Journal:

Article Title: Identifying Functional Neighborhoods within the Cell Nucleus: Proximity Analysis of Early S-Phase Replicating Chromatin Domains to Sites of Transcription, RNA Polymerase II, HP1?, Matrin 3 and SAF-A

doi: 10.1002/jcb.21834

Figure Lengend Snippet: Plot shows the distribution of percentage proximity values of TS, pol II, HP1γ and PCNA to each other. Segmentation and centroid detection was followed by the proximity analysis for two components at a time. X axis represents the distance in pixels (1 pixel = 0.07 microns) and Y axis represents the percent proximity. Y axis error bars denote the SEM. The proximity relationship between any two components was divided based on their centroid to centroid distance into two categories (1–3 pixels & 4–7 pixels). The red and green circles illustrate sites and indicate the two categories mentioned above which translate to approximate center-to-center distances of 0–0.2 microns and 0.2–0.5 microns. (B & C) proximity analysis matrix in which the centroid distances between the labeled sites were split into zones of ’1–3’ and ‘1–7’ pixels, respectively. In each zone the first column represents the reference sites, while the second, third and fourth columns in each row show sites that are arranged in decreasing order of proximity to the reference.

Article Snippet: Primary antibodies used in the immuno-labeling experiments were rabbit polyclonal antibodies to HP1γ (ab10480, Abcam Inc., Cambridge, MA) and SAF-A/hnRNP-U [ Fackelmayer et al., 1994 ], monoclonal mouse antibodies (IgG) to RNA polymerase II (ARNA-3, RDI division of Fitzgerald industries) and polyclonal chicken antibodies to matrin 3 [ Belgrader et al., 1991 ].

Techniques: Labeling

Journal:

Article Title: Identifying Functional Neighborhoods within the Cell Nucleus: Proximity Analysis of Early S-Phase Replicating Chromatin Domains to Sites of Transcription, RNA Polymerase II, HP1?, Matrin 3 and SAF-A

doi: 10.1002/jcb.21834

Figure Lengend Snippet: Representative mid plane sections of HeLa cell nuclei labeled for HP1γ - DAPI (A), TS - DAPI (B), PCNA - DAPI (C) and pol II - DAPI (D) were pseudo-colored as indicated by the legend (DAPI in green, and functional/protein factor sites in red; primary antibodies against HP1γ and TS were labeled with anti-rabbit-IgG-Alexa594, anti-rat-IgG-Alexa647 respectively; and primary antibodies against PCNA and pol II were labeled with anti-rabbit-IgG-Alexa488 and anti-mouse-IgG-Alexa594 respectively in separate experiments). Images of individual channels were pseudo-colored and overlaid for line profile analysis. Areas of the nucleus that are intensely stained with DAPI are considered to be enriched in heterochromatin. The green and the red peaks indicated in the line (yellow line across the image) profile correspond to the distribution of DAPI and functional sites respectively. The areas showing complete separation of red and green channel are indicated in the line profile by respective colored arrows. Regions of complete or large degree of overlap are indicated by yellow colored arrows.

Article Snippet: Primary antibodies used in the immuno-labeling experiments were rabbit polyclonal antibodies to HP1γ (ab10480, Abcam Inc., Cambridge, MA) and SAF-A/hnRNP-U [ Fackelmayer et al., 1994 ], monoclonal mouse antibodies (IgG) to RNA polymerase II (ARNA-3, RDI division of Fitzgerald industries) and polyclonal chicken antibodies to matrin 3 [ Belgrader et al., 1991 ].

Techniques: Labeling, Functional Assay, Staining

Journal:

Article Title: Identifying Functional Neighborhoods within the Cell Nucleus: Proximity Analysis of Early S-Phase Replicating Chromatin Domains to Sites of Transcription, RNA Polymerase II, HP1?, Matrin 3 and SAF-A

doi: 10.1002/jcb.21834

Figure Lengend Snippet: A representative HeLa cell nucleus is shown multi-labeled for (A) PCNA, TS, HP1γ and DAPI. For (A) PCNA, TS, HP1γ and DAPI are pseudocolored in green (anti-mouse-IgG-Alexa488), cyan (anti-rat-IgG-Alexa647), red (anti-rabbit-IgG-Alexa594) and blue respectively. (B, C & D) show pair wise combination of different channels pseudocolored in red and green. Similarly, (E) shows a representative HeLa cell nucleus multi-labeled for pol II, TS, HP1γ and DAPI and subsequent pair wise combinations in green and red (F-H). Anti-mouse-IgG-Alexa488 is used as secondary antibody for pol II labeling. (I) to (N) represent magnified regions within the yellow boxes in panels (B) to (H). Line profile analysis of colocalization for the magnified regions (J) to (N) is shown in (O) to (S). White scale bars (A) to (H) indicate 5 microns.

Article Snippet: Primary antibodies used in the immuno-labeling experiments were rabbit polyclonal antibodies to HP1γ (ab10480, Abcam Inc., Cambridge, MA) and SAF-A/hnRNP-U [ Fackelmayer et al., 1994 ], monoclonal mouse antibodies (IgG) to RNA polymerase II (ARNA-3, RDI division of Fitzgerald industries) and polyclonal chicken antibodies to matrin 3 [ Belgrader et al., 1991 ].

Techniques: Labeling