Journal: Asian Journal of Andrology

Article Title: RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

doi: 10.4103/aja202368

Figure Lengend Snippet: RNF187 knockdown inhibits the proliferation and migration and promotes the apoptosis of GC-2 cells. ( a ) Relative expression levels of Rnf187 were measured by qRT-PCR in GC-2 cells transfected with si-NC, si- Rnf187 -#1, or si- Rnf187 -#2 ( n = 3 for each group). The internal control was 18S rRNA . ( b ) CCK-8 assays were conducted to assess the viability of RNF187-knockdown GC-2 cells ( n = 6 for each group). ( c ) Colony formation assays were performed to assess the proliferative ability of RNF187-knockdown GC-2 cells ( n = 3 for each group). Scale bars = 2 mm. ( d ) Quantitative analysis of the data in c . ( e ) Transwell assays were performed to evaluate the migration of RNF187-knockdown GC-2 cells ( n = 3 for each group). Scale bars = 100 µm. ( f ) Quantitative analysis of the results in e . ( g ) EdU incorporation assays were carried out to investigate cell proliferation ( n = 3 for each group). The EdU-positive cells are labeled in purple, and the cell nuclei are labeled in blue. Scale bars = 20 µm. ( h ) Quantitative analysis of the data in g . ( i ) TUNEL assays were performed to investigate the apoptotic capacity of RNF187-knockdown GC-2 cells ( n = 3 for each group). The TUNEL-positive cells are labeled in purple, and the cell nuclei are labeled in blue. Scale bars = 20 µm. ( j ) Quantitative analysis of the data in i . * P < 0.05; ** P < 0.01; *** P < 0.001; all compared with si-NC. RNF187: RING finger 187; GC-2 cells: mouse spermatocyte-derived cell line; si-NC: negative control siRNA; si- Rnf187 -#1 or #2: small interfering RNA targeting RNF187 (si- Rnf187 -#1: 5’-GCAUGGUCAGGCAGAAUCA-3’; si- Rnf187 -#2: 5’-GGGCAUGUUAUGGACCGAA-3’); qRT-PCR: quantitative real-time polymerase chain reaction; OD: optical density; CCK-8: Cell Counting Kit-8; EdU: 5-ethynyl-2’-deoxyuridine; DAPI: 4’,6-diamidino-2-phenylindole; TUNEL: terminal deoxynucleotidyl transferase-dUTP nick-end labeling.

Article Snippet: GC-2 spd cells, a cell line derived from mouse spermatocytes (GC-2), were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 ( v / v ).

Techniques: Knockdown, Migration, Expressing, Quantitative RT-PCR, Transfection, Control, CCK-8 Assay, Labeling, TUNEL Assay, Derivative Assay, Negative Control, Small Interfering RNA, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Asian Journal of Andrology

Article Title: RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

doi: 10.4103/aja202368

Figure Lengend Snippet: Overexpression of RNF187 increases the proliferation and migration of GC-2 cells but does not impact their apoptosis. ( a ) Western blot analysis of RNF187 expression in GC-2 cells transfected with the Flag-RNF187 plasmid or the corresponding empty vector. ( b ) CCK-8 assays were conducted to assess the viability of GC-2 cells overexpressing RNF187 ( n = 6 for each group). ( c ) Colony formation assays were performed to assess the proliferative ability of GC-2 cells overexpressing RNF187 ( n = 3 for each group). Scale bars = 2 mm. ( d ) Quantitative analysis of the results in c . ( e ) Transwell assays were carried out to evaluate the migration of GC-2 cells transfected with the Flag-RNF187 plasmid or the corresponding empty vector ( n = 3 for each group). Scale bars = 100 µm. ( f ) Quantitative analysis of the results in e . ( g ) EdU incorporation assays were executed to evaluate the proliferation of GC-2 cells transfected with the Flag-RNF187 plasmid or the corresponding empty vector ( n = 3 for each group). The EdU-positive cells are labeled in purple, and the cell nuclei are labeled in blue. Scale bars = 20 µm. ( h ) Quantitative analysis of the data in g . ( i ) TUNEL assays were performed to investigate the apoptotic capacity of GC-2 cells overexpressing RNF187 ( n = 3 for each group). The TUNEL-positive cells are labeled in purple, and the cell nuclei are labeled in blue. Scale bars = 20 µm. ( j ) Quantitative analysis of the data in i . * P < 0.05; ** P < 0.01; *** P < 0.001; all compared with EV. RNF187: RING finger 187; GC-2 cells: mouse spermatocyte-derived cell line; Flag-RNF187: recombinant plasmid pcDNA3.1-Flag-RNF187; EV: negative control pcDNA3.1 empty vector; TUBULIN: β-tubulin; OD: optical density; CCK-8: Cell Counting Kit-8; EdU: 5-ethynyl-2’-deoxyuridine; DAPI: 4’,6-diamidino-2-phenylindole; TUNEL: terminal deoxynucleotidyl transferase-dUTP nick-end labeling; NS: nonsignificant.

Article Snippet: GC-2 spd cells, a cell line derived from mouse spermatocytes (GC-2), were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 ( v / v ).

Techniques: Over Expression, Migration, Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Labeling, TUNEL Assay, Derivative Assay, Recombinant, Negative Control, Cell Counting

Journal: Asian Journal of Andrology

Article Title: RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

doi: 10.4103/aja202368

Figure Lengend Snippet: Interaction between RNF187 and histone H3 in GC-2 cells. ( a ) The procedures used to identify proteins that interact with RNF187 in GC-2 cells. The Flag-RNF187 plasmid or the corresponding empty vector was transfected into GC-2 cells. Total protein was harvested and subjected to IP experiments with anti-Flag beads, PAGE of the immunoprecipitated products, in-gel digestion, and LC-MS/MS analysis. ( b ) The iBAQ algorithm embedded in MaxQuant was used to estimate protein expression levels. ( c ) The Flag-RNF187 and GFP-H3-WT overexpression plasmids as well as the Flag-RNF187 and pcDNA3.1-GFP overexpression plasmids were transfected separately into GC-2 cells. Protein extracts were incubated with anti-Flag beads or anti-GFP beads for co-IP assays, followed by western blot analysis with anti-Flag or anti-GFP antibodies. ( d ) Visualization of the RNF187-H3 docking interface showing the docked protein complex formed between RNF187 (blue) and H3 (yellow) as viewed from the front and the back. The backbone and secondary structures of the associated proteins are shown in cartoon mode. The surface mode shows a representation of the solvent-accessible surface area. The merged mode shows an integration of the cartoon view with the surface view. Binding residues at the interface between RNF187 and H3 (in stick mode) are shown in the interface view. In RNF187 and H3, the interacting sites are shown in red and purple, respectively. The dashed yellow lines indicate molecular contacts between interacting sites. RNF187: RING finger 187; H3: histone H3; GC-2 cells: mouse spermatocyte-derived cell line; Flag-RNF187: recombinant plasmid pcDNA3.1-Flag-RNF187; EV: negative control pcDNA3.1 empty vector; GFP-H3-WT: recombinant plasmid pEGFP-H3-WT; pcDNA3.1-GFP: empty vector containing GFP; IP: immunoprecipitation; LC-MS/MS: liquid chromatography-mass spectrometry; m/z: mass-to-charge ratio; Atp5f1 : ATP synthase F(0) complex subunit B1; Slc39a7 : zinc transporter SLC39A7; Tubb2a : tubulin beta-2A chain; Hdgf : hepatoma-derived growth factor; Cacybp : calcyclin-binding protein; Rab5a : ras-related protein Rab-5A; Gnb2 : guanine nucleotide-binding protein subunit beta-2; Psmb7 : proteasome subunit beta type-7; GFP: green fluorescent protein; iBAQ: intensity-based absolute quantification.

Article Snippet: GC-2 spd cells, a cell line derived from mouse spermatocytes (GC-2), were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 ( v / v ).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing, Over Expression, Incubation, Co-Immunoprecipitation Assay, Western Blot, Solvent, Binding Assay, Derivative Assay, Recombinant, Negative Control, Liquid Chromatography, Mass Spectrometry, Quantitative Proteomics

Journal: Asian Journal of Andrology

Article Title: RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

doi: 10.4103/aja202368

Figure Lengend Snippet: RNF187 ubiquitinates histone H3 at lysine 57/80. ( a ) RNF187 knockdown abolished H3 ubiquitination. Control and RNF187-knockdown GC-2 cells were transfected with GFP-H3-WT and HA-Ub plasmids for ubiquitination assays; the data were subsequently verified by western blot analysis. ( b ) Flag-RNF187 ubiquitinates H3 through its RING domain. GC-2 cells were transfected with the HA-Ub plasmid and the Flag-RNF187 or Flag-RNF187-RM plasmid for ubiquitination assays and were then harvested for IP; the results were subsequently verified by western blot analysis. ( c ) In vitro ubiquitination assays were performed to compare the ability of Flag-RNF187 and Flag-RNF187-RM to ubiquitinate H3. ( d ) Ubiquitination sites in histone H3. ( e ) The K57R and K80R mutations in H3 abolished RNF187-induced H3 ubiquitination. WT, K57R or K80R GFP-H3 plasmids were expressed in GC-2 cells, and the cells were subjected to immunoprecipitation followed by western blot analysis. RNF187: RING finger 187; H3: histone H3; GC-2 cells: mouse spermatocyte-derived cell line; si-NC: the protein expression in the cells treated with negative control siRNA; si-RNF187-#1 or #2: small interfering RNA targeting RNF187 (si- Rnf187 -#1: 5´-GCAUGGUCAGGCAGAAUCA-3´; si- Rnf187 -#2: 5´-GGGCAUGUUAUGGACCGAA-3´); Flag-RNF187: recombinant plasmid pcDNA3.1-Flag-RNF187; EV: negative control pcDNA3.1 empty vector; Flag-RNF187-RM: recombinant plasmid pcDNA3.1-Flag-RNF187-RING-Mutant; GFP-H3-WT: recombinant plasmid pEGFP-H3-WT; GFP-H3-K57R: recombinant plasmid pEGFP-H3-K57R; GFP-H3-K80R: recombinant plasmid pEGFP-H3-K80R; HA-Ub: recombinant plasmid pRK5-HA-Ub; IP: immunoprecipitation; E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; ATP: adenosine triphosphate; WT: wild-type; GFP: green fluorescent protein.

Article Snippet: GC-2 spd cells, a cell line derived from mouse spermatocytes (GC-2), were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 ( v / v ).

Techniques: Knockdown, Ubiquitin Proteomics, Control, Transfection, Western Blot, Plasmid Preparation, In Vitro, Immunoprecipitation, Derivative Assay, Expressing, Negative Control, Small Interfering RNA, Recombinant, Mutagenesis

Journal: Asian Journal of Andrology

Article Title: RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

doi: 10.4103/aja202368

Figure Lengend Snippet: The impact of H3 residues K57 and K80 on the behavior of GC-2 cells. ( a ) CCK-8 assays were performed to assess the viability of GC-2 cells transfected with various H3 constructs ( n = 6 for each group). ( b ) Colony formation assays were conducted to assess the proliferative ability of GC-2 cells transfected with various H3 constructs ( n = 3 for each group). Scale bars = 2 mm. ( c ) Quantitative analysis of the results in b . ( d ) Transwell assays were carried out to evaluate the migration of GC-2 cells transfected with the WT, K57R or K80R GFP-H3 plasmids ( n = 3 for each group). Scale bars = 100 µm. ( e ) Quantitative analysis of the results in d . ( f ) EdU incorporation assays were used to evaluate the proliferation of GC-2 cells transfected with the WT, K57R or K80R GFP-H3 plasmid ( n = 3 for each group). The EdU-positive cells are labeled in purple, and the cell nuclei are labeled in blue. Scale bars = 20 µm. ( g ) Quantitative analysis of the data in f . * P < 0.05; ** P < 0.01; *** P < 0.001. H3: histone H3; GC-2 cells: mouse spermatocyte-derived cell line; EV: negative control pcDNA3.1 empty vector; GFP-H3-WT: recombinant plasmid pEGFP-H3-WT; GFP-H3-K57R: recombinant plasmid pEGFP-H3-K57R; GFP-H3-K80R: recombinant plasmid pEGFP-H3-K80R; OD: optical density; CCK-8: Cell Counting Kit-8; EdU: 5-ethynyl-2’-deoxyuridine; DAPI: 4’,6-diamidino-2-phenylindole; WT: wild-type; GFP: green fluorescent protein.

Article Snippet: GC-2 spd cells, a cell line derived from mouse spermatocytes (GC-2), were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 ( v / v ).

Techniques: CCK-8 Assay, Transfection, Construct, Migration, Plasmid Preparation, Labeling, Derivative Assay, Negative Control, Recombinant, Cell Counting

Journal: Asian Journal of Andrology

Article Title: RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80

doi: 10.4103/aja202368

Figure Lengend Snippet: RNF187-mediated H3 ubiquitination at K57/80 increases transcriptional activity in GC-2 cells. ( a ) EU incorporation assays in GC-2 cells transfected with the Flag-RNF187 plasmid or the corresponding empty vector ( n = 3 for each group). The EU-positive cells are labeled in green, and the cell nuclei are labeled in blue. Scale bars = 50 µm. ( b ) Quantitative analysis of fluorescence intensity in a ( n = 100 cells for each group). ( c ) EU incorporation assays in GC-2 cells transfected with the WT, K57R, or K80R GFP-H3 plasmid ( n = 3 for each group). The EU-positive cells are labeled in green, and the cell nuclei are labeled in blue. Scale bars = 50 µm. ( d ) Quantitative analysis of fluorescence intensity in c ( n = 100 cells for each group). * P < 0.05; ** P < 0.01; *** P < 0.001. ( e ) Schematic diagram of a functional model showing the role of RNF187 in the GC-2 cell line. RNF187 ubiquitinates H3 at K57 and K80, thereby driving gene transcription and cell growth. RNF187: RING finger 187; H3: histone H3; GC-2 cells: mouse spermatocyte-derived cell line; Flag-RNF187: recombinant plasmid pcDNA3.1-Flag-RNF187; EV: negative control pcDNA3.1 empty vector; GFP-H3-WT: recombinant plasmid pEGFP-H3-WT; GFP-H3-K57R: recombinant plasmid pEGFP-H3-K57R; GFP-H3-K80R: recombinant plasmid pEGFP-H3-K80R; EU: 5-ethynyl uridine; DAPI: 4’,6-diamidino-2-phenylindole; GFP: green fluorescent protein.

Article Snippet: GC-2 spd cells, a cell line derived from mouse spermatocytes (GC-2), were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 ( v / v ).

Techniques: Ubiquitin Proteomics, Activity Assay, Transfection, Plasmid Preparation, Labeling, Fluorescence, Functional Assay, Derivative Assay, Recombinant, Negative Control

Journal: Human Reproduction (Oxford, England)

Article Title: Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate

doi: 10.1093/humrep/dey373

Figure Lengend Snippet: Germ cell differentiation in ectopic marmoset testis tissue xenotransplants at 4 months post-transplantation . Morphological identification of spermatogonia and spermatocytes by H/PAS and VIMENTIN, and confirmation by immunostaining for MAGE-A4 and BOLL expression. Transplants were negative for the post-meiotic marker CREM-1. Arrows indicate spermatocytes in the lumen of marmoset seminiferous tubules; arrowheads indicate spermatogonia at the basal membrane.

Article Snippet: Following another washing step, non-specific adhesion sites were blocked with 4% normal goat serum (Tebu-bio, Boechout, Belgium) diluted in PBS or with Cas-block (Life Technologies) (only for CREM-1 antibody) for 30 min. Next, primary antibodies against VIMENTIN (marmoset specific), MAGE-A4 (mouse monoclonal anti-melanoma-associated antigen 4; dilution 1:200; gift from Dr Spagnoli; marker for spermatogonia and primary spermatocytes, marmoset specific), BOLL (mouse monoclonal anti-BOLL; dilution 1:400; H00066037-M03; R&D system, Abingdon, UK; marker for spermatocytes (meiotic cells)), CREM-1 (rabbit polyclonal anti-CREM-1; dilution 1:400; sc-440; Santa-Cruz Biotechnology, Heidelberg, Germany; marker for round spermatids (post-meiotic cells)) and ACROSIN (rabbit polyclonal anti-ACROSIN; dilution 1:500; sc-67151; Santa-Cruz; marker for acrosome visualisation in round, elongating and elongated spermatids (post-meiotic cells), marmoset specific), were applied to the sections and incubated in a humidified chamber overnight at 4°C.

Techniques: Cell Differentiation, Transplantation Assay, Immunostaining, Expressing, Marker, Membrane