterochromatin protein 1 (HP1) protein isoforms and the KAP1 protein. (a) Analysis of a GFP-tagged HP1a-KAP1, b] GFP-tagged HP1ß-KAP1, and c] GFP-tagged HP1?-KAP1. (b) An example of a nuclear distribution of HP1? and the KAP1 protein. HP1?-positive regions of nucleoli (green) were absent of KAP1. Abbreviation Nu means nucleoli; arrows show HP1? foci absent of KAP1 and red frames show HP1? foci colocalizing with KAP1. (c) Example of STED analysis showing the location of KAP1 inside the cell nucleoli decorated by the HP1 isoforms (see HP1ß in white frame). (d) FLIM-FRET analysis of a] GFP-tagged HP1ß ?CD/Alexa 594-KAP1, b] GFP-tagged HP1ß ?CSD/ Alexa 594-KAP1, and c] GFP-tagged HP1ß ?Hinge/Alexa 594-KAP1. E [%] means FLIM-FRET efficiency. Scale bars represent 4 µm.

Fig 5: Binding of HP1? to STAT3 is enhanced by BDNF signaling in neurons. a Representative Western Blot image of coimmunoprecipitation assays using whole-cell extracts of 9-DIV primary cortical neurons treated (30 min) with Water or 0.7 nM BDNF. Protein was precipitated with a specific HP1? antibody (Cell Signaling Technologies #2616) and the elution analyzed by SDS-PAGE. Membrane was immunoblotted with an anti-STAT3 antibody (Cell Signaling Technologies #4904) to detect co-association of HP1? with STAT3, (n = 4). b Quantitation of STAT3 association with HP1? from dataset in “A”. c Representative Western Blot image: Inhibitors of JAK/STAT signaling (WP1066 (WP) and Ruxolitinib 2 (RX2)) reduce HP1a antibody precipitation of STAT3. Data was normalized to ßactin levels which did not change upon BDNF treatment, * = p < 0.05