Journal: Molecules and Cells

Article Title: TSPAN12 Precedes Tumor Proliferation by Cell Cycle Control in Ovarian Cancer

doi: 10.14348/molcells.2019.0015

Figure Lengend Snippet: (A–D) Stable A2780, SKOV3 cells with TSPAN12 silencing were analyzed by western blot analysis of Cyclin A2, Cyclin E2, Cyclin D1, CDK2, and CDK4. (E-H) OVCAR3 and SKOV3 cells with TSPAN12 overexpression were analyzed by western blot analysis after being treated with or without CDK inhibitor, AT7519 (Inh, 100 nM) for 72 h. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data are presented as the mean ± standard deviation (SD) of three independent experiments.

Article Snippet: Blots were incubated with antibodies against TSPAN12 (1:400, anti-rabbit, Av46887; Sigma), Cyclin D1, Cyclin E2, CDK2, CDK4 (1:400, anti-rabbit, Cell Cycle Antibody Sampler kit #9932, #9870; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000, anti-mouse, 60004-1; Proteintech, USA).

Techniques: Western Blot, Over Expression, Standard Deviation

Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: Ecotoxicology and environmental safety

Article Title: The influence of phenolic environmental estrogen on the transcriptome of uterine leiomyoma cells: A whole transcriptome profiling-based analysis.

doi: 10.1016/j.ecoenv.2021.111945

Figure Lengend Snippet: Fig. 4. The effects of 10.0 μmol/L BPA, 32.0 μmol/L NP on genes and proteins expression in uterine leiomyoma cells. (a) Hierarchically clustered heatmap of differentially expressed genes associated with cell cycle after BPA and NP exposure. (b) E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression by q-PCR presented as relative changes. There were increased E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression in uterine leiomyoma cells treated with10.0 μmol/L BPA, 32.0 μmol/L NP for 48 h compared to control group (*P < 0.05). (c) Representative western blots and (d) band intensity bar graphs of proteins. There were significantly (*P < 0.05) higher expression levels of E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 in the cells treated with BPA and NP for 48 h compared to controls. The western band images shown are representative of three independent experiments and the data were expressed as mean ± SE done in three independent experiments.

Article Snippet: The following primary antibodies were used for the western blotting: E2F1 Monoclonal Antibody (66515-1-Ig; Proteintech; diluted 1:1000); Cyclin D1 Monoclonal Antibody (60186-1-Ig; Proteintech; diluted 1:5000); Cyclin E2 Polyclonal Antibody (11935-1-AP; Proteintech; diluted 1:500); MCM2 Polyclonal Antibody (10513-1-AP; Proteintech; diluted 1:1500); MCM3 Polyclonal Antibody (15597-1-AP; Proteintech; diluted 1:1000); MCM4 Polyclonal Antibody (13043-1-AP; Proteintech; diluted 1:600); MCM5 Polyclonal Antibody (11703-1-AP; Proteintech; diluted 1:1000); MCM6 Polyclonal Antibody (13347-2-AP; Proteintech; diluted 1:8000); phospho-Rb monoclonal (ab184796; abcam; diluted 1:1000); phospho-PI3K polyclonal (ab182651; abcam; diluted 1:500); phospho- AKT polyclonal (ab38449; abcam; diluted 1:500).

Techniques: Expressing, Control, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Graphene Quantum Dot Sensitized Heterojunctions Induce Tumor-Specific Cuproptosis to Boost Sonodynamic and Chemodynamic Enhanced Cancer Immunotherapy.

doi: 10.1002/advs.202410606

Figure Lengend Snippet: Figure 6. a) CRT levels in 4T1 cells were measured following various treatments. b) Following different treatments, Western blot analysis was used to examine the presence of DLAT and DLAT oligomers in 4T1 cells. c,d) By utilizing flow cytometry, we determined the expression of CD80 and CD86 in DCs. e) WB analysis of FDX1 and LIAS in 4T1 cells after different treatments. f) Bio-TEM images of 4T1 cells before and after GQD/Cu2O + US treatment. Statistical significance between the experimental group and the control group is calculated with a two-tailed Student’s t-test. Data are presented as the mean ± SD. (n = 3). *p < 0.05 and ***p < 0.001.

Article Snippet: After incubation with antibodies to DLAT (1:20 000, proteintech), LIAS (1:1000, Abcam), FDX1 (1:1000, Abcam), γ-H2AX (Abcam), or α- tubulin (1:1000, Abcam), the cell lysates were analyzed and detected with a chemiluminescent imaging system.

Techniques: Western Blot, Cytometry, Expressing, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: Maintenance of Long-Range DNA Interactions after Inhibition of Ongoing RNA Polymerase II Transcription

doi: 10.1371/journal.pone.0001661

Figure Lengend Snippet: Binding of (A) EKLF, (B) GATA-1, (C) NF-E2 and (D) CBP at β-globin regulatory elements. Blue bars depict untreated fetal liver samples, red bars DRB treated fetal liver samples and green bars α-amanitin treated fetal liver samples. Enrichment is relative to amylase. Error bars indicate standard error of mean.

Article Snippet: Antibodies used: RNAPII (N-20; sc-899), NF-E2 (C-19; sc-291), GATA-1 (N6; sc-265) and CBP (A22; sc-369) from Santa Cruz Biotechnology, Ac-H3 (#06-599) and anti-tri-methyl Histone H3 K4 (#07-473) from Upstate, PanH3 (#ab1791) and anti-di-methyl Histone H3 K9/K27 (#ab7312) from Abcam.

Techniques: Binding Assay