Journal: International journal of oncology

Article Title: Atrazine promotes RM1 prostate cancer cell proliferation by activating STAT3 signaling.

doi: 10.3892/ijo.2016.3433

Figure Lengend Snippet: Figure 3. Atrazine accelerates the cell cycle in vitro. (A) PI staining and flow cytometry analysis. (B) Flow cytometry analysis comparing cell numbers between control and atrazine treated cells over a period of 48 h. Mean ± SEM of three independent experiments are presented. (C) qRT‑PCR analysis of p53, p21, cyclin D1 and cyclin B1 mRNA levels, **P<0.01. (D) Western blot analysis of P53, Cyclin D1 and Cyclin B1 protein amounts. (E) The P53, Cyclin D1 and Cyclin B1 protein levels from three independent experiments. Columns, mean; bars, SE (*P<0.05, **P<0.01 versus control).

Article Snippet: Anti-Grim-19, Stat3, MMP9, MMP2, VEGF, PCNA, P53, c-myc, Cyclin B1, Cyclin D1, Bcl-2, Bax, and Caspase-3 primary antibodies, as well as secondary antibodies were acquired from Proteintech Group (USA).

Techniques: In Vitro, Staining, Flow Cytometry, Control, Western Blot

Journal: International journal of oncology

Article Title: Atrazine promotes RM1 prostate cancer cell proliferation by activating STAT3 signaling.

doi: 10.3892/ijo.2016.3433

Figure Lengend Snippet: Figure 4. Atrazine accelerates the cell cycle in vivo. (A) qRT‑PCR analysis of p53, p21, cyclin D1 and cyclin B1 mRNA expression. (B) Western blot analysis of P53, P21, Cyclin D1 and Cyclin B1 protein amounts. (C) Quantitation of P53, P21, Cyclin D1 and Cyclin B1 protein levels from three independent experiments. Columns, mean; bars, SE (*P<0.05, **P<0.01 versus control).

Article Snippet: Anti-Grim-19, Stat3, MMP9, MMP2, VEGF, PCNA, P53, c-myc, Cyclin B1, Cyclin D1, Bcl-2, Bax, and Caspase-3 primary antibodies, as well as secondary antibodies were acquired from Proteintech Group (USA).

Techniques: In Vivo, Expressing, Western Blot, Quantitation Assay, Control

Journal: The Journal of biological chemistry

Article Title: Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation.

doi: 10.1074/jbc.M701847200

Figure Lengend Snippet: FIGURE 1. KLF4 interacts with and is acetylated by p300/CBP. A, KLF4 represses cyclin B1 and activates p21 expression in an LS174T doxycycline- inducible cell line. LS174T-Tet-On-KLF4 cells were treated with 1 g/ml doxy- cycline (Dox) for various time periods prior to harvest. Western blot was then performed on the cell lysate. B, RT-PCR for several KLF4 target genes in LS174T-Tet-On-KLF4 cells after treatment with doxycycline for 48 h. C, KLF4 interacts with the CH3 domain of p300. GST-p300 (CH3) protein was incu- bated with FLAG-tagged KLF4 overnight. After pulldown with GST beads, KLF4 was detected via Western blot with FLAG antibody. D, KLF4 is acetylated by p300/CBP. KLF4 and full-length p300 or CBP were overexpressed in 293T cells. After IP with FLAG antibody, a 55-kDa protein was detected via Western blot using an acetyl-lysine-specific (Ac-Lys) antibody. Western blots con- firmed similar levels of expression of KLF4 in each condition and the expres- sion of p300 and CBP where indicated. E, endogenous KLF4 is acetylated. Endogenous KLF4 was immunoprecipitated from HCT116 cells using KLF4 antibody and probed via Western blot with anti-KLF4 and anti-acetylated lysine antibody. Upper band is KLF4. Anti-KLF4 antibody alone (without cell lysate) and mouse IgG (with cell lysate) were included as negative controls.

Article Snippet: Then Western blot was performed using a p21Cip1/WAF1 (Cell Signaling, catalog number 4135), cyclin B1 (Cell Signaling, catalog number 4135), -actin (Sigma, catalog number A1978), or KLF4 antibody (14).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation.

doi: 10.1074/jbc.M701847200

Figure Lengend Snippet: FIGURE 3. Acetylation by p300 regulates KLF4-mediated transactivation. A, lysine mutant KLF4 is less effective at activating the IAP-luciferase reporter. Empty vector, wild-type, or lysine mutant KLF4 was co- transfected into 293T cells with IAP luciferase plasmid. After 48 h, luciferase activity was measured. Co-trans- fection of KLF4 with E1A, which also binds the CH3 domain of p300, sequesters p300, abolishes KLF4-mediated activation of the reporter. B, addition of p300 results in super-activation of the IAP reporter. Full-length p300 plasmid was co-transected into 293T cells, and luciferase activity measured as in A. C, lysine mutant KLF4 is still a strong repressor of the cyclin B1 luciferase reporter. Wild-type or lysine mutant KLF4 was co-transfected into 293T cells. After 48 h, luciferase activity was measured. D, addition of p300 had no effect on KLF4-mediated repression of the cyclin B1 gene. Same experiment as in C, but with the addition of full-length p300.

Article Snippet: Then Western blot was performed using a p21Cip1/WAF1 (Cell Signaling, catalog number 4135), cyclin B1 (Cell Signaling, catalog number 4135), -actin (Sigma, catalog number A1978), or KLF4 antibody (14).

Techniques: Mutagenesis, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Activation Assay

Journal: The Journal of biological chemistry

Article Title: Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation.

doi: 10.1074/jbc.M701847200

Figure Lengend Snippet: FIGURE5.KLF4 differentially modulates histone acetylation at the pro- moters of its target genes. A, KLF4 physically binds the proximal pro- moter of IAP and cyclin B1 genes and modulates local histone H4 acetyla- tion. LS174T-Tet-On-KLF4 cells were treated with doxycycline for 48 h, DNA was cross-linked and sonicated, and IP was performed using the labeled antibodies. Eluted DNA-protein complexes were decross-linked, and PCR was performed using primers targeting the proximal promoter of the labeled genes. KLF4 did not bind an unrelated gene, GAPDH. B, KLF4 recruits p300 to the IAP promoter but not the cyclin B1 promoter. The same experiment as in A was performed, but a p300-specific antibody was used instead. Dox, doxycycline.

Article Snippet: Then Western blot was performed using a p21Cip1/WAF1 (Cell Signaling, catalog number 4135), cyclin B1 (Cell Signaling, catalog number 4135), -actin (Sigma, catalog number A1978), or KLF4 antibody (14).

Techniques: Sonication, Labeling

Journal: The Journal of biological chemistry

Article Title: Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation.

doi: 10.1074/jbc.M701847200

Figure Lengend Snippet: FIGURE 6. KLF4 recruits HDAC3 to repress cyclin B1 expression. A, treatment with butyrate, a histone deacetylase inhibitor, results in increased alkaline phosphatase enzymatic activity. HT29 cells were treated with sodium butyrate for various time periods, and then cell lysate was incubated with p-nitro- phenyl phosphate, an artificial substrate of IAP that produces a yellow color when cleaved. Absorbance was then measured at 405 nm. B, inhibition of deacetylation produces a similar pattern of expression as KLF4. LS174T cells were treated with butyrate for various time periods, and Western blot was performed using labeled antibodies. Actin was included as a loading control. C, KLF4 synergizes with HDAC3 in repressing the cyclin B1 reporter. Empty vector (clear bars) or KLF4 (shaded bars) was co-transfected with the cyclin B1-luciferase reporter, along with various HDAC plasmids. Co-transfection of HDAC3 with KLF4 in synergistic repression of the reporter. No synergy was seen with other HDACs. D, KLF4 recruits HDAC3 to the cyclin B1 promoter. ChIP was performed as in Fig. 5, but using an HDAC3-specific antibody. Over- expression in KLF4 in LS174T cells resulted increased occupancy of HDAC3 on the cyclin B1 promoter. GAPDH was included as a negative control.

Article Snippet: Then Western blot was performed using a p21Cip1/WAF1 (Cell Signaling, catalog number 4135), cyclin B1 (Cell Signaling, catalog number 4135), -actin (Sigma, catalog number A1978), or KLF4 antibody (14).

Techniques: Expressing, Histone Deacetylase Assay, Activity Assay, Incubation, Inhibition, Western Blot, Labeling, Control, Plasmid Preparation, Transfection, Luciferase, Cotransfection, Over Expression, Negative Control