Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2004 Mar 15;18(6):673–686. doi: 10.1101/gad.1180204

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2004, Cold Spring Harbor Laboratory Press

PMC Copyright notice

Figure 4. — Knockdown of TopBP1 derepresses E2F1 transcriptional activity. (A) HEK293 cells were transfected with a Flag-tagged TopBP1-expressing plasmid, increasing amounts of pSUPER-siTopBP1 and pEGFP (left panel), or with either pSUPER or pSUPER-siTopBP1 (right panel). The expression of GFP protein in each transfectant serves as a control. The expression of endogenous TopBP1, E2F1, and PCNA was analyzed by immunoblotting. (B) E2F transcriptional activities were determined by p14^ARF promoter-driven luciferase assay in HEK293 cells transfected with E2F and TopBP1 siRNA expression plasmids. Cells were left untreated or treated with NCS for 3 h before analysis. (^*) p < 0.001 (t test) compared with their counterpart control groups. (C) The endogenous E2F1 activity was assayed in HEK293 cells transfected with TopBP1 siRNA expression plasmids. Cells were treated with NCS for different periods of time before analysis. The expression of endogenous TopBP1 and E2F1 was analyzed by immunoblotting. (^*) p < 0.001 (t test) compared with their counterpart control groups. (D) The endogenous E2F1 transcriptional activity was assayed in HEK293 cells transfected with the expression vectors of dominant-negative mutants of Brg1 and Brm. Cells were treated with NCS for different periods of time as indicated before analysis. The expression of E2F1, Brg1, and Brm in the whole-cell lysates was detected with their specific antibodies, respectively. (#) p < 0.05 (t test) compared with vector control; (^*) p < 0.01 (t test) compared with vector control. (E) E2F transcriptional activity assay as described in B was performed in T98G cells in the presence of TopBP1 siRNA. The expression of endogenous TopBP1 was analyzed by immunoblotting. (^*) p < 0.001 (t test) compared with their counterpart control groups. (F) E2F transcriptional activity assay as described in B was performed in NIH3T3 cells in the presence of mTopBP1 siRNA. The expression of endogenous TopBP1 was analyzed by immunoblotting. (^*) p < 0.001 (t test) compared with their counterpart control groups. (G) T98G cells were transfected with an empty vector or pSUPER-siTopBP1. Two days later, cells were either left untreated or treated with NCS (300 ng/mL) or adriamycin (1 μM) for 5 h before harvesting. RNA was then extracted and RT-PCR analysis was performed using primers specific for selected E2F1 target genes or GAPDH as indicated. Mock RT-PCR represents a control reaction without addition of RNA.

Figure 4. — Knockdown of TopBP1 derepresses E2F1 transcriptional activity. (A) HEK293 cells were transfected with a Flag-tagged TopBP1-expressing plasmid, increasing amounts of pSUPER-siTopBP1 and pEGFP (left panel), or with either pSUPER or pSUPER-siTopBP1 (right panel). The expression of GFP protein in each transfectant serves as a control. The expression of endogenous TopBP1, E2F1, and PCNA was analyzed by immunoblotting. (B) E2F transcriptional activities were determined by p14^ARF promoter-driven luciferase assay in HEK293 cells transfected with E2F and TopBP1 siRNA expression plasmids. Cells were left untreated or treated with NCS for 3 h before analysis. (^*) p < 0.001 (t test) compared with their counterpart control groups. (C) The endogenous E2F1 activity was assayed in HEK293 cells transfected with TopBP1 siRNA expression plasmids. Cells were treated with NCS for different periods of time before analysis. The expression of endogenous TopBP1 and E2F1 was analyzed by immunoblotting. (^*) p < 0.001 (t test) compared with their counterpart control groups. (D) The endogenous E2F1 transcriptional activity was assayed in HEK293 cells transfected with the expression vectors of dominant-negative mutants of Brg1 and Brm. Cells were treated with NCS for different periods of time as indicated before analysis. The expression of E2F1, Brg1, and Brm in the whole-cell lysates was detected with their specific antibodies, respectively. (#) p < 0.05 (t test) compared with vector control; (^*) p < 0.01 (t test) compared with vector control. (E) E2F transcriptional activity assay as described in B was performed in T98G cells in the presence of TopBP1 siRNA. The expression of endogenous TopBP1 was analyzed by immunoblotting. (^*) p < 0.001 (t test) compared with their counterpart control groups. (F) E2F transcriptional activity assay as described in B was performed in NIH3T3 cells in the presence of mTopBP1 siRNA. The expression of endogenous TopBP1 was analyzed by immunoblotting. (^*) p < 0.001 (t test) compared with their counterpart control groups. (G) T98G cells were transfected with an empty vector or pSUPER-siTopBP1. Two days later, cells were either left untreated or treated with NCS (300 ng/mL) or adriamycin (1 μM) for 5 h before harvesting. RNA was then extracted and RT-PCR analysis was performed using primers specific for selected E2F1 target genes or GAPDH as indicated. Mock RT-PCR represents a control reaction without addition of RNA.