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: FEBS Open Bio

Article Title: Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon

doi: 10.1002/2211-5463.13001

Figure Lengend Snippet: Enriched GO terms related to cell‐cycle control for the genes down‐regulated by MTA.

Article Snippet: The membrane was then sequentially incubated with the following solutions at room temperature: 5% skim milk in PBS containing 0.1% Tween 20 for 1 h; the primary antibodies against human CCNE2 (1 : 2000, RRID: AB_1847388; Merck KGaA), human CDC25A (1 : 100, RRID: AB_627226; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or chicken actin (1 : 2000, RRID: AB_63314; Life Technologies) for 2 h; and the horseradish peroxidase‐conjugated secondary antibodies (1 : 1500–2500) against anti‐chicken IgY for CCNE2 detection (RRID: AB_228385; Life Technologies), and anti‐mouse IgG for CDC25A and actin detection (RRID: AB_2687537; SeraCare Life Sciences, Inc., Milford, MA, USA) for 1 h. The immunoreactive CCNE2 protein was visualized on X‐ray film using the enhanced chemiluminescence detection system (GE Healthcare).

Techniques: Control

Journal: FEBS Open Bio

Article Title: Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon

doi: 10.1002/2211-5463.13001

Figure Lengend Snippet: MTA down‐regulated genes related to cell‐cycle control.

Article Snippet: The membrane was then sequentially incubated with the following solutions at room temperature: 5% skim milk in PBS containing 0.1% Tween 20 for 1 h; the primary antibodies against human CCNE2 (1 : 2000, RRID: AB_1847388; Merck KGaA), human CDC25A (1 : 100, RRID: AB_627226; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or chicken actin (1 : 2000, RRID: AB_63314; Life Technologies) for 2 h; and the horseradish peroxidase‐conjugated secondary antibodies (1 : 1500–2500) against anti‐chicken IgY for CCNE2 detection (RRID: AB_228385; Life Technologies), and anti‐mouse IgG for CDC25A and actin detection (RRID: AB_2687537; SeraCare Life Sciences, Inc., Milford, MA, USA) for 1 h. The immunoreactive CCNE2 protein was visualized on X‐ray film using the enhanced chemiluminescence detection system (GE Healthcare).

Techniques: Control

Journal: FEBS Open Bio

Article Title: Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon

doi: 10.1002/2211-5463.13001

Figure Lengend Snippet: RT‐PCR verification of the MTA‐mediated down‐regulation of CCNE2 and CDC25A . RT‐PCR was performed to confirm the reduction in CCNE2 and CDC25A mRNA expression that was screened in the microarray analysis. After MTA treatment for the indicated hours, the total RNA was subjected to RT‐PCR. The electrophoretically separated PCR bands were stained with ethidium bromide, and representative gel images (A) of three replicates are shown. The numbers under the photographs show the fluorescence intensity of each band relative to an initial value (= 1.00), which was calculated after normalization against GAPDH as a reference. The bar graphs show the fluctuations of the CCNE2 (B) and CDC25A (C) expression levels obtained independently by the RT‐PCR and microarray analyses. The relative mRNA levels determined by RT‐PCR represent mean ± SD of three experiments. Values with different letters are significantly different by one‐way ANOVA followed by Tukey–Kramer HSD test ( P < 0.01). The characters in parentheses indicate the probe set ID of the microarray.

Article Snippet: The membrane was then sequentially incubated with the following solutions at room temperature: 5% skim milk in PBS containing 0.1% Tween 20 for 1 h; the primary antibodies against human CCNE2 (1 : 2000, RRID: AB_1847388; Merck KGaA), human CDC25A (1 : 100, RRID: AB_627226; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or chicken actin (1 : 2000, RRID: AB_63314; Life Technologies) for 2 h; and the horseradish peroxidase‐conjugated secondary antibodies (1 : 1500–2500) against anti‐chicken IgY for CCNE2 detection (RRID: AB_228385; Life Technologies), and anti‐mouse IgG for CDC25A and actin detection (RRID: AB_2687537; SeraCare Life Sciences, Inc., Milford, MA, USA) for 1 h. The immunoreactive CCNE2 protein was visualized on X‐ray film using the enhanced chemiluminescence detection system (GE Healthcare).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Microarray, Staining, Fluorescence

Journal: FEBS Open Bio

Article Title: Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon

doi: 10.1002/2211-5463.13001

Figure Lengend Snippet: MTA‐induced reduction of the CCNE2 and CDC25A proteins in RCM‐1 cells. Following MTA treatment for the indicated hours, proteins extracted from the cells were applied to immunoblotting with the respective amounts of 3.75 µg (per each lane) for detecting CCNE2 (A) and 30 µg for CDC25A (B). The photographs show the immunologically detected proteins together with actin as a reference.

Article Snippet: The membrane was then sequentially incubated with the following solutions at room temperature: 5% skim milk in PBS containing 0.1% Tween 20 for 1 h; the primary antibodies against human CCNE2 (1 : 2000, RRID: AB_1847388; Merck KGaA), human CDC25A (1 : 100, RRID: AB_627226; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or chicken actin (1 : 2000, RRID: AB_63314; Life Technologies) for 2 h; and the horseradish peroxidase‐conjugated secondary antibodies (1 : 1500–2500) against anti‐chicken IgY for CCNE2 detection (RRID: AB_228385; Life Technologies), and anti‐mouse IgG for CDC25A and actin detection (RRID: AB_2687537; SeraCare Life Sciences, Inc., Milford, MA, USA) for 1 h. The immunoreactive CCNE2 protein was visualized on X‐ray film using the enhanced chemiluminescence detection system (GE Healthcare).

Techniques: Western Blot

Journal: FEBS Open Bio

Article Title: Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon

doi: 10.1002/2211-5463.13001

Figure Lengend Snippet: CCNE2 accumulation in various mammalian cells. Immunoblotting was performed to examine CCNE2 accumulation in different nontransformed and transformed mammalian cell lines. The following cell lines were used: WB‐F344, WB‐F344 rat liver epithelial cells; WB‐Ha‐ras, v‐Ha‐ras‐transformed WB; WB‐neu, neu‐transformed WB; WBsrc‐neo2, v‐src‐transformed WB; WB‐myc/ras, myc/ras‐transformed WB; Ehrlich, Ehrlich ascite tumor cells; HPD1NR, transplantable pancreatic ductal cancer cells; HaPT1, hamster pancreatic cancer cells; MSU‐2, human foreskin fibroblast cells; KNC, human well‐differentiated colon cancer cells; RCM‐1, human well‐differentiated rectal cancer cells; CoCM‐1, human moderately differentiated colon cancer cells; HCT116, human poorly differentiated colon cancer cells; BxPC3 and Ming, human pancreatic cancer cells.

Article Snippet: The membrane was then sequentially incubated with the following solutions at room temperature: 5% skim milk in PBS containing 0.1% Tween 20 for 1 h; the primary antibodies against human CCNE2 (1 : 2000, RRID: AB_1847388; Merck KGaA), human CDC25A (1 : 100, RRID: AB_627226; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or chicken actin (1 : 2000, RRID: AB_63314; Life Technologies) for 2 h; and the horseradish peroxidase‐conjugated secondary antibodies (1 : 1500–2500) against anti‐chicken IgY for CCNE2 detection (RRID: AB_228385; Life Technologies), and anti‐mouse IgG for CDC25A and actin detection (RRID: AB_2687537; SeraCare Life Sciences, Inc., Milford, MA, USA) for 1 h. The immunoreactive CCNE2 protein was visualized on X‐ray film using the enhanced chemiluminescence detection system (GE Healthcare).

Techniques: Western Blot, Transformation Assay

Journal: Cancer discovery

Article Title: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

doi: 10.1158/2159-8290.CD-19-1390

Figure Lengend Snippet: Clinical vignettes including treatment sequence, timing of metastatic progression, and available biopsies with key genomic findings are provided for the following - (a) four patients with biallelic alterations in RB1, including a patient with multiple biopsies and convergent evolution toward RB1 disruption (top, phylogenetic analysis for this patient is provided in Figure 2A). (b) Three patients with acquired alterations in AKT1 following progression on CDK4/6i. In the first (top), a new mutation in AKT1 W80R was identified. In the second (middle), a baseline alteration (AKT1 L52H) was identified at the time of diagnosis; at the time of progression on CDK4/6i, two biopsies were obtained – both demonstrating the baseline AKT1 L52H mutation, one also demonstrating an acquired amplification of the wild-type AKT1 protein (phylogenetic analyses for these patients are provided in Figure 2B and ​andD).D). (c) Three patients with resistance to CDK4/6i and RAS-family alterations (including two instances of KRAS G12D and one instance of HRAS mutation). (d) Three patients with intrinsic resistance to CDK4/6i and amplification events in CCNE2.

Article Snippet: AKT1 (BRDN0000464992), KRASG12D (BRDN0000553331), AURKA (TRCN0000492002), CCNE2 (ccsbBroadEn_11340), and GFP bacterial streaks were obtained from the Genetic Perturbation Platform, Broad Institute, MA.

Techniques: Sequencing, Disruption, Mutagenesis, Biomarker Discovery, Amplification

Journal: Cancer discovery

Article Title: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

doi: 10.1158/2159-8290.CD-19-1390

Figure Lengend Snippet: (a) T47D cells were modified via CRISPR-mediated downregulation (RB1) or lentiviral overexpression (AKT1, KRAS G12D, AURKA, CCNE2) to interrogate potential resistance mediators identified in patient biopsy samples. Western blotting with the indicated antibodies is included. (b-f) Modified T47D cells were exposed to escalating doses of CDK4/6i (palbociclib – left, abemaciclib – right) and viability was estimated via cell-titer-glo (CTG) assay. Control (CRISPR non-targeting guide or GFP) cells are plotted along with the resistance driver of interest (RB1 – b, AKT1 – c, KRAS G12D – d, AURKA – e, CCNE2 – f). Parental and variant cell lines are normalized to vehicle control and viability is plotted as a function of increasing CDK4/6i (graphed as triplicate average +/− standard deviation). All variants provoke CDK4/6i resistance (to both palbociclib and abemaciclib) in vitro in T47D cells. Corresponding IC50 values are included in Supplemental Table 7.

Article Snippet: AKT1 (BRDN0000464992), KRASG12D (BRDN0000553331), AURKA (TRCN0000492002), CCNE2 (ccsbBroadEn_11340), and GFP bacterial streaks were obtained from the Genetic Perturbation Platform, Broad Institute, MA.

Techniques: Modification, CRISPR, Over Expression, Western Blot, CTG Assay, Control, Variant Assay, Standard Deviation, In Vitro

Journal: Cancer discovery

Article Title: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

doi: 10.1158/2159-8290.CD-19-1390

Figure Lengend Snippet: (a) Breast cancer cell lines (T47D, MCF7, MDA-MB-361) were cultured long-term to resistance in the presence of CDK4/6i (palbociclib, abemaciclib). The resulting cell lines which emerged were subjected to western blotting for putative mediators of drug resistance (RB1, AKT1, KRAS/ERK, AURKA, and CCNE2). (b-c) T47D cells cultured to resistance in the presence of abemaciclib demonstrated low levels of RB1 expression (T47D-AR1) and increased sensitivity to the AURKA inhibitor LY3295668. MDA-MB-361 cells cultured to resistance in the presence of abemaciclib demonstrated high levels of ERK activation (361-AR1) and increased sensitivity to the ERK inhibitor LY3214996. MDA-MB-361 cells cultured to resistance in the presence of palbociclib demonstrated high levels of AURKA (361-PR1) and increased sensitivity to the AURKA inhibitor LY3295668. MCF7 cells cultures to resistance in the presence of palbociclib demonstrated increased levels of CCNE2 (MCF7-PR1) and increased sensitivity to the CHEK1 inhibitor prexasertib.

Article Snippet: AKT1 (BRDN0000464992), KRASG12D (BRDN0000553331), AURKA (TRCN0000492002), CCNE2 (ccsbBroadEn_11340), and GFP bacterial streaks were obtained from the Genetic Perturbation Platform, Broad Institute, MA.

Techniques: Cell Culture, Western Blot, Expressing, Activation Assay

Journal: Cancer discovery

Article Title: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

doi: 10.1158/2159-8290.CD-19-1390

Figure Lengend Snippet: (a) A patient with locally advanced HR+/HER2- breast cancer developed metastatic recurrence on adjuvant tamoxifen. She received CDK4/6i and letrozole in the first line setting with prolonged clinical benefit (>3 years). At progression, she was placed on trial with the AURKA inhibitor LY3295668; she subsequently experienced prolonged disease control ~11 months. Baseline staging studies following progression on CDK4/6i in the patient described are included (top); she had osseous metastatic disease and visceral disease limited to the foci noted in the liver. Two interval restaging studies (top) demonstrate disease stability/mild response. Liver biopsy obtained at the time of progression on CDK4/6i and prior to LY3295668 demonstrated high Ki67 and high RB1 protein expression via immunohistochemistry (IHC, 10x) (bottom). (b) Schematic diagram demonstrating the potential utility of next-generation sequencing following progression on CDK4/6i; actionable alterations in RB1, ERBB2, FGFR2, AKT1, RAS, AURKA, and CCNE2 could dictate informed selection of targeted therapies as indicated.

Article Snippet: AKT1 (BRDN0000464992), KRASG12D (BRDN0000553331), AURKA (TRCN0000492002), CCNE2 (ccsbBroadEn_11340), and GFP bacterial streaks were obtained from the Genetic Perturbation Platform, Broad Institute, MA.

Techniques: Adjuvant, Control, Expressing, Immunohistochemistry, Next-Generation Sequencing, Selection