plasmid 32727  (Addgene inc)

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: Cyclin D1 is regulated by members of the SREBP family of transcription factors. (A) HepG2 cells were transduced with lentiviruses encoding GFP or FLAG-tagged versions of the nuclear forms of SREBP1a ( nS1a ), SREBP1c ( nS1c ), or SREBP2 ( nS2 ). The levels of cyclin D1, HMG-CoA synthase, the nuclear forms of SREBP1a, SREBP1c and SREBP2 ( FLAG ), and β-actin in total lysates were determined by Western blotting. The quantifications of cyclin D1 and HMG-CoA synthase across three independent experiments are included in Supplementary Figure S1 . (B) HepG2 cells were transduced as in (A) , and mRNA was isolated and used to generate cDNA. The expression of cyclin D1 ( CCND1 ) was determined by real-time qPCR using GAPDH for normalization. (C) HepG2 cells were transduced with lentiviruses encoding shRNAs, either non-targeted (C) or targeting SREBP1 ( S1 ) or SREBP2 ( S2 ). The levels of cyclin D1 and the precursor forms of SREBP1 ( pSREBP1 ) and SREBP2 ( pSREBP2 ) were detected by Western blotting. β-Actin was used as a loading control. (D) HepG2 cells were transduced with shRNA as in (C) and the expression of cyclin D1 was determined by real-time qPCR. (E) MCF7 cells were transduced with shRNA as in (C) , and the levels of cyclin D1 and the precursor forms of SREBP1 ( pSREBP1 ) and SREBP2 ( pSREBP2 ) were detected by Western blotting. (F) MCF7 cells were transduced with shRNA as in (C) , and the expression of cyclin D1 was determined by real-time qPCR. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons adjustment (B, D, and F) . p-values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS , not significant.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Transduction, Western Blot, Isolation, Expressing, Control, shRNA

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: The expression of cyclin D1 responds to physiological changes in SREBP activity. (A) HepG2 cells were grown in media supplemented with regular serum ( FBS ) or lipoprotein-deficient serum ( LPDS ). Where indicated, the LPDS was supplemented with 25-hydroxycholesterol ( 25-HC , 5 μg/ml) for 8 h before the end of the experiment. The levels of cyclin D1, nuclear SREBP1 ( nSREBP1 ), and β-actin were determined by Western blotting ( upper panel ). The relative levels of cyclin D1 were quantified and are presented as fold change ( lower panel ). (B) HepG2 cells grown in regular media were left untreated or treated with 1% HPCD (w/v) for the indicated times. The levels of cyclin D1, nuclear SREBP1 ( nSREBP1 ), and β-actin were determined by Western blotting ( upper panel ). The relative levels of cyclin D1 in untreated cells ( − ) and cells exposed to HPCD for 2 h ( HPCD ) were quantified and are presented as fold change ( lower panel ). Significance was determined by paired t-tests (B) or one-way ANOVA with Tukey’s multiple comparisons adjustment (A) . p-values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS , not significant.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: The human cyclin D1 promoter is SREBP-responsive. (A) HEK293 cells were transfected with the CCND1-luc promoter-reporter gene ( CCND1-Luc ) together with either empty vector ( pcDNA3 ) or expression vectors for the nuclear forms of SREBP1a ( nS1a ), SREBP1c ( nS1c ), or SREBP2 ( nS2 ). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. (B) HepG2 cells were transfected with the CCND1-luc promoter-reporter gene and treated as indicated. Cells in regular media were either left untreated or treated with 1% HPCD (w/v) for 4 h ( left ). Cells in LPDS were either left untreated or treated with 25HC (5 μg/ml) for 8 h ( right ). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. (C) Schematic illustration of the human cyclin D1 promoter with the location of the E-box and putative SRE elements. (D) HepG2 cells were transfected with the CCND1-luc promoter–reporter gene, either wild type ( WT ) or the indicated deletion mutant ( ΔSRE , ΔE-box or ΔΔ ) together with either empty vector ( pcDNA3 ) or an expression vector for the nuclear form of SREBP1a ( nS1a ). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. (E) HepG2 cells were transfected with the CCND1-luc promoter–reporter gene, either wild type ( WT ) or the ΔE-box mutant, together with expression vectors for either non-targeted (C) or SREBP1-targeted ( S1 ) shRNA. Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. Significance was determined by paired t-tests (B, D, E) or one-way ANOVA with Tukey’s multiple comparisons adjustment (A) . p-values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p< 0.0001. NS , not significant.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Mutagenesis, shRNA

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: SREBP1 interacts with the human cyclin D1 promoter. (A) 6xMyc-tagged nuclear SREBP1a ( nS1a) , SREBP1c ( nS1c ), and SREBP2 ( nS2 ) were expressed in HEK293 cells and used in DNAP assays using a biotinylated DNA probe corresponding to the human cyclin D1 promoter. The three proteins were expressed at similar levels as monitored by Western blotting ( left ). Increasing amounts of the three proteins were incubated with the promoter probe, captured on streptavidin beads, washed, separated on SDS-PAGE gels, and analyzed by Western blotting ( Myc , right ). (B) Nuclear SREBP1a ( nS1a , 10 μl), SREBP1c ( nS1c , 10 μl), and SREBP2 ( nS2 , 50 μl) were incubated with the biotinylated cyclin D1 probe in the absence or presence of non-biotinylated competitor DNA corresponding to the SREBP-binding site in the human LDL receptor promoter (fivefold excess) and processed as in (A) . (C) Nuclear SREBP1a ( nS1a ) was incubated with the biotinylated cyclin D1 probe, either wild-type ( WT ) or the indicated deletion mutant ( ΔSRE , ΔE-box or ΔΔ ) and processed as in (A) . (D) FLAG-tagged nuclear SREBP1a ( nS1a ) was purified from transfected HEK293 cells and incubated with unlabeled DNA probes corresponding to the human cyclin D1 promoter, either wild-type ( WT ) or ΔE-box, separated on native PAGE gels and stained with SYBR Safe. The shifted SREBP1a–DNA complex is indicated by an arrow. The two probes alone were also run on the same gel ( left two lanes ). (E) FLAG-tagged nuclear SREBP1a ( nS1a ) was incubated with an unlabeled DNA probe corresponding to the human cyclin D1 promoter, separated on a native PAGE gel, and stained with SYBR Safe. Monoclonal anti-FLAG antibodies were added to the sample in lane 2 prior to loading it on the gel. The shifted SREBP1a–DNA complex and the supershifted SREBP1a–DNA–antibody complexes are indicated by arrows.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Western Blot, Incubation, SDS Page, Binding Assay, Mutagenesis, Purification, Transfection, Clear Native PAGE, Staining

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: The recruitment of SREBP1 to the cyclin D1 promoter correlates with enhanced expression of the cyclin D1 gene. (A) MCF7 cells were serum starved for 24 h and then left untreated or treated with insulin for an additional 2 h. The cells were collected, permeabilized, incubated with SREBP1 or preimmune rabbit (IgG) antibodies, followed by the protein A/G-MNase fusion protein. Endogenous SREBP1 and its bound DNA was subsequently isolated using protein A magnetic beads and used for PCR with primers specific for the E-box in the human cyclin D1 promoter. The same primers were also used for PCR of the same region from genomic DNA isolated from the same cells ( Input ). The PCR products were separated on PAGE gels and stained with SYBR Safe ( upper panel ). The intensity of the signals in the samples and the input were quantified ( lower panel ). (B) mRNA was isolated from the cells in (A) and used to determine the expression of cyclin D1 by real-time qPCR, using GAPDH as a reference. (C) MCF7 cells were transfected with the CCND1-luc promoter–reporter gene, either wild-type ( WT ) or the ΔE-box, serum starved for 24 h followed by an additional 2-h incubation in the absence or presence of insulin, after which the cells were lysed, and luciferase activity was measured. Significance was determined by paired t-tests (A–C) . p-values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p< 0.0001. NS , not significant.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Expressing, Incubation, Isolation, Magnetic Beads, Staining, Transfection, Luciferase, Activity Assay

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: The SREBP pathway regulates the proliferation of MCF7 cells. (A) MCF7 cells were transduced with lentiviruses expressing non-targeted shRNA (C) or shRNA targeting SREBP1 ( S1 ) or SREBP2 (S2) . After selection, an equal number of cells were seeded in 12-well plates and cells counted over a 72-h period. (B) MCF7 cells were transduced with lentiviruses expressing non-targeted shRNA (C) or shRNA targeting SREBP1 ( S1 ) or SREBP2 ( S2 ) and metabolically labeled with BrdU, and the incorporation of BrdU was determined by an ELISA assay. (C) MCF7 cells were transduced with lentiviruses expressing non-targeted shRNA (C) or shRNA targeting SREBP1 ( S1 ) or SREBP2 ( S2 ). The cells were collected, fixed, and stained with propidium iodine, and the DNA content was analyzed by FACS. (D) The same cells as in (C) were lysed and the levels of cyclin D1, Rb, and the precursor form of SREBP1 ( pSREBP1 ), and the phosphorylation of Rb on serine 780 ( pRb-S780 ) were analyzed by Western blotting. β-Actin was used as loading control. (E) MCF7 cells were transduced with lentiviruses expressing non-targeted shRNA (C) or shRNA targeting SREBP1 ( S1 ). After selection, the cells were serum-starved for 24 h, followed by an additional 20-h incubation in the absence or presence of serum. The cells were collected and processed as in (C) . (F) The same cells as in (E) were lysed and the levels of cyclin D1, Rb and the precursor form of SREBP1 ( pSREBP1 ), and the phosphorylation of Rb on serine 780 ( pRb-S780 ) were analyzed by Western blotting. β-Actin was used as loading control. Significance was determined by paired t-tests (E) or one-way ANOVA with Tukey’s multiple comparisons adjustment (A–C) . p-values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS , not significant.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Transduction, Expressing, shRNA, Selection, Metabolic Labelling, Labeling, Enzyme-linked Immunosorbent Assay, Staining, Phospho-proteomics, Western Blot, Control, Incubation

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: The SREBP1-dependent induction of cyclin D1 promotes the phosphorylation of Rb. (A) HepG2 cells were transduced with lentiviruses expressing GFP or nuclear SREBP1a ( nS1a ), and 48 h following transduction, the cells were treated with or without the cdk4/6 inhibitor palbociclib (10 μM) for 6 h. The levels of cyclin D1, Rb, and the nuclear form of SREBP1 ( nSREBP1 ), and the phosphorylation of Rb on serine 780 ( pRb-S780 ) were analyzed by Western blotting. β-Actin was used as loading control. (B) HepG2 cells were transduced with lentiviruses expressing GFP or nuclear SREBP1a ( nS1a ) together with non-targeted (C) or cyclin D1 ( CCND1 ) shRNA. Forty-eight hours after transduction, the levels of cyclin D1, Rb, CDK4, and nuclear SREBP1 ( nSREBP1 ), and the phosphorylation of Rb on serine 780 ( pRb-S780 ) were analyzed by Western blotting. β-Actin was used as loading control. (C) HepG2 cells were transduced with lentiviruses expressing GFP or cyclin D1 together with non-targeted (C) or SREBP1 ( S1 ) shRNA. Forty-eight hours after transduction, the levels of cyclin D1, Rb, CDK4 and the precursor form of SREBP1 ( pSREBP1 ), and the phosphorylation of Rb on serine 780 ( pRb-S780 ) were analyzed by Western blotting. β-Actin was used as loading control. (D) The same cells as in (C) were also fixed and stained with propidium iodine, and the DNA content was analyzed by FACS. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons adjustment (D) . p-values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Phospho-proteomics, Transduction, Expressing, Western Blot, Control, shRNA, Staining

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet: The SREBP1-dependent expression of cyclin D1 links insulin signaling to Rb phosphorylation. (A) MCF7 cells were transduced with lentiviruses expressing non-targeted (C) or SREBP1 ( S1 ) shRNA. After selection, the cells were serum-starved for 24 h, followed by an additional 2-h incubation in the absence or presence of insulin. The levels of cyclin D1, Rb, and nuclear ( nSREBP1 ) and precursor SREBP1 ( pSREBP1 ) and CDK4, and the phosphorylation of Rb on serine 780 ( pRb-S780 ) were analyzed by Western blotting. (B) MCF7 cells were transduced with lentiviruses expressing non-targeted (C) or cyclin D1 ( CCND1 ) shRNA and treated and processed as in (A) . (C) MCF7 cells were serum-starved for 24 h followed by a 2-h stimulation with insulin in the absence or presence of palbociclib (10 μM). The cells were processed and analyzed as in (A) . (D) Model. We propose that cells respond to growth-promoting signals, such as insulin or EGF, by activating SREBP1. The active SREBP1 molecules bind to an E-box in the promoter of cyclin D1, thereby enhancing the expression of the corresponding gene. The enhanced expression of cyclin D1 promotes the phosphorylation of Rb by activating cdk4/6, thereby promoting G1 progression. At the same time, SREBP1, especially SREBP1a, has the capacity to enhance the expression of lipogenic genes to support cell growth. Thus, the SREBP-dependent regulation of cyclin D1 could help coordinate cell cycle progression with increased lipid synthesis to support cell growth. This may be especially important in tumor cells, which express the most potent member of the SREBP family of transcription factors, SREBP1a.

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques: Expressing, Phospho-proteomics, Transduction, shRNA, Selection, Incubation, Western Blot

Journal: Frontiers in Oncology

Article Title: The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis

doi: 10.3389/fonc.2022.942386

Figure Lengend Snippet:

Article Snippet: The human cyclin D1 promoter in pGL3Basic was a gift from Frank McCormick (Addgene plasmid #32727). pHAGE-CCND1 was a gift from Gordon Mills and Kenneth Scott (Addgene plasmid #116721).

Techniques:

Journal: PLoS Biology

Article Title: G1/S cell cycle regulators mediate effects of circadian dysregulation on tumor growth and provide targets for timed anticancer treatment

doi: 10.1371/journal.pbio.3000228

Figure Lengend Snippet: (A) Western blot analysis of phosphorylated RB at multiple sites (pRB-S807/811, pRB-S795, pRB-S780, pRB-S612), total RB, CCNB1, cyclin D1, and CDK4 with the specific antibodies as indicated in the control (CTL) and jet lag (JL) cells collected every 6 hours (24 hours, 30 hours, 36 hours, 42 hours, 48 hours) for 24 hours after the final dex stimulation, as depicted with black arrowheads in . GAPDH (αGAPDH) is loading control. Representative images were taken from n = 3 independent experiments. (B) Statistical analysis of the WB data in (A) showing time-dependent variation of protein abundance as indicated in CTL (grey circle) and JL (brown circle) cells. GAPDH was used to normalize protein levels. * p < 0.05, *** p < 0.001; two-way ANOVA and Sidak multiple comparisons test. p MetaCycle < 0.05 denotes results of MetaCycle analysis, identifying a 24-hour rhythm in the expression of pRB (S807/S811) ( p = 0.0481) and CCND1 protein ( p = 0.006) in CTL cells (grey circle). Data normalized are represented as mean ± SEM from n = 3 independent experiments. (C) Schematic representation of the elements of the human cyclin D1 promoter (upper). Elements of human cyclin D1 promoter are represented by different colors. The transcriptional start site is identified by a black line and arrow. The data show log 2 fold-change values for gene expression of signaling-dependent transcriptional activators or repressors that directly target enhancer elements in the cyclin D1 promoter, as indicated by the corresponding color codes (See ). (D) Carcinogen (MCA)-induced tumor bearing mice were exposed to a chronic jet-lag schedule (see ), following which tumor and liver tissues were harvested from Jet-lag or Control mice at the indicated time points (ZT3, ZT9, ZT15) and subjected to western blot analysis using the indicated antibodies. Representative images from n = 3 independent experiments are shown. (E) Immunoblot analysis using liver extracts prepared from mouse liver tissues collected at 4-hour intervals as indicated for 24 hours in constant darkness. Specific antibodies were used for detecting endogenous pRB-S807/811, cyclin D1, CDK4, and CCNB1 proteins, as indicated. Anti-GAPDH (αGAPDH) was used for loading control. Similar results were obtained in two independent experiments. (F) HEK293T cells were transiently transfected with a cyclin D1-Luc reporter construct (p cyclin D1 (−1748)-Luc) alone or co-transfected with plasmids expressing CLOCK, BMAL1, and CRY1, as indicated. After 24 hours, the cells were lysed and cyclin D1 promoter-driven luciferase activity was measured and normalized with pRL-TK activity. Representative results from three independent experiments performed are shown with the means ± SEM; n = 3. *** p < 0.0001, one-way ANOVA and Tukey multiple comparisons test. Underlying data for this figure can be found in . AP-1, activator protein 1; BMAL1, brain and muscle Arnt-like protein-1; CCD, chronic circadian desynchrony; CCNB1, cyclin B1; CCND1, cyclin D1; CDK, cyclin dependent kinase; CLOCK, circadian locomotor output cycles protein kaput; CREB/ATF, cAMP response element-binding protein/activating transcription factor; CRY1, Cryptochrome1; CSL, CBF1, Suppressor of Hairless, Lag-1; CT, circadian time; dex, dexamethasone; E-box, enhancer box; Ets, E26 transformation-specific transcription factor; E2F, E2 transcription factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jmj, jumonji and AT-rich interaction domain containing 2 (Jarid2); LEF/TCF, lymphoid enhancer-binding factor/T-cell factor; Luc, luciferase; MAR, matrix-associated region; MCA, methylcholanthrene; NFKB, nuclear factor kappa-light-chain-enhancer of activated B cells; Oct-1, POU domain, class 2, transcription factor 1; pRB, phosphorylated RB; pRL-TK, renilla luciferase reporter of the HSV-thymidine kinase promoter; RB, retinoblastoma; SP1/EgR, specificity protein 1 (SP1)/early growth response (EgR) transcription factor; STAT3/5B, signal transducer and activator of transcription 3/5B; WB, western blot; YY-1, yin yang 1 transcription factor; Z-box, Z-DNA forming sequence; ZT, zeitgeber time.

Article Snippet: For cyclin D1 promoter analysis, a −1748 human cyclin D1 promoter pGL3 basic (32726) and a −962 human cyclin D1 promoter pGL3 basic (32727) were purchased from Addgene.

Techniques: Western Blot, Control, Quantitative Proteomics, Expressing, Gene Expression, Transfection, Construct, Luciferase, Activity Assay, Binding Assay, Transformation Assay, Sequencing

Journal: PLoS Biology

Article Title: G1/S cell cycle regulators mediate effects of circadian dysregulation on tumor growth and provide targets for timed anticancer treatment

doi: 10.1371/journal.pbio.3000228

Figure Lengend Snippet: (A) Western blot analysis of the dose dependency of PD-0332991 action on RB phosphorylation in U2OS cells. After a 48-hour incubation with increasing doses of PD-0332991, cell extracts were harvested for immunoblot analysis of protein abundances of phosphorylated RBs at multiple sites (pRB-S807/811, pRB-S795, pRB-S780, pRB-S612), total RB, cyclin D1, CDK4, and CDK6 using specific antibodies. Anti-GAPDH (αGAPDH) was used for loading control. (B) Schematic of the experimental schedule to determine time dependence of the antiproliferative effect of PD-0332991 on U2OS cells. After 24 hours of dex (100 nM, yellow bolt) synchronization, the cells were treated with vehicle or PD-033291 (0.01–5 μM, red bolt) at 6-hour intervals over the course of 24 hours and subjected to an MTT cell proliferation assay at the indicated time points 48 hours later. (C) Graph of experimental procedures in (B) to show time-dependent antiproliferative effects of PD-0332991 at various doses. ** p < 0.001, *** p < 0.0001; two-way ANOVA and Tukey multiple comparisons test. p MetaCycle < 0.05 denotes results of MetaCycle analysis, identifying a 24-hour rhythm in drug sensitivity of cells treated with 0.1 μM PD-0332991 ( p = 0.0146). Data were normalized to represent the average ± SD; n = 3 in all time points. The result is representative of three independent experiments. (D) Twenty-four hours after the final dex stimulation depicted in , Control (CTL) and Jet lag (JL) U2OS cells stably expressing siRNA against GFP (si-GFP stable ) or BMAL1 (si-BMAL1 stable ) were subject to a time course of vehicle or PD-0332991 (0.1 μM, 1 μM) treatment, and subsequent MTT analysis was performed as described in (B). Data were normalized to represent the average ± SD; n = 3 in all time points. p MetaCycle < 0.001 denotes results of MetaCycle analysis, identifying a 24-hour rhythm in drug sensitivity of cells treated with 1 μM PD-0332991 ( p = 0.00019). The result is representative of two independent experiments. Underlying data for this figure can be found in . BMAL1, brain and muscle arnt-like protein-1; CCD, chronic circadian desynchrony; CDK, cyclin dependent kinase; dex, dexamethasone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; MTT, thiazolyl blue tetrazolium bromide; RB, retinoblastoma; siRNA, small interference RNA U2OS, human U2 osteosarcoma.

Article Snippet: For cyclin D1 promoter analysis, a −1748 human cyclin D1 promoter pGL3 basic (32726) and a −962 human cyclin D1 promoter pGL3 basic (32727) were purchased from Addgene.

Techniques: Western Blot, Phospho-proteomics, Incubation, Control, MTT Cell Proliferation, Stable Transfection, Expressing

Journal: Molecular cancer research : MCR

Article Title: ETV4 Facilitates Cell Cycle Progression in Pancreatic Cells through Transcriptional Regulation of Cyclin D1

doi: 10.1158/1541-7786.MCR-17-0219

Figure Lengend Snippet: List of selective cell cycle regulatory genes with symbol, functions, and folds change in high and low ETV4-expressing PC cells

Article Snippet: The construct -962 human cyclin D1 promoter pGL3Basic (Addgene plasmid #32727) was from Frank McCormick laboratory and was procured through Addgene.

Techniques:

Journal: Molecular cancer research : MCR

Article Title: ETV4 Facilitates Cell Cycle Progression in Pancreatic Cells through Transcriptional Regulation of Cyclin D1

doi: 10.1158/1541-7786.MCR-17-0219

Figure Lengend Snippet: (A) Cyclin D1 cDNA construct (pCMV6-CCND1) was transfected in ETV4-silenced Colo357 and ASPC1 cells for its overexpression and confirmed by immunoblot analyses. (B) After 48 h of transfection, cells further transfected with Cyclin D1 promoter reporter construct and transcriptional activity of Cyclin D1 was examined. Data are presented as normalized relative luciferase activity (mean ± SD; n = 3, *p < 0.05). (C-D) Overexpression of cyclin D1 in ETV4-silenced cells rescued the inhibitory effect of ETV4-silencing on (C) growth and (D) cell cycle progression of PC cells.

Article Snippet: The construct -962 human cyclin D1 promoter pGL3Basic (Addgene plasmid #32727) was from Frank McCormick laboratory and was procured through Addgene.

Techniques: Construct, Transfection, Over Expression, Western Blot, Activity Assay, Luciferase