ccnd1 promoter luciferase reporter construct  (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: