Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Cell cycle analysis of VP40 clones. 293T, V2CL, and V2CH cells were seeded in a 96-well plate at 5 × 10 5 cells in 100 μL of fresh media. Cells were blocked at G 0 (starvation; 0.1% fetal bovine serum DMEM), G 1 /S (20 mM of hydroxyurea), and G 2 /M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour, and subsequent treatment with 50 ng/mL of nocodazole) for 2 days. Blocked cells were then imaged (A) and assayed for cell viability with CellTiter-Glo (B). The same experiment was repeated; however, cells were allowed to incubate for 5 days after blocking. Cells were subsequently imaged (C) and assayed for cell viability via CellTiter-Glo (D). Statistical analysis by Student’s 2-tailed t test compares V2CL and V2CH cell cycle-blocked groups with corresponding 293T groups (†, P < .05; ††, P < .01; †††, P < .001). Additional Student’s 2-tailed t test compares cell cycle-blocked groups with controls of their own cell type (**, P < .01; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Cell Cycle Assay, Clone Assay, Blocking Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Effect of nuclear VP40 on cyclin and cdk regulation and activity. (A) Log phase 293T, V2CL, V2CI, and V2CH cells were harvested, lysed, and subjected to SDS/PAGE for western blot analysis of cyclin D1 (CycD1), cyclin E (CycE), cyclin A (CycA), cyclin B1 (CycB1), cdk4, cdk6, cdk2, cdk1, and actin levels. (B) Five hundred micrograms of 293T or V2CH whole-cell extracts were used for IP with 10 µg of either normal rabbit immunoglobulin G (IgG) or α-CycD1. IPed material was incubated with 50 µL 30% of Protein A/G for 2 hours, followed by two 1× PBS washes and 1 kinase buffer wash. Pellets were resuspended in kinase buffer, and 15 µL samples were incubated with 50 µg of either no peptide, 780S, or 780A peptide, along with 2 µL of [γ- 32 P] ATP. Some samples were also treated with Fascaplysin ([Fascap.] 1 µM). All samples incubated for 1 hour, followed by dotting onto Whatman glass microfiber filters and drying for 30 minutes. Filters were incubated in 1× TE buffer with gentle agitation for 2 days, dried, and then quantified with a scintillation counter. Background levels of CycD1 kinase activity (α-CycD1 IP with 780A peptide substrate) are indicated by black bar. (C) Log phase 293T and V2CH cells were harvested for separation of cytoplasmic (Cyt) and nuclear (Nuc) compartments with the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific). Western blot analysis for levels of VP40, CHMP6, HDAC1, and Actin was performed. (D) Log phase 293T and V2CH cells (3 × 10 6 ) were harvested and cross-linked with 1% formaldehyde for 1 hour followed by quenching with 1.25 glycine (9:1 cell suspension/glycine). Samples were sonicated, and 100 µL of each sample (corresponding to approximately 5 × 10 5 cells) was used for IP with 1 µg of anti-Pol II, α-p300, or α-VP40 at 4°C overnight. The next day, Protein A/G (30% slurry) was added and incubated for 2 hours at 4°C. Complexes were washed once with each TNE300 + 0.1% NP40, TNE150 + 0.1% NP40, TNE50 + 0.1% NP40, and IP wash buffer before the addition of proteinase K (800 units/mL). Samples were incubated for 15 minutes at 65°C, reversing solution was added, and samples were incubated for an additional 90 minutes at 65°C. DNA was purified, and qPCR was performed with 2 μL of undiluted DNA with primers for the CycD1 promoter region (spanning −180 to +238 from the messenger RNA start site at +1). The absolute quantification of the samples was determined based on the cycle threshold value relative to the standard curve generated from serial dilutions of DNA from 293T cells. Data are presented as percentage (%) of the input (DNA purified from sonicated samples before IP with specific antibodies). Student’s 2-tailed t test compares V2CH ChIPed DNA with corresponding 293T ChIP samples (**, P < .01).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Activity Assay, SDS Page, Western Blot, Incubation, Sonication, Purification, Generated

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Alteration of cell viability by VP40 in multiple cell types. Cells including (A) U937, HeLa, and (B) 3 peripheral blood mononuclear cells (PBMCs) log-phase cultures (~1.65 × 10 5 cells) were transfected with attractene and 1.5 µg of cytomegalovirus-VP40 plasmid. The PBMCs received a 1-time treatment of 50 IU/mL of interleukin-2 the day before transfection. Control cells received attractene treatment alone. Cell viability was assayed 3 days post-transfection. Statistical analysis by Student’s 2-tailed t test compares control cells with transfected cells (*, P < .05; **, P < .01).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Transfection, Plasmid Preparation

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Differential biogenesis of exosomes at different phases of the cell cycle. 293T and V2CL cells were blocked at G 0 (starvation; 0.1% fetal bovine serum DMEM), G 1 /S (20 mM of hydroxyurea), and G 2 /M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour and subsequent treatment with 50 ng/mL of nocodazole) for 5 days. Control (unsynchronized) cells were also incubated for 5 days. Black arrows point to bands of noticeable difference between V2CL and 293T cells. (A) Blocked cells and controls were harvested, washed twice in 1× PBS, and lysed. Samples were run on a 4–20% Tris-glycine gel and analyzed by western blot for the presence of ESCRT pathway proteins (VPS4, EAP45, TSG101, CHMP6, and EAP20), exosomal markers (Alix and CD63), and Actin. (B) Cell-free supernatants from blocked cells were harvested and passed through a 0.22-µm filter. One milliliter of filtered supernatant was incubated with 30 μL of NT80/82 particles overnight at 4°C. The next day, the NT pellet was washed once in 1× PBS and resuspended in 10 μL Laemmli buffer, followed by SDS/PAGE and western blot analysis for VP40 protein, exosomal markers CD63 and Alix, and Actin. (C) One milliliter of filtered supernatant from blocked and control cells was incubated with 30 μL of NT80/82 particles overnight at 4°C. The next day, the NT pellets were isolated, washed, and subjected to AChE assay for quantification of exosomes. Statistical analysis was completed by Student’s 2-tailed t test (*, P < .05; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Incubation, Western Blot, SDS Page, Isolation, AChE Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Extracellular vesicles released by VP40-producing cells at different phases of the cell cycle. 293T, V2CL, and V2CH cells were blocked at G 0 (starvation; 0.1% fetal bovine serum DMEM), G 1 /S (20 mM of hydroxyurea), and G 2 /M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour and subsequent treatment with 50 ng/mL of nocodazole) for 5 days. Supernatants were harvested, filtered (0.22 μm), and analyzed by ZetaView for size (peak [mode] diameter) (A) and concentration of particles (B). Statistical analysis by Student’s 2-tailed t test compares V2CL and V2CH cell cycle-blocked groups with corresponding 293T groups (†, P < .05; ††, P < .01; †††, P < .001). Additional Student’s 2-tailed t test compares cell cycle-blocked groups with controls of their own cell type (*, P < .05; **, P < .01; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Concentration Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Iodixanol gradient separation of extracellular vesicles (EVs) from 293T and VP40-producing cells. 293T and V2CI cells were grown in exosome-free media for 5 days, followed by harvesting of the supernatant and incubation with ExoMAX (1:1 reagent/filtered supernatant) reagent overnight at 4°C. The EVs were pelleted, resuspended in 300 µL of sterile 1× PBS, and loaded onto a 6–18% iodixanol density gradient (1.2% increments). Samples were ultracentrifuged for 90 minutes at 100000 × g , followed by harvesting and isolation of each fraction, and incubation with 30 µL of NT80/82 particles overnight at 4°C. The NT pellets were washed in 1× PBS, resuspended in 12 µL of Laemmli buffer, and loaded onto a 4–20% Tris-glycine gel. Western blot of 293T (A) and V2CI (B) fractions were analyzed for levels of VP40, CD63, CD81, CD9, and Actin. Major groups of EVs or exosome type are indicated by black boxes.

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Incubation, Isolation, Western Blot

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: The presence of VP40 in exosomes in in vitro and in vivo EBOV-infected cells. (A) HUVECs were cultured and infected with EBOV (MOI of 1) and incubated for 3 days under BSL-4 containment. Two milliliters supernatant were harvested, passed through a 0.22-µm filter, and incubated with ExoMAX (1:1 reagent/filtered supernatant) reagent overnight at 4°C. EVs were pelleted, resuspended in 0.5 mL 1× PBS, and loaded on qEV columns. Fraction numbers 7–10 (0.5 mL each) were collected and separately incubated with 30 μL NT80/82 at room temperature for 1 hour. The EV-bound NTs were washed with 1× PBS, followed by resuspension in 10 μL 2× NuPAGE LDS sample buffer, heating at 95°C for 10 minutes, and loading onto a 4–12% Tris-glycine gel for subsequent western blot analysis for VP40, GP, NP, and Actin levels. Negative control (Null) samples consisted of purified exosomes from uninfected HUVECs. (B) Gamma-irradiated and inactivated NHP (rhesus monkey) serum samples were obtained. NHP 1: day 0 prebleed sample. NHP 2: pool of day 4 and day 5 postinfection (pi); NHP 2 died on day 7 post-EBOV infection. NHP 3: pool of day 8–11 pi; NHP 3 died on day 12 post-EBOV infection. One hundred microliters of serum were diluted with 400 μL sterile 1× PBS and filtered (0.22 µm). Twenty-five microliters NT80/82 particles were incubated with the filtered samples at 4°C overnight. The next day, NT pellets were washed once in 1× PBS, resuspended in 12 μL Laemmli buffer, run on 4–20% SDS/PAGE, and analyzed by western blot for VP40 protein and exosomal markers CD81 and CD9.

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: In Vitro, In Vivo, Infection, Cell Culture, Incubation, Western Blot, Negative Control, Purification, Irradiation, SDS Page

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Induction of recipient T-cell death by purified VP40 EVs. (A) 293T and V2CH cells were grown in exosome-free media for 5 days, followed by harvesting of cell-free supernatants and filtration through 0.22 μm. Supernatants were then spun at 100000 × g for 90 minutes to pellet EVs, followed by resuspension in sterile 1× PBS. Concentrations of resulting ultracentrifuged EVs were determined with ZetaView analysis, followed by treatment of CEM cells with increasing concentrations of EVs (10000, 25000, or 75000 particles/cell) from V2CH cell type. Controls included CEM cells that were left untreated and CEM cells that received a treatment of the highest concentration of 293T cells (75000 particles/cell). Cells were incubated for 3 days followed by analysis of cell viability by CellTiter-Glo. (B) 293T and V2CI cells were grown in exosome-free media for 5 days. Cell-free supernatants were harvested and incubated with equal volumes of ExoMAX overnight at 4°C. The EVs were pelleted, resuspended in 400 µL sterile 1× PBS, and loaded onto a 6–18% iodixanol density gradient (1.2% increments). Samples were ultracentrifuged for 90 minutes at 100000 × g , followed by harvesting and isolation of each fraction. Select fractions (13.2 + 14.4 and 16.8 + 18.0) were pooled and subjected to a second ultracentrifuge spin for 90 minutes at 100000 × g diluted in 1× PBS to pellet the EVs away from residual iodixanol. Resulting EV pellets were resuspended in 100 µL sterile 1× PBS and used to treat recipient CEM cells at a concentration of 10000 particles per cell (concentrations determined by ZetaView analysis). Cells were incubated for 5 days followed by analysis of viability by CellTiter-Glo assay. Statistical analysis by Student’s 2-tailed t test compares groups treated with EVs from V2C cells to those treated with EVs from 293T cells (*, P < .05; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Purification, Filtration, Concentration Assay, Incubation, Isolation, Glo Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Effect of cdk4/6 inhibitors on VP40-producing cell viability and extracellular vesicle biogenesis. 293T and V2CH cells were treated with low, medium, and high concentrations of Fascaplysin (0.1, 0.5, 1.0 µM) or Ribociclib (0.1, 1.0, 10.0 µM) for 5 days. DMSO was also used at a final concentration of 1%. (A) Supernatants were harvested, filtered (0.22 µm), and analyzed by ZetaView for concentration of particles. (B) Cell viability was also measured by CellTiter-Glo. Statistical analysis by Student’s 2-tailed t test compares drug-treated groups to untreated controls of their own cell type (*, P < .05; **, P < .01; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Concentration Assay

Journal: The AAPS journal

Article Title: Pharmacodynamics of Cyclin D1 Degradation in Ovarian Cancer Xenografts with Repeated Oral SHetA2 Dosing.

doi: 10.1208/s12248-023-00874-7

Figure Lengend Snippet: Fig. 2 A PBPK/PD model for SHetA2 in tumor-bearing mice. The PBPK model includes 14 perfusion-limited tissue compartments, elimination of SHetA2 from liver (CLliv), intes- tine (CLGI) and plasma (CLPL), and oral absorption kinetics (ka, ka1, FAGG). The PD model is based on the SHetA2 concentra- tion in the tumor and depicts the stimulation of cyclin D1 deg- radation through three transit compartments (TC1-3). Cyclin D1 synthesis and degradation are depicted by the zero-order constant kin and the first-order constant kout, respectively. The initial effect E induced by drug concentration (Cdrug) is described by the maximum effect (Smax), the drug concen- tration for 50% effect (SC50), and the slope factor (γ). The transit compartments account for events such as cyclin D1 phosphorylation, ubiquitination, and proteasomal degradation, with τ denoting the transit time among compartments

Article Snippet: PathScan® Total Cyclin D1 Sandwich ELISA Kit was purchased from Cell Signaling Technology (Boston, MA).

Techniques: Clinical Proteomics, Concentration Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: The AAPS journal

Article Title: Pharmacodynamics of Cyclin D1 Degradation in Ovarian Cancer Xenografts with Repeated Oral SHetA2 Dosing.

doi: 10.1208/s12248-023-00874-7

Figure Lengend Snippet: Fig. 5 Dose- and time-depend- ent effects of SHetA2 on cyclin D1 in vitro. Cyclin D1 protein levels were quantified in cell lysates using both ELISA (a, c, e, and f), which are presented as mean ± SD, and western blot- ting (b, d, and g). Two human ovarian cancer cell lines, A2780 and SKOV3, were treated with varying concentrations of SHetA2 (1, 2.5, 5, 7.5, 10, 15 μM) for 24 h (a and b). Time- dependent changes in cyclin D were monitored at different time points (0, 2, 4, 6, 8, 12, 24 h) after treating SKOV3 cells with 2.5 or 10 μM SHetA2 (c and d). The recovery of drug-induced cyclin D1 reduction upon drug removal was assessed at a dose of 10 μM SHetA2 (e, f, and g). After A2780 or SKOV3 cells were treated with control or SHetA2 for 24 h, the drug- containing culture medium was removed, cultures were washed, and the cells were incubated with fresh medium for an additional 24 h. The levels of cyclin D1 were monitored at 4, 8, and 24 h after drug removal. Representative western blots for mortalin and cyclin D1, along with GAPDH or cyclophilin G as the loading control (b, d, and g). Statistical analyses were performed in GraphPad Prism using one-way ANOVA tests

Article Snippet: PathScan® Total Cyclin D1 Sandwich ELISA Kit was purchased from Cell Signaling Technology (Boston, MA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Incubation

Journal: The AAPS journal

Article Title: Pharmacodynamics of Cyclin D1 Degradation in Ovarian Cancer Xenografts with Repeated Oral SHetA2 Dosing.

doi: 10.1208/s12248-023-00874-7

Figure Lengend Snippet: Fig. 6 The time profiles of cyclin D1 after treatment with SHetA2 in vitro and in vivo. a Changes in cyclin D1 protein levels over time in SKOV3 treated with different SHetA2 concentrations (1–15 μM) dur- ing a 24-h incubation followed by drug removal (the data are from Fig. 5). Different time points (0, 2, 4, 6, 8, 12, 24 h) were monitored for the 2.5 and 10 μM groups, and only 24-h time point was measured for the other doses. The recovery of drug-induced cyclin D1 reduc- tion upon drug removal was evaluated at 10 μM by washing the drug- containing culture medium and incubating with fresh medium for an additional 24 h. b The time profile of cyclin D1 levels in SKOV3 tumors in mice treated with a single (light blue circle) and 7-day (blue circles) oral dose of 60 mg/kg SHetA2 once daily. The sym- bols represent the observed cyclin D1 protein levels in cancer cells or tumors, quantified using the ELISA method, with data presented as mean ± SD (n = 3). Both in vitro and in vivo data were used to estimate the PD model parameters, and the lines represent the best- fit curves. c Sensitivity analysis for each parameter. Sensitivity coef- ficients are determined through the division of changes in the model’s output by variations in the parameters

Article Snippet: PathScan® Total Cyclin D1 Sandwich ELISA Kit was purchased from Cell Signaling Technology (Boston, MA).

Techniques: In Vitro, In Vivo, Incubation, Enzyme-linked Immunosorbent Assay

Journal: The AAPS journal

Article Title: Pharmacodynamics of Cyclin D1 Degradation in Ovarian Cancer Xenografts with Repeated Oral SHetA2 Dosing.

doi: 10.1208/s12248-023-00874-7

Figure Lengend Snippet: Fig. 7 The simulated time pro- files of SHetA2 concentration (a) and cyclin D1 (b) in tumors in mice treated with different dosage regimens of SHetA2

Article Snippet: PathScan® Total Cyclin D1 Sandwich ELISA Kit was purchased from Cell Signaling Technology (Boston, MA).

Techniques: Concentration Assay

Journal: Endocrine Related Cancer

Article Title: Array-CGH identifies cyclin D1 and UBCH10 amplicons in anaplastic thyroid carcinoma

doi: 10.1677/erc-08-0018

Figure Lengend Snippet: Figure 2 (A) Array-CGH profiles of chromosome 11 for case 7 (upper) carrying amplification in 11q22.1 and gain in 11q13, and for case 4 (below) harboring an 11q13 amplicon. (B) Western blot analyses showing cyclin D1 expression in positive control cells (C), and ATCs 21, 25, and 27, while normal thyroid (N) and ATCs 22, 23, and 24 are negative. Incubation of the same filter with a-actinin served as loading control. (C) Fluorescence in situ hybridization (FISH) of CCND1 (Cyclin D1, red) and centromere 11 (CEP11, green) copy numbers. Two signals are observed in normal metaphase and interphase nuclei, while ATC cases 4 and 7, and HTh 7 cells show relative gain of CCND1.

Article Snippet: The CCND1 plasmid was obtained from Addgene (Rc/CMV-CCND1 #8962, Cambridge, MA, USA) and the control plasmid Rc/CMV was kindly provided by Dr Sue Firth at the Kolling Institute of Medical Research, NSW, Australia.

Techniques: Amplification, Western Blot, Expressing, Positive Control, Incubation, Control, Fluorescence, In Situ Hybridization

Journal: Endocrine Related Cancer

Article Title: Array-CGH identifies cyclin D1 and UBCH10 amplicons in anaplastic thyroid carcinoma

doi: 10.1677/erc-08-0018

Figure Lengend Snippet: Figure 5 Analysis of CCND1 (cyclin D1) effect on thyroid cell growth. Comparison plot for proliferation (MTS) assay (average of three independent experiments) of (A) HTh 7 ATC cells transfected against CCND1 siRNA and its reference control cells at 0, 24, 48, and 72 h after transfection, and (B) Nthy-ori 3-1 normal thyroid cells transfected with CCND1 or empty vector (pRC/CMV). Standard errors are indicated at each time point. Efficiency of siRNA as well as CCND1 transfection was validated by western blot analysis and qRT-PCR. Quantification was performed against a-tubulin for the western blot analysis and against 18S for qRT-PCR.

Article Snippet: The CCND1 plasmid was obtained from Addgene (Rc/CMV-CCND1 #8962, Cambridge, MA, USA) and the control plasmid Rc/CMV was kindly provided by Dr Sue Firth at the Kolling Institute of Medical Research, NSW, Australia.

Techniques: Comparison, MTS Assay, Transfection, Control, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: Journal of Hepatocellular Carcinoma

Article Title: Exploration of the Mechanism of Action of Dendrobium officinale in the Treatment of Liver Cancer Based on Network Pharmacology, Molecular Docking and in vitro Validation

doi: 10.2147/JHC.S527095

Figure Lengend Snippet: The aqueous extract of Dendrobium officinale exerts an anti-liver cancer effect by down regulating the protein expression levels of p-PI3K/PI3K, AKT1, EGFR, and CCND1. The protein expression levels of ( A ) p-PI3K/PI3K, ( B ) AKT1, ( C ) EGFR, and ( D ) CCND1 were determined by Western blotting. DNL, DNM, DNH vs Control, * p <0.05, ** p <0.01, and *** p <0.001.

Article Snippet: CCK-8 (Elabsciencec, model: E-CK-A362); incubator and pipette gun were purchased from Thermo; AKT1 (model: 20584-1-AP), EGFR (model: 18986-1-AP), CCND1 (model: 26939-1-AP) antibodies were purchased from proteintech; PI3K (proteintech, model: 10176-2-AP); PI3K (proteintech, model: 10176-2-AP) antibodies were purchased from proteintech.

Techniques: Expressing, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 2. Cyclin D1 inhibits liganded PPAR transactivation function. (A) The (AOX)3 luciferase reporter (1 g) was transfected into HeLa cells with the expression vector encoding the human PPAR in either the presence or absence of cyclin D1 (pCMV-cyclin D1). Comparison was made with the effect of the expression of equal amounts of empty expression vector cassette (pRC/CMV). 15d-PGJ2 (10 M) was added as indicated. The results are shown as mean standard error of the mean throughout. (B) (AOX)3LUC reporter activity in HeLa cells transfected with tetracycline- inducible vector pcz-cyclin D1. (C). PPAR1 promoter activity in cyclin D1/ or cyclin D1/ 3T3 cells cotransfected with pCMV-cyclin D1 or control vector as indicated. (E) Semiquantitative RT-PCR for PPAR1 from mRNA of livers of cyclin D1/ or cyclin D1/ mice. The PPAR LBD construct linked to the Gal4 DNA binding domain was assessed for activity using the heterologous reporter (UAS)5E1BTATA LUC in the presence or absence of the expression vectors for cyclin D1 in cyclin D1/ 3T3 cells. The relative transactivation level was shown as luciferase activity represented by light units measured in cells cotransfected with a specific receptor expression plasmid. Reporter gene activity was normalized to prl-TK LUC activity.

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Luciferase, Transfection, Expressing, Plasmid Preparation, Comparison, Activity Assay, Control, Reverse Transcription Polymerase Chain Reaction, Construct, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 3. PPAR and cyclin D1 abundance in mammary tumors induced by distinct oncogenes and human benign and malignant breast disease. (A) Expression of PPAR and cyclin D1 in murine mammary tumors. Western blotting of normal mammary epithelium or mammary tumors of MMTV-directed oncogene mice (MMTV-Ras [Ras], MMTV-Src [Src], or MTV-ErbB2 [ErbB2]) is shown. Abundance of PPAR, cyclin D1, or the loading control GDI is shown. MMTV-ErbB2 mammary tumors (T) and adjacent normal mammary epithelium (N) are represented below. (B). The mean levels of cyclin D1 and PPAR protein are shown for n 7 tumors induced by each oncogene, compared with normal mammary epithelium. (C). Representative immunostaining of normal mammary epithelium, benign breast disease, and ER infiltrating breast cancers (n 40) (magnification, 388). (D). The percent immunopositive staining for PPAR and cyclin D1 is shown for premalignant lesions (n 36) with comparison to normal mammary epithelium.

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Expressing, Western Blot, Control, Immunostaining, Staining, Comparison

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 4. Cyclin D1 deficiency enhances PPAR function. (A) MEFs derived from either cyclin D1/ or cyclin D1/ mice were treated with vehicle, BRL-49653 (1 M), and troglitazone (5 M), and lipid accumulation was assessed. cyclin D1/ MEFs exhibit lipid accumulation by Oil

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 5. Transgenic inducible cyclin D1 antisense regulates PPAR abundance in vivo. Western blot of cyclin D1/ 3T3 (A) and cyclin D1/

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Transgenic Assay, In Vivo, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 6. Enhanced PPAR-responsive gene expression in cyclin D1/ cells. (A) MEFs derived from either cyclin D1/ or cyclin D1/ mice were treated with BRL-49653 (1 M). Western blotting was conducted for PPAR and the PPAR-responsive genes, ACRP30 (11) and S3/12. Levels of cyclin D1, C/EBP, C/EBP, and the loading control (GDI) are shown. (B) Western blot analysis of cyclin D1/ MEFs or cyclin D1/

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Gene Expression, Derivative Assay, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 7. Morphological analysis of hepatic cells from cyclin D1 wt and cyclin D1/ mice. Hepatic tissues were stained for catalase, CMP, methyl-green pyronin (MPG), and Oil Red-O for staining of neutral lipids as described in Materials and Methods.

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Staining

Journal: Molecular and Cellular Biology

Article Title: Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation

doi: 10.1128/mcb.23.17.6159-6173.2003

Figure Lengend Snippet: FIG. 8. Prediction model of cyclin D1 helix-loop-helix region binding to PPAR. (A) Ribbon model of left panel: nuclear corepressor (red) binding to PPAR (grey, with helix 12 in magenta and coactivator binding region of PPAR in green); right panel, nuclear receptor coactivator (yellow) binding to PPAR (grey, with helix 12 in magenta and coactivator binding region of PPAR in green). (B). Ribbon model of PPAR and cyclin D1 helix-loop-helix region. Predicted cyclin D1 helix-loop-helix structure is in yellow with component of hydrophobic cluster in red. (C). PPAR (grey, with helix 12 in magenta and coactivator binding region of PPAR in green) and cyclin D1 hydrophobic cluster of helix-loop-helix motif colored red. (The position alignment, determined by energy minimization using the AMBER force field, displays the cyclin D1 helix-loop- helix motif in an alignment similar to that of a nuclear receptor corepressor).

Article Snippet: The antibodies were polyclonal cyclin D1 antibody Ab3 for Western blot analysis, anti-PPAR polyclonal antibody H100, and monoclonal E8 (Santa Cruz Biotechnology), anti-S3/12 (46), and anti-guanine dissociation inhibitor (GDI) (25) as a protein loading control.

Techniques: Binding Assay