Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A) Schematic drawing of CHD3, CHD4 and CHD5 proteins showing conserved domains and the CHD5 specific sequence. (B) Table showing sequence identity between different domains of CHD3, CHD4 and CHD5. (C) Expression of recombinant CHD5. Recombinant CHD5-Flag was purified from extracts of baculovirus-infected Sf9 cells. Extracts were incubated with anti-Flag affinity resin, washed and eluted with an excess of Flag peptide. Purified protein was seperated by SDS-PAGE followed by Western blotting with CHD5 antibody (lane 1) or silver staining (lane 2). Asterisk indicates degradation products with intact anti-CHD5 epitope. (D) CHD5 is a nucleosome-stimulated ATPase. Purified recombinant CHD5-Flag was subjected to ATPase assay as described in Materials and Methods. Different amounts of recombinant human CHD5-Flag, naked DNA or in vitro assembled nucleosomes were used as indicated at the bottom of the panel. Reaction products were separated by thin layer chromatography and the amount of released 32 P i was quantitated. The percentage of ATP hydrolysis was calculated for each reaction. The percentage of ATP hydrolysis of control reactions (without enzyme) was set to one. Error bars depict the standard deviation of 3 independent experiments.

Article Snippet: For detection of CHD5, polyclonal rabbit anti human-CHD5 (Santa Cruz Biotechnology (H-185) sc-68390) which is also reactive with mouse CHD5 was used.

Techniques: Sequencing, Expressing, Recombinant, Purification, Infection, Incubation, SDS Page, Western Blot, Silver Staining, ATPase Assay, In Vitro, Thin Layer Chromatography, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A) CHD5 protein is expressed in mouse brain and testis. Western blot was performed with CHD5 antibody on lysates from different mouse tissues. Extracts from HEK cells transiently transfected with CHD5-Flag served as a positive control. Lamin B was used as a loading control. (B) Two different CHD5 isoforms are expressed in mouse brain and testis. Panel 1: schematic representation of the location of the PCR primers used to detect CHD5 cDNA of isoforms 1 and 2. Panel 2: schematic representation of part of the CHD5 gene structure showing the two different putative splicing variants. Exons 23, 24 and 25 are indicated. Horizontal lines are introns. Diagonal lines indicate splicing. Panel 3: RT-PCR detecting expression of different CHD5 isoforms in mouse testis and brain. H 2 O: negative control. Asterisk: primer dimers.

Article Snippet: For detection of CHD5, polyclonal rabbit anti human-CHD5 (Santa Cruz Biotechnology (H-185) sc-68390) which is also reactive with mouse CHD5 was used.

Techniques: Western Blot, Transfection, Positive Control, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: Mouse brain sections were immunostained with antibodies against CHD5 or CHD3/4 and antibody binding was detected using a peroxidase labeled secondary antibody and 3,3'-diaminobenzidine (DAB) as a substrate. (1) CHD5 positive neurons in the hippocampus (20× magnification). (2) 200× magnification of CHD5 positive hippocampal neurons. (3) CHD5 positive neurons in the cortex of the cerebellum (100× magnification). (4) 400× magnification of CHD5 positive cerebellar neurons. (5) and (6) consecutive sections of cerebellum showing CHD3/4 (5) and CHD5 (6) expression.

Article Snippet: For detection of CHD5, polyclonal rabbit anti human-CHD5 (Santa Cruz Biotechnology (H-185) sc-68390) which is also reactive with mouse CHD5 was used.

Techniques: Binding Assay, Labeling, Expressing

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A) Western blot analysis CHD3/4, CHD5 and lamin expression in lysates from 3 week-old and from adult mice. Extracts from HEK cells expressing recombinant CHD5-Flag and extracts from murine brain were used as positive controls for CHD5 expression. Antibodies used for Western blot are indicated. (B) CHD5 protein is maximally expressed in round spermatids at Stages VII–VIII of the spermatognenic cycle. Upper images show results of the immunostaining and the bottom images show the PAS staining pattern of a section directly adjacent to the section shown above it. Left panel: low magnification (scale bars indicate 0.5 mm) of a mouse testis containing seminiferous tubules in different spermatogenic stages. Middle and right: higher magnification of the areas (scale bars indicate 0.05 mm) marked in the left panel, roman numerals indicate the stages of the spermatogenic cycle. (C) CHD5 is expressed in postmeiotic cells. Immunofluorescence analysis was performed using CHD5 antibody and visualized with confocal microscopy. Panel 1: Nuclei stained with DAPI. Panel 2: CHD5 staining, the outline of the tubule as seen in panel 1 is indicated with white lines. Panel 3: phosphorylated H2AX (γH2AX) staining. Spermatocytes in late meiotic prophase (pachytene and diplotene), show intense staining in a subregion of the nucleus (XY body) (arrow). Earlier spermatocytes (at the more basal part of the tubule) show nucleus wide γH2AX staining (arrowhead). Panel 4: overlay of the DAPI, CHD5 and γH2AX channels. (D) CHD5 and CHD3/4 expression at different stages of spermatogenesis. Panel 1: CHD5 is expressed in post-meiotic cells (100× magnification). Panel 2: CHD3/4 is expressed in the area where spermatogonia, Sertoli cells and spermatocytes are present (100× magnification). Panel 3: CHD5 immunostaining (250× magnification). Panel 4: CHD3/4 immunostaining (250x). Formalin-fixed paraffin-embedded tissue sections were immunostained with the antibodies indicated.

Article Snippet: For detection of CHD5, polyclonal rabbit anti human-CHD5 (Santa Cruz Biotechnology (H-185) sc-68390) which is also reactive with mouse CHD5 was used.

Techniques: Western Blot, Expressing, Recombinant, Immunostaining, Staining, Immunofluorescence, Confocal Microscopy, Formalin-fixed Paraffin-Embedded

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A, B) Spread nuclei of spermatids immunostained for CHD5 and counterstained with DAPI. An example of a round spermatid nucleus that is positive for CHD5 expression and displays the high intensity focus in the chromocenter is shown in A and such an example is also indicated with a white arrow in B, the yellow arrow indicates a spermatid nucleus that expresses CHD5, but does not have the extra CHD5 focus. The green arrow indicates a CHD5-negative round spermatid nucleus. (C) Spread nuclei of spermatids immunostained for CHD5 and H3K9me2, and counterstained with DAPI. The nuclear area encompassing the chromatin of the sex chromosome is indicated with a dashed line H3K9me2 marks the chromocenter and the sex chromosome in the left (early) round spermatid nucleus, and mainly the sex chromosome in the right (late) round spermatid nucleus. CHD5 level is very low in the left nucleus, but clearly enriched in the chromocenter and the focus (indicated with a white arrow) adjacent to the chromocenter and Y chromosome chromatin in the right nucleus.

Article Snippet: For detection of CHD5, polyclonal rabbit anti human-CHD5 (Santa Cruz Biotechnology (H-185) sc-68390) which is also reactive with mouse CHD5 was used.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A,B) Spread wild type (A) and Spo11 null ( Spo11 YF/YF ) (B) spermatocyte nuclei stained for MacroH2A.1 and CHD3/4. Colocalisation on the PAR and the X centromere is observed in pachytene, but not in diplotene nuclei of wild type spermatocytes. In the absence of functional SPO11, some zygotene-like nuclei show enrichment of both CHD4 and macroH2A.1 in the pseudo XY body. (C) Spread spermatid nucleus stained for CHD5, and MacroH2A, colocalisation of the CHD5/MacroH2A focus in the chromocenter is clear in the merge with DAPI (right image). (D) Spread spermatid nuclei stained with macroH2A (green) and CREST (red, left panel) or with CHD5 and CREST (right panel). The enlargements in the middle of each panel clearly show that the macroH2A.1/CHD5 spot in the chromocenter does not colocalise with CREST marking of the centromeres. (E) Spread spermatid nuclei costained for CHD5 and RNA polymerase II. Both high and low levels of RNA polymerase II can be observed in nuclei that express CHD5.

Article Snippet: For detection of CHD5, polyclonal rabbit anti human-CHD5 (Santa Cruz Biotechnology (H-185) sc-68390) which is also reactive with mouse CHD5 was used.

Techniques: Staining, Functional Assay

Journal: PLoS ONE

Article Title: MicroRNA-211 Expression Promotes Colorectal Cancer Cell Growth In Vitro and In Vivo by Targeting Tumor Suppressor CHD5

doi: 10.1371/journal.pone.0029750

Figure Lengend Snippet: (A) The levels of cell growth-associated proteins in HCT-116 vec and HCT-116 miR-211 cell lines were analyzed by Western blot and (B) semi-quantified based on targeted protein/β-actin relative intensities. The levels of p53-related pathway proteins in HCT-116 vec and HCT-116 miR-211 cell lines were analyzed by Western blot (C) and semi-quantified based on targeted protein/β-actin relative intensities (D).

Article Snippet: Primary antibodies used for Western blot analysis included rabbit anti-human CHD5 (Abcam; 1∶5,000 dilution), mouse anti-human Bcl-2 (Sigma; 1∶1,000 dilution), rabbit anti-human Bcl-xL (Sigma; 1∶1,000 dilution), rabbit anti-human Bad (Santa Cruz; 1∶500 dilution), rabbit anti-human Bax (Sigma; 1∶500 dilution), rabbit anti-human p53 (Sigma; 1∶1,000 dilution), mouse anti-human MDM-2 (Sigma; 1∶1,000 dilution), and mouse anti-human β-actin (Sigma; 1∶5,000 dilution) and secondary antibodies included goat anti-rabbit IgG-HRP (Santa Cruz; 1∶2,000 dilution) and goat anti-mouse IgG-HRP (Santa Cruz; 1∶2,000 dilution).

Techniques: Western Blot