Journal: Cells

Article Title: Weighted Correlation Network Analysis Reveals CDK2 as a Regulator of a Ubiquitous Environmental Toxin-Induced Cell-Cycle Arrest

doi: 10.3390/cells9010143

Figure Lengend Snippet: E2F1 and E2F4 were downregulated by OTA exposure. ( A ) E2F1 and E2F4 were significantly downregulated following 100 nM OTA exposure according to RNA-sequencing data in both cell lines, while E2F7 was found to be upregulated only in HEK293-T cells. Due to the consistent results between the two cell lines, the expression of E2F1 and E2F4 only was verified by RT-qPCR ( B ) and Western blot ( C ) (mean ± SD, * p < 0.05, N = 3).

Article Snippet: CDKN1A/p21 (Cell Signaling Technology, cat. no. 2946, Frankfurt, Germany), CDK2 (Cell Signaling Technology, cat. no. 2546), and E2F4 (R&D, cat. no. AF5139) were detected using IRDye-coupled fluorescent secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) and an Odyssey imaging system from LI-COR Biosciences.

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: EGF-mediated inhibition of ubiquitin-specific peptidase 24 expression has a crucial role in tumorigenesis

doi: 10.1038/onc.2016.445

Figure Lengend Snippet: USP24 inhibits the G1–S transition by increasing the E2F4 level. ( A ) Total RNA was isolated from USP24-silenced A549 cells to investigate the CCNA2, E2F1 and GAPDH mRNA levels by reverse transcription–PCR (RT–PCR). ( B ) A549 cells with USP24 knockdown were collected for flow cytometry. After three independent experiments, the ratio of G0/G1 and S-phase was quantified. ( C ) The mRNA was isolated from U2OS cells with USP24 knockdown or E2F4 overexpression to investigate CCNA2, E2F1 and GAPDH mRNA expression with RT–PCR (a). The proteins was isolated from U2OS cells with USP24 knockdown or E2F4 overexpression to investigate USP24, E2F4 and actin protein expression by western blotting (b). The CCNA2, E2F1 and GAPDH mRNA levels were quantified after three independent experiments (c). ( D ) Cells with USP24 knockdown or with E2F4 overexpression were collected for the flow cytometry assay. The G1 and S-phase ratio was quantified after three independent experiments. ( E ) Cells with USP24 knockdown or with E2F4 overexpression were collected for the colony assay (a). The colony number was calculated after three independent experiments (b). ( F ) Total mRNA and proteins were collected from A549 cells with USP24 knockdown to study the USP24, E2F4 and GAPDH mRNA levels by RT–PCR and the USP24, E2F4 and actin protein levels by western blotting. The protein and mRNA levels were quantified after three independent experiments. ( G ) A549 (a) and U2OS (b) were collected for the immunoprecipitation assay with an anti-USP24 antibody. Immunoprecipitated (IP) samples were used for western blotting with antibodies against the indicated proteins. ( H ) A549 cells with USP24 kockdown were collected after cycloheximide treatment. The USP24, E2F4 and actin levels were studied by western blotting with antibodies against the indicated proteins. The E2F4 level was quantified for the statistical analysis after three independent experiments. ( I ) Samples were collected from A549 cells for immunoprecipitation with an anti-E2F4 antibody. IP samples were used to perform the in vitro deubiquitination assay with recombinant human USP24 (50 μg/ml) and then evaluate the USP24, E2F4 and ubiquitinated E2F4 levels with antibodies against the indicated proteins (a). The ubiquitinated E2F4 level was quantified for the statistical analysis after three independent experiments (b). A549 cells with USP24 knockdown were collected for IP with an anti-E2F4 antibody. The IP samples were used for western blotting with antibodies against the indicated proteins (c).

Article Snippet: Cell lysates with MG132 (5 μ m ) treatment were immunoprecipitated with Bax, p300, securin or E2F4 antibody for 4 h, and then incubated with protein A-Sepharose for 1 h. After washing, recombinant human USP24 (Origene, Rockville, MD, USA) was added to the substrates for 2 h at 37 °C in deubiquitination buffer (50 m m Tris (pH 8.0), 10 m m dithiothreitol and 5 μ m MG132).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Over Expression, Expressing, Western Blot, Colony Assay, Immunoprecipitation, In Vitro, Recombinant

Journal: Oncogene

Article Title: EGF-mediated inhibition of ubiquitin-specific peptidase 24 expression has a crucial role in tumorigenesis

doi: 10.1038/onc.2016.445

Figure Lengend Snippet: USP24 expression correlated with its substrates in clinical lung cancer samples. ( A – D ) USP24 expression correlated with its substrates, p300 ( A ), Bax ( B ), Securin ( C ) and E2F4 ( D ), in clinical lung cancer samples. ( E ) The USP24 level was examined in the specimens from normal lung tissue and lung cancer patients using immunohistochemistry with an anti-USP24 antibody (a) or IgG as an internal control (b). USP24 expression between lung cancer patients and the corresponding normal lung tissue (c).

Article Snippet: Cell lysates with MG132 (5 μ m ) treatment were immunoprecipitated with Bax, p300, securin or E2F4 antibody for 4 h, and then incubated with protein A-Sepharose for 1 h. After washing, recombinant human USP24 (Origene, Rockville, MD, USA) was added to the substrates for 2 h at 37 °C in deubiquitination buffer (50 m m Tris (pH 8.0), 10 m m dithiothreitol and 5 μ m MG132).

Techniques: Expressing, Immunohistochemistry