Journal: Cell Chemical Biology
Article Title: Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G 0 /G 1 Arrest by Inhibiting Deubiquitinase USP2a
doi: 10.1016/j.chembiol.2017.03.002
Figure Lengend Snippet: Impact of LCAHA on the Expression and Stability of Cyclin D1 (A) The expression of cyclin D1-encoding mRNA ( CCND1 ) was evaluated by RT-PCR. The cells were treated with DMSO or 10 μM LCAHA for 48 hr. (B and C) The cells were treated with DMSO or LCAHA for 48 hr, and cycloheximide (CHX) was added for the last 15–60 min, followed by western blot analysis of cyclin D1 expression. The graph in (C) presents densitometry analysis of the expression level of cyclin D1 (B) and shows mean ± SEM values from three independent experiments. The statistical significance was evaluated using a t test on mean t 1/2 values from these three experiments. (D and E) Western blot analysis of the expression of cyclin D1, phospho-Akt, and phospho-GSK-3β in HCT116 p53 wt cells after the treatment with 5 μM LCAHA for the indicated time periods. (D) Western blot images from the representative experiment. (E) Densitometry analysis of the expression level of cyclin D1, P-Akt(S473), and P-GSK-3β(S9) normalized to α-tubulin. The graphs show mean ± SD from three independent experiments. Statistical significance was evaluated using a t test: *p < 0.05, **p < 0.01. (F) HCT116 cells were treated with DMSO (marked with D), or 5 or 20 μM LCAHA for 48 hr, followed by western blot analysis of the expression of Aurora A and cyclin A1. The graphs present densitometry analysis of the proteins' expression and show mean ± SEM from three independent experiments. Statistical significance was evaluated using one-way ANOVA with Tukey's post hoc test: *p < 0.05, **p < 0.01.
Article Snippet: The following antibodies and dilutions were used: rabbit polyclonal anti-cyclin D1 (1:200, Santa Cruz Biotechnology (SCBt), cat. sc-753), mouse monoclonal anti-Cyclin D3 (1:1 000, Cell Signaling Technology (CST), cat. 2936), polyclonal anti-p53 (1:200, SCBt, cat. sc-6243), rabbit monoclonal anti-p21 (1:1 000, CST, cat. 2947), rabbit monoclonal anti-p27 (1:1 000, CST, cat. 3686), mouse monoclonal anti-Cyclin A2 (1:1 000, CST, cat. 4656), mouse monoclonal anti-Cyclin E1 (1:1 000, CST, cat. 4129), rabbit monoclonal anti-p-Akt(S473) (1:1 000, CST, cat. 4060), rabbit monoclonal anti-p-GSK-3β(S9) (1:1 000, CST, cat. 5558), mouse monoclonal anti-Aurora A (1:100, SCBt, cat. sc-373856), rabbit polyclonal anti-cyclin A1 (1:200, SCBt, cat. sc-7252), rabbit monoclonal anti-GAPDH (1:4 000, CST, cat. 2118), rabbit monoclonal anti-α-Tubulin (1:4 000, CST, cat. 2125), goat peroxidase-conjugated anti-rabbit (1:2 000, CST, cat. 7074), horse peroxidase-conjugated anti-mouse (1:2 000, Cell Signaling, cat. 7076).
Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot
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