Journal: Cell Death & Disease

Article Title: Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells

doi: 10.1038/cddis.2014.232

Figure Lengend Snippet: E2F3 in mouse SCs is developmentally controlled by the RB family. ( a ) Immunolocalization of E2F3 in SC (white arrows), preleptotene spermatocytes (black arrowheads), and spermatogonia (Spg, white arrowheads) of adult control and SC-RbKO mice. NC: negative control, secondary antibody only. Scale bar, 25 μ m. ( b ) mRNA levels of E2f3 in control and SC-RbKO mice testes at 6 and 10–12 weeks of age relative to housekeeping genes (L19 and Ppia) and normalized to the control group. ( c ) Both E2F3 isoforms a and b are expressed at high levels in 10–12-week-old control (WT) and KO testes. E2F3++TM4; E2F3-overexpressing TM4 mouse-SC line (positive control); liver, negative control. ( d ) Co-immunoprecipitation using E2F3 antibody from whole-testis lysate from PND10 and PND40 control and SC-RbKO mice. WB analysis of RB family members shows that E2F3 interacts with RB at PND10 in the wild-type testes. Following RB deletion, E2F3 forms complexes with both p107 and p130 in the PND40 SC-RbKO testes. NC: negative control is immunoprecipitation with the same E2F3 antibody in the presence of a specific blocking peptide (sc-878 P)

Article Snippet: The lysate was then incubated with 2 μ g of E2F3 C-18 antibody (sc-878, Santa Cruz) overnight at 4 °C.

Techniques: Control, Negative Control, Positive Control, Immunoprecipitation, Blocking Assay

Journal: Cell Death & Disease

Article Title: Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells

doi: 10.1038/cddis.2014.232

Figure Lengend Snippet: Partial rescue of spermatogenesis in SC-RbKO mouse testes following in vivo E2F3 silencing. ( a ) A significant recovery of testis weight following in vivo knockdown of E2F3ab in the 12-week-old SC-RbKO testes. Control: non-transfected WT testis, SC-RbKO: non-transfected mutant, pSuperEGFP: mutant transfected with empty pSUPER.gfp/neo plasmid, SiE2F3ab: mutant transfected with shRNA-E2F3ab plasmid. Values are mean±S.E.M. of n =4. ( b ) GFP expression introduced by the in viv o transfection was confirmed 10 weeks after injection by a squash preparation of seminiferous tubule segment under fluorescence microscopy containing transfected SCs (red arrowhead). ( c ) H&E staining revealed normal spermatogenesis in control mouse testes at the age of 12 weeks (upper left panel) and disorganized seminiferous tubules in testes from SC-RbKO (upper middle panel) and SC-RbKO-transfected with control plasmid (KO/pSuperEGFP) at the same age (upper right panel). Testicular sections of SC-RbKO mice transfected with shRNA-E2F3ab plasmid (lower panels) show elongated spermatids and a restored seminiferous tubule architecture. ( d ) Testis architecture, ectoplasmic specializations (espin: green, left panels) and tight junctions (claudin11: red, right panels) were restored in SC-RbKO after knockdown of E2F3 (Magnification × 400)

Article Snippet: The lysate was then incubated with 2 μ g of E2F3 C-18 antibody (sc-878, Santa Cruz) overnight at 4 °C.

Techniques: In Vivo, Knockdown, Control, Transfection, Mutagenesis, Plasmid Preparation, shRNA, Expressing, Injection, Fluorescence, Microscopy, Staining

Journal: Cell Death & Disease

Article Title: Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells

doi: 10.1038/cddis.2014.232

Figure Lengend Snippet: Hypothetical model of the RB family and E2F3 dynamics during mouse SC development. Wild-type: In the juvenile mouse SC, which are actively proliferating and have not yet formed an intact BTB, RB and E2F3 interact physically, to either activate or repress E2F3-mediated gene expression. In the terminally differentiated SCs, there is a tightly regulated balance of RB and E2F3 action. The RB family member p130 is expressed in wild-type SC in addition to RB. SC-RbKO: The juvenile SC proliferate normally and exhibit no apparent phenotype beyond altered gene expression profile in the absence of RB. Neither RB nor p130 interact with E2F3 in the juvenile SC-RbKO SC. The adult SC-RbKO SC gradually resume proliferation, become dedifferentiated, apoptotic, and fail to support spermatogenesis. Simultaneously, SC-RbKO SC assume an ectopic expression of the RB family member p107, and p107 and p130 both begin to interact with E2F3; possibly attempting to suppress a deregulated E2F3 action. Rescued SC-RbKO: E2F3 was knocked down in vivo using a shRNA in PND15 SC-RbKO testes to assess the contribution of E2F3 to the SC-RbKO phenotype. This resulted in partial rescue of the SC-RbKO phenotype with a patchy restoration of spermatogenesis and seminiferous tubule architecture

Article Snippet: The lysate was then incubated with 2 μ g of E2F3 C-18 antibody (sc-878, Santa Cruz) overnight at 4 °C.

Techniques: Gene Expression, Expressing, In Vivo, shRNA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDM2, MDMX, and p73 regulate cell-cycle progression in the absence of wild-type p53.

doi: 10.1073/pnas.2102420118

Figure Lengend Snippet: Fig. 3. MDM2 is required for maintaining E2F1 and E2F3a levels. H1299 cells were transfected with increasing amounts of MDM2 siRNA as stated in the figure (siRNA concentration was balanced up to 40 nM with siLuc in each sample) (A–C), with increasing concentrations of Nutlin-3 for 20 h (D and F), or with control or MDM2 siRNA (each at 15 nM) for 24 h (E) as indicated. (A) Protein levels of MDM2, E2F1, p21, and actin were measured by immunoblot. (B) E2F1 mRNA levels were quantitated by qRT-PCR analysis. (C and D) Following either MDM2 siRNA (C) or Nutlin-3 (D) treatment, the cells were then either mock treated or treated with MG132 (25 μM) for an additional 4 h. Protein levels were analyzed by immunoblot using antibodies against MDM2, E2F1, E2F2, E2F3, and actin. (E and F) mRNA levels of E2F3a or E2F3b following MDM2 siRNA (15 nM) treatment (E) or Nutlin-3 treatment (F) were quanti- fied by qRT-PCR. *P < 0.05 (one-way ANOVA), **P < 0.01 (one-way ANOVA); ns, not significant (one-way ANOVA). All data represent at least three bio- logical replicates (error bars represent SEM).

Article Snippet: Commercially obtained antibodies used in this study were as follows: pRB (IF-8) SC-102, p107 (C-18) SC-318, p130 (C-20) SC-317, E2F1 (KH95) SC-251, E2F2 (C-20) SC-318, E2F3 (C-18) SC-878, p21 (C-19) SC-397 (all SC are antibodies from Santa Cruz Biotechnology), p21 (12D1) (Cell Signaling Technology; 2947), p73 EP436Y (Abcam; ab40658), GAPDH 14C10 (Abcam; 2118), mouse HA-Tag (Cell Signaling Technology; C29F4), rabbit HA-Tag (BioLegend; 905102), actin (Sigma-Aldrich; A2066), and MDM2 DIV2Z (Cell Signaling Technology; 86934).

Techniques: Transfection, Concentration Assay, Control, Western Blot, Quantitative RT-PCR

Journal: Oncotarget

Article Title: The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1

doi: 10.18632/oncotarget.20765

Figure Lengend Snippet: (A) Oncoprint analysis of the 8 indicated genes using the cBioPortal program mining the TCGA database. Top panel: The analysis indicates the percentage of total alterations in 971 cases and the specific alteration (amplification, deep deletion, missense mutation, truncating mutation, and mRNA upregulation). Bottom panel: overexpression of mRNAs as determined by RNA seq. Kaplan-Meier graphs generated with KM Plotter displaying the relation between overexpression of the indicated proteins (red) and probability of overall (B) or relapse-free (C) survival relative to patients that do not overexpress the indicated proteins (black). Significance (log-rank P) for (B) is as follows: E2F1 was P = 1.6e-07, E2F2 was P = 0.92, E2F3 was P = 0.0036, E2F1 and E2F2 was P = 0.19, E2F1 and E2F3 was P = 5.1e-16, E2F2 and E2F3 was P = 0.47, E2F1 & E2F2 & E2F3 was P = 0.12, BubR1 was P = 1.6e-08, Hec1 was P = 4.8e-05, Nek2 was P = 1.3e-06, Mps1 was P = 4.8e-05, SgoI was P = 0. 2, and P = 0.036 for all genes. Log-rank P for (C) is as follows: E2F1 was P = 3.6e-13, E2F2 was P = 0.12, E2F3 was P = 8.9e-10, E2F1 and E2F2 was P = 0.042, E2F1 and E2F3 was P = 1e-13, E2F2 and E2F3 was P = 0.017, E2F1 and E2F2 and E2F3 was P = 0.0082, BubR1 was P < 1e-16, Hec1 was P < 1e-16, Nek2 was P < 1e-16, Mps1 was P < 1e-16, Sgo1 was P < 1.3e-05, and P < 5.2e-11 for all genes. (D) Kaplan-Meier curves generated in cBioPortal based on the METABRIC database displaying percentage of patient survival (y axis) indicated by months (x axis) that overexpress (red) E2F1, E2F2, and E2F3 versus patients who do not overexpress any of the genes (blue). P value for percent overall survival = 0.949, P value for relapse-free survival = 0.0250.

Article Snippet: The following primary antibodies were used in this experiment: E2F1 (3742, Cell Signaling), E2F2 (sc-633, Santa Cruz Biotechnology), E2F3 (sc-878, Santa Cruz Biotechnology), cyclin B1 (sc-245, Santa Cruz Biotechnology), Mad2 (ab70383, Abcam), Bcl2 (2870, Cell Signaling), pBcl2 (Ser70) (2827, Cell Signaling), pSer/Thr/Phe (9631, Cell Signaling), cleaved caspase 3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), Hec1 (GTX70268, GeneTex, Irvine, CA), pBad (Ser136) (4366, Cell Signaling), Bad (9268, Cell Signaling), Mps1/TTK (3255, Cell Signaling), PP2Ac (2259, Cell Signaling), and Sgo1 (ab58023, Abcam). β-actin antibody (4970, Cell Signaling) was used as a loading control.

Techniques: Amplification, Mutagenesis, Over Expression, RNA Sequencing, Generated

Journal: Oncotarget

Article Title: The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1

doi: 10.18632/oncotarget.20765

Figure Lengend Snippet: Co-occurrences between the indicated genes (C-BIOPORTAL/TCGA Analysis)

Article Snippet: The following primary antibodies were used in this experiment: E2F1 (3742, Cell Signaling), E2F2 (sc-633, Santa Cruz Biotechnology), E2F3 (sc-878, Santa Cruz Biotechnology), cyclin B1 (sc-245, Santa Cruz Biotechnology), Mad2 (ab70383, Abcam), Bcl2 (2870, Cell Signaling), pBcl2 (Ser70) (2827, Cell Signaling), pSer/Thr/Phe (9631, Cell Signaling), cleaved caspase 3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), Hec1 (GTX70268, GeneTex, Irvine, CA), pBad (Ser136) (4366, Cell Signaling), Bad (9268, Cell Signaling), Mps1/TTK (3255, Cell Signaling), PP2Ac (2259, Cell Signaling), and Sgo1 (ab58023, Abcam). β-actin antibody (4970, Cell Signaling) was used as a loading control.

Techniques:

Journal: Oncotarget

Article Title: The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1

doi: 10.18632/oncotarget.20765

Figure Lengend Snippet: Network that includes the 8 query genes (E2F1, E2F2, E2F3, BUBR1, TTK, NDC80, Nek2, Sgo1) and the 50 most frequently altered neighbor genes (out of 279)

Article Snippet: The following primary antibodies were used in this experiment: E2F1 (3742, Cell Signaling), E2F2 (sc-633, Santa Cruz Biotechnology), E2F3 (sc-878, Santa Cruz Biotechnology), cyclin B1 (sc-245, Santa Cruz Biotechnology), Mad2 (ab70383, Abcam), Bcl2 (2870, Cell Signaling), pBcl2 (Ser70) (2827, Cell Signaling), pSer/Thr/Phe (9631, Cell Signaling), cleaved caspase 3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), Hec1 (GTX70268, GeneTex, Irvine, CA), pBad (Ser136) (4366, Cell Signaling), Bad (9268, Cell Signaling), Mps1/TTK (3255, Cell Signaling), PP2Ac (2259, Cell Signaling), and Sgo1 (ab58023, Abcam). β-actin antibody (4970, Cell Signaling) was used as a loading control.

Techniques: Amplification, Mutagenesis, Ubiquitin Proteomics, Binding Assay, Migration, Negative Control

Journal: Oncotarget

Article Title: The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1

doi: 10.18632/oncotarget.20765

Figure Lengend Snippet: (A) Colony formation assay was done in MCF10A cells overexpressing E2F1, E2F2, and E2F3a transfected with control or siSgo1. (B) Colonies were quantified using Image J. (C) Nuclei were detected with DAPI, and the percentage of cells undergoing DNA fragmentation was calculated (D) (P≤0.05). (E) The BrdU/7-AAD assay was performed in MCF10A cells overexpressing E2F1, E2F2, and E2F3 transfected with control or siSgo1, and percentages of specific cell cycle phases were calculated (F) . (G) Annexin V staining was performed in MCF10A cells expressing E2F1, E2F2, and E2F3a with cells transfected with control or siSgo1, and percentages of Annexin V + /7-AAD + cells were calculated from three independent experiments (H) . (I) Western blots were performed with protein extracts from MCF10A cells expressing E2F1, E2F2, and E2F3a transfected with control or siSgo1 to measure levels of the indicated apoptotic markers.

Article Snippet: The following primary antibodies were used in this experiment: E2F1 (3742, Cell Signaling), E2F2 (sc-633, Santa Cruz Biotechnology), E2F3 (sc-878, Santa Cruz Biotechnology), cyclin B1 (sc-245, Santa Cruz Biotechnology), Mad2 (ab70383, Abcam), Bcl2 (2870, Cell Signaling), pBcl2 (Ser70) (2827, Cell Signaling), pSer/Thr/Phe (9631, Cell Signaling), cleaved caspase 3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), Hec1 (GTX70268, GeneTex, Irvine, CA), pBad (Ser136) (4366, Cell Signaling), Bad (9268, Cell Signaling), Mps1/TTK (3255, Cell Signaling), PP2Ac (2259, Cell Signaling), and Sgo1 (ab58023, Abcam). β-actin antibody (4970, Cell Signaling) was used as a loading control.

Techniques: Colony Assay, Transfection, Control, Staining, Expressing, Western Blot