Journal: Nucleic Acids Research

Article Title: Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

doi: 10.1093/nar/gkab019

Figure Lengend Snippet: K134 acetyl mimic expression reduces G1/S checkpoint resolution. ( A, C ) To determine which stage of the cell cycle LMNB1 K134 acetylation may affect in the presence of DSBs, cell cycle analysis was performed by flow cytometry of MRC5 cells stably expressing WT, K134Q or K134R mCherry-LMNB1 that were treated with bleomycin to induce DSBs. Representative contour plots are shown with each cell cycle stage indicated. ( B, D ) Average percentage of cells ± SEM in each phase of the cell cycle. ( B ) Left: G1 phase, middle: S phase, right: G2 phase ( D ) M phase. ( E ) Average percentage of cells ± SEM in (left) early S phase and (right) mid/late S phase, n = 3 biological replicates, one-way ANOVA, Bonferroni post-hoc test, average is indicated in each bar. ( F ) Representative western blot showing pRb (S780) phosphorylation levels in cells stably expressing WT, K134Q or K134R mCherry-LMNB1 across a time course of recovery from bleomycin treatment. ( G ) Proposed hypothesis that LMNB1 acetylation may affect progression through the G1/S checkpoint. As the G1/S checkpoint is regulated by assessing DNA damage, we propose that LMNB1 K134 acetylation triggers a stall in this checkpoint.

Article Snippet: The following primary antibodies were used: ICP27 (1:1000, Ms, abcam, ab31631), ICP8 (1:1000, Ms, abcam, ab20194), gC (1:1000, Ms, abcam, ab6509), pRb-S780 (1:1000, Rb, clone D59B7, Cell Signaling Technology, #8180), LMNB1 (1:1000, Rb, abcam, ab16048), RFP (reactive against variants of RFP, 1:1000, Rb, Rockland, #600-401-379) and tubulin (1:5000, Ms, clone DM1A, Sigma, #T6199).

Techniques: Expressing, Cell Cycle Assay, Flow Cytometry, Stable Transfection, Western Blot, Phospho-proteomics

Journal: Oncology Letters

Article Title: PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis

doi: 10.3892/ol.2014.1957

Figure Lengend Snippet: Cell cycle proteins are overexpressed in human colorectal carcinoma. (A) Western blotting of human colorectal carcinoma tissues (T) and matched adjacent normal colorectal tissues (N) (above the panel) of the expression of the cell cycle proteins (left of the panel). (B) Western blot analysis of the expression of these cell cycle proteins (left of the panel) in eight colorectal carcinoma cell lines (above the panel). The glioblastoma cell line LN229 was included as the positive control and β-actin (Actin) was used as protein loading control. CDK, cyclin-dependent kinases; pRB, phosphorylated retinoblastoma protein.

Article Snippet: Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Positive Control, Control

Journal: Oncology Letters

Article Title: PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis

doi: 10.3892/ol.2014.1957

Figure Lengend Snippet: PD-0332991 blocks RB phosphorylation in colorectal carcinoma cells. (A) The DLD-1 and (B) COLO320 cells were treated at various concentrations of PD-0332991 as indicated (top of the panel) for 48 h. The total proteins were extracted from these treated and untreated cells (controls) and subjected to western blotting using the antibodies as indicated (left of panel). CDK, cyclin-dependent kinases; pRB, phosphorylated retinoblastoma protein.

Article Snippet: Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: Oncology Letters

Article Title: PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis

doi: 10.3892/ol.2014.1957

Figure Lengend Snippet: CDK6 phosphorylates RB for cancer cell growth. (A) COLO320 cells were transduced with lentiviral vectors encoding CDK4 shRNA sequences (CDK4-20, CDK4-64) and (B) CDK6 shRNA sequences (CDK6-893, CDK6-747) and were examined by western blotting using the antibodies as indicated (left of panel). The scrambled shRNA-encoded vector was used as the control (CONT) in the two experiments. (C) The cells transduced with CDK4-shRNA and (D) CDK6-shRNA vector were examined by a cell proliferation assay. The data show the mean ± standard deviation of three independent experiments. Student’s t-test was used to analyze the data. ** P<0.001. CDK, cyclin-dependent kinases; pRB, phosphorylated retinoblastoma protein.

Article Snippet: Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Techniques: Transduction, shRNA, Western Blot, Plasmid Preparation, Control, Proliferation Assay, Standard Deviation