Journal: mBio

Article Title: STAT-5 Regulates Transcription of the Topoisomerase IIβ-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote Human Papillomavirus Replication

doi: 10.1128/mBio.02006-15

Figure Lengend Snippet: Activation of ATR pathway in HPV-positive cells. (A) Western blot analysis of ATR, p-ATR, CHK1, p-CHK1, and GAPDH levels in HFK, HFK-16, HFK-18, and HFK-31 cells grown in monolayer cultures. (B) Western blot analysis for ATR, p-ATR, CHK1, p-CHK1 (S296) (p-CHK1), p-CHK1 (S345), involucrin, and GAPDH in HFK, HFK-31, and HPV31-positive CIN612 cells differentiated in high-calcium medium for the indicated times (in hours). (C) Sections of organotypic raft cultures generated from HFKs (top) and CIN612 (bottom) were stained with hematoxylin and eosin (H&E), p-ATR, and CHK1, as well as 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. (D) Western blot analysis for ATR, p-ATR, CHK1, p-CHK1, and GAPDH levels in HFK cells expressing LXSN vector, HPV31E6, HPV31E7, and HPV31E6E7. All results are representative of observations from three independent experiments.

Article Snippet: The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA).

Techniques: Activation Assay, Western Blot, Generated, Staining, Expressing, Plasmid Preparation

Journal: mBio

Article Title: STAT-5 Regulates Transcription of the Topoisomerase IIβ-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote Human Papillomavirus Replication

doi: 10.1128/mBio.02006-15

Figure Lengend Snippet: Suppression of ATR phosphorylation by VE822 blocks HPV genome amplification upon keratinocyte differentiation. (A) Western blot analysis of ATR, p-ATR, ATM, p-ATM, and GAPDH levels in CIN612 cells treated with VE822 at various concentrations for 48 h. (B) Western blot analysis of Bcl-XL, PARP, and GAPDH levels in CIN612 cells treated with VE822 (5 µM) for the indicated times. (C) Western blot analysis of ATR, p-ATR, CHK1, p-CHK1, and GAPDH proteins in differentiated CIN612 cells in the presence or absence of VE822 (5 µM) in high-calcium medium for the indicated times. (D) Southern blot analysis for HPV31 episomes in monolayer cultures of CIN612 cells untreated or treated with VE822 (5 µM) for the indicated times. Band densities, as determined by ImageJ software, are shown underneath. (E) Southern blot analysis for HPV31 episomes in CIN612 cells that were untreated or treated with VE822 (5 µM) following differentiation in high-calcium medium for indicated times (in hours). All results are representative of observations from three independent experiments.

Article Snippet: The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA).

Techniques: Amplification, Western Blot, Southern Blot, Software

Journal: mBio

Article Title: STAT-5 Regulates Transcription of the Topoisomerase IIβ-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote Human Papillomavirus Replication

doi: 10.1128/mBio.02006-15

Figure Lengend Snippet: Suppression of CHK1 phosphorylation by UCN01 blocks HPV genome amplification upon keratinocyte differentiation. (A) Western blot analysis of p-CHK1, CHK1, p-CHK2, CHK2, p-ATM, ATM, and GAPDH levels in CIN612 cells treated with UCN01 (100 nM) or CHK2i (5 µM) in high-calcium medium for the indicated times (in hours). (B) Southern blot analysis for HPV31 episomes in monolayer cultures of CIN612 cells that were untreated or treated with UCN01 (100 nM) for the indicated times. (C) Southern blot analysis for HPV31 episomes in CIN612 cells that were untreated or treated with UCN01 (100 nM) or CHK2i (5 µM) following differentiation in high-calcium medium for the indicated times (in hours). Band densities determined by ImageJ software are shown below. (D) Northern blot analysis for HPV31 early and late gene expression in CIN612 cells in the presence or absence of UCN01 (100 nM) following differentiation in high-calcium medium for the indicated times (in hours). (E) Western blot analysis of p-AKT, AKT, CDK4, CDK6, and GAPDH levels in CIN612 cells treated with UCN (100 nM) and CHK2i (5 µM) for 48 h. All results are representative of observations from two or more independent experiments.

Article Snippet: The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA).

Techniques: Amplification, Western Blot, Southern Blot, Software, Northern Blot, Expressing

Journal: mBio

Article Title: STAT-5 Regulates Transcription of the Topoisomerase IIβ-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote Human Papillomavirus Replication

doi: 10.1128/mBio.02006-15

Figure Lengend Snippet: Knockdown of STAT-5 suppresses ATR DNA damage responses. (A) Western blot analysis of ATR, p-ATR, CHK1, p-CHK1, and GAPDH levels in CIN612 cells treated with pimozide (10 µM) in high-calcium medium for the indicated times (in hours). (B) Western blot analysis of STAT-5, ATR, p-ATR, CHK1, p-CHK1, and GAPDH protein levels in shRNA control and shRNA lentivirus-infected CIN612 cells upon differentiation in high-calcium medium for 72 h. All results are representative of observations from two or more independent experiments.

Article Snippet: The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA).

Techniques: Western Blot, shRNA, Infection

Journal: mBio

Article Title: STAT-5 Regulates Transcription of the Topoisomerase IIβ-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote Human Papillomavirus Replication

doi: 10.1128/mBio.02006-15

Figure Lengend Snippet: TopBP1 knockdown results in a loss of differentiation-dependent HPV genome amplification. (A) Western blot analysis of TopBP1 and GAPDH proteins in CIN612 cell monolayers infected with multiple shRNA lentiviruses targeting TopBP1 for the indicated times. “Scram” refers to the scrambled vector controls for TopBP1. (B) Western blot analysis of TopBP1, p-ATR, ATR, p-CHK1, CHK1, and GAPDH proteins following differentiation of CIN612 cells that had been infected with combined shRNA lentiviruses targeting TopBP1 for the indicated times. (C) Southern blot analysis of HPV31 genomes in CIN612 cells following infection with pooled TopBP1 shRNA lentiviruses and differentiation in high-calcium medium for indicated times. All results are representative of observations from two or more independent experiments.

Article Snippet: The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA).

Techniques: Amplification, Western Blot, Infection, shRNA, Plasmid Preparation, Southern Blot