Journal: Nature

Article Title: Reduced cyclin D3 expression in erythroid cells protects against malaria

doi: 10.1038/s41586-026-10110-9

Figure Lengend Snippet: a , Top, vector content and expression of luciferase reporter genes driven by the extreme allele combinations: on the one hand the WT rs9349205-G|rs112233623-C (G|C) alleles on the other hand the derived rs9349205-A| rs112233623-T (A|T) alleles of the CCND3 enhancer in the erythroid cell line HUDEP-2, with empty vector as a control construct. Below, histograms show averages of the relative luminescence activity of the combinations of the two extreme alleles at rs9349205 and rs112233623 described above in absence (-) or presence of GATA1 and FOG-expressing plasmids; SMAD3 and p300 expressing plasmids; SMAD3 expressing plasmid alone; or empty expression vector. All values are plotted relative to the WT (G|C) construct. Note that the activity of the empty pGL4 vector is barely detectable. The mean ± s.e.m is shown (n = 7 (vectors); n = 3 (vectors + SMAD3), n = 3 (vectors+SMAD3 + p300), n = 4 (vectors + GATA1 + FOG) biologically independent experiments). A two-sided Student’s t -test was used, with level of significance indicated by asterisks (*P < .05;**P < .01; ****P < .0001, ns, not significant). The statistical results for all comparisons are provided in Supplementary Table . b , In silico prediction of binding to rs112233623 allele variants, showing derived allele T hindering binding of SMAD3 while favouring GATA1 binding. c , d , Representative electrophoretic mobility shift assays (EMSA) showing binding of SMAD3 and GATA1 proteins with labelled oligonucleotide probes (*) containing the WT (C) or derived (T) allele of rs112233623 (n = 3 biologically independent experiments). Competitor unlabelled oligonucleotides were used at the indicated fold excess to demonstrate specificity of binding. c , SMAD3 binds to the WT rs112233623-C allele (lanes 2 and 8); is weakly supershifted by anti-SMAD3 antibody (lanes 3 and 9); and is competed away by an excess of unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 10-12, at 25X, 50X and 100X respectively), but not by an excess of oligonucleotide containing the derived rs112233623-T allele (lanes 13-15, at 25X, 50X and 100X respectively). d , GATA1 binds to the rs112233623-T derived allele (lanes 2, 8 and 9), is supershifted by anti-GATA1 antibody (lane 3); and is competed away by an excess of unlabelled DNA oligonucleotides containing the derived rs112233623-T allele (lanes 10-12, at 25X, 50X and 100X respectively) compared to unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 13-15, at 25X, 50X and 100X respectively). GATA1 does not appear to bind to the WT rs112233623-C allele (lane 5). The samples derive from the same experiment and, the gels were processed in parallel. For gel source data, see Supplementary Fig. . e , f , ChIP–qPCR for SMAD3 or GATA1 binding to the CCND3 enhancer region surrounding rs112233623 ( CCND3 ) and to a SimpleChIP Human α Satellite as negative control in erythroblasts derived from individuals homozygous for the rs112233623-T decrease of expression (DoE) allele versus the WT rs112233623-C allele (WT). e , ChIP was performed using an antibody against SMAD3, results are represented as percentage of input and nonspecific IgG used as negative control. The mean ± s.e.m is shown (n = 3 biologically independent experiments). A two-sided two-Sample t-test was used; significant differences are indicated (*P < .05; ns, not significant). The statistical results for the comparisons are provided in Supplementary Table . f , ChIP assays were conducted with an antibody against GATA1, results are represented as percentage of input and nonspecific IgG used as negative control (n = 2 biologically independent experiments).

Article Snippet: ChIP DNA was purified and subsequently quantified by qPCR using primers designed to amplify a region surrounding rs112233623 in the CCND3 enhancer and SimpleChIP Human α Satellite Repeat Primers (4486, Cell Signaling Technology).

Techniques: Plasmid Preparation, Expressing, Luciferase, Derivative Assay, Control, Construct, Activity Assay, In Silico, Binding Assay, Electrophoretic Mobility Shift Assay, ChIP-qPCR, Negative Control

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Effect of cyclin D1 and Id1 on breast cancer cell protein expression and migration . Actively cycling MDA-MB-231 and ZR75-1 cells were monitored 20 h post-transfection with the indicated siRNA (cyclin D1/CDK4/6/Id1) or vector (Id1) for changes in gene or protein expression, and migration. Blots are representative, and plots are mean values from at least three independent experiments. Error bars represent standard deviation. MDA-MB-231 cells: (A) Microarray analysis. Left panel: CCND1 gene expression, right panel: ID1 gene expression (B) Western blot for cyclin d1, Id1 and Actin protein, (C) Cell migration as measured by Boyden chamber assay, dots indicate total number of migrated cells. (D) ChIP assay for Id1 promoter region following cyclin D1 pull down. ZR75-1: (E) Western blot for cyclin d1 and Actin protein (F) Cell migration- Boyden chamber assay. ***P ≥ 0.001, **P ≥ 0.01, *P ≥ 0.05 vs. control, two-tailed student's t-test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Standard Deviation, Microarray, Gene Expression, Western Blot, Boyden Chamber Assay, Control, Two Tailed Test

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Effect of cyclin d1 and Id1 on EMT markers . MDA-MB-231 cells were monitored 20 h post-transfection with the indicated siRNA (cyclin D1/CDK4/6/Id1) or vector (Id1) for changes in EMT-related gene expression by microarray analysis. Additionally, MDA-MB-231 and ZR75-1 gene expression was examined by qPCR assay. Plots are mean values from at least three independent experiments Error bars represent standard deviation. (A) Microarray analysis of SNAI1 , SNAI2 , CDH11 , TWIST1 and VIM gene expression. (B-D) CCND1, ID1 and SNAI2 in MDA-MB-231 cells. (E, F) qPCR analysis of CCND1 and SNAI2 in ZR75-1 cells. ***P ≥ 0.001, **P ≥ 0.01, *P ≥ 0.05 vs. control, two-tailed student's t-test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Transfection, Plasmid Preparation, Gene Expression, Microarray, Standard Deviation, Control, Two Tailed Test

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Distribution of CCND1 and ID1 gene expression according to clinico- pathological parameters in breast cancer patients

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Gene Expression

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Correlation of CCND1 , ID1 , SNAI1 and SNAI2 expression to recurrence free survival . Expression of our genes of interest in relation to recurrence-free survival was examined in a breast cancer database containing 1,107 tumours from Sims et al. (2008). Gene expression intensity was quartiled as 1-low, 2- medium low, 3- medium high and 4- high, and assessed in all patients, ER-positive and ER-negative patients, respectively (A) CCND1 quartiles (B) ID1 quartiles (C) SNAI1 quartiles (D) SNAI2 quartiles. P-value is based on log-rank test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Expressing, Gene Expression

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: EMT-related gene expression intensity and recurrence free survival in CCND1 / ID1 high and low tumours . A breast cancer database was employed to examine (A) Mean-centered average expression of EMT-related genes of interest in CCND1 / ID1 subgroups, and (B, C) Recurrence-free survival of ER-positive and negative patients in CCND1 / ID1 subgroups. P-value is based on log-rank test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Gene Expression, Expressing

Journal: BMC Cancer

Article Title: Cyclin D1, Id1 and EMT in breast cancer

doi: 10.1186/1471-2407-11-417

Figure Lengend Snippet: Gene expression of Cyclin d1/Id1 and EMT markers in breast cancer cell lines and claudin-low tumours . Expression of EMT and related genes from the Neve et al cell line (A-C) and Herschkowitz et al. claudin-low (D-F) studies. (A and D) Heatmaps showing relative expression of genes of interest, Red = high, Green = low. (B and E) Scatterplots showing the relationship between breast cancer subgroups and CCND1 / ID1 expression (C and F) Proportion of CCND1- low/ ID1 -high cell lines and tumours in breast cancer subgroups. ***P ≥ 0.001 , Chi 2 test.

Article Snippet: 3 μg of anti-cyclin d1 antibody (DCS-6, DAKO, Denmark) was used to pull down cyclin D1, with subsequent detection of Id1 using SimpleChIPTM Human Id1 Promoter Primers (Cell Signalling Technology, Danvers, MA) and Mrg1 as positive control (Eurofins Laboratories Ltd., Manchester, UK).

Techniques: Gene Expression, Expressing