Journal: Clinical Science (London, England : 1979)

Article Title: Unravelling the effects of selective estrogen receptor modulators on colorectal cancer: a prognostic role for insulin-like growth factor binding protein-5

doi: 10.1042/CS20258451

Figure Lengend Snippet: ( A ) Representative microscopic images showing the dose-dependent effects of 27-OHC and E2 on cell migration in HCT-116 cells after 48 h of treatment. Cells were exposed to four concentrations of 27-OHC (0.001, 0.01, 0.1, and 1 μM) and E2 (0.625, 1.25, 2.5, and 5 nM). Images were captured prior to the application of 1% SDS and reflect differences in cell migration across doses. ( B ) Quantitative analysis of 27-OHC-induced migration changes across four colon cancer cell lines. Cell migration was assessed using a trans-well assay following 48-h treatment with 27-OHC at the indicated concentrations. Experiments were performed in triplicate across three biological replicates ( n = 3). ( C ) Quantitative evaluation of E2-induced migration responses in colon cancer cells. Similarly, cell migration was assessed after 48 h of E2 treatment using the same four concentrations. Data were normalised to phenol red-free SFM controls and presented as fold changes relative to the untreated group. All graphs represent the mean ± SD. Statistical significance was determined using one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: HCT-116 and HT-29 cells (3 × 10 5 cells/well) were seeded into six-well plates and transfected with 50 nM siRNA (Dharmacon, ON-TARGET plus Human ESR2 (2100) siRNA-SMART pool, 20 nmol (L-003402-00-0020)) as performed previously [ ] using RNAiMAX Lipofectamine (Invitrogen) in phenol red-free Opti-MEM medium.

Techniques: Migration

Journal: Clinical Science (London, England : 1979)

Article Title: Unravelling the effects of selective estrogen receptor modulators on colorectal cancer: a prognostic role for insulin-like growth factor binding protein-5

doi: 10.1042/CS20258451

Figure Lengend Snippet: Panels ( A , B ) illustrate the effect of varying concentrations of 27-OHC and E2 on the abundance of ERβ and IGFBP-5 in HCT-116 and HT-29 cell lines. The experiment was conducted in three biological repeats ( n = 3), with four different concentrations of 27-OHC (0.001, 0.1, 1, and 10 μM) or E2 (0.625, 1.25, 2.5, and 5 nM) used for treatment. The raw data from each Western blot experimental group were analysed through Image J and normalised against the phenol red-free SFM control group and were presented as fold changes relative to the control. Statistical analysis of differences was conducted using a one-way ANOVA with Dunnett's post hoc test showing with mean ± SD.

Article Snippet: HCT-116 and HT-29 cells (3 × 10 5 cells/well) were seeded into six-well plates and transfected with 50 nM siRNA (Dharmacon, ON-TARGET plus Human ESR2 (2100) siRNA-SMART pool, 20 nmol (L-003402-00-0020)) as performed previously [ ] using RNAiMAX Lipofectamine (Invitrogen) in phenol red-free Opti-MEM medium.

Techniques: Western Blot, Control

Journal: Clinical Science (London, England : 1979)

Article Title: Unravelling the effects of selective estrogen receptor modulators on colorectal cancer: a prognostic role for insulin-like growth factor binding protein-5

doi: 10.1042/CS20258451

Figure Lengend Snippet: ( A ) Effects of short-term exposure to E2 and 27-OHC on cell proliferation in HT-29 colon cancer cells. Cells were treated with E2 (5, 10, and 20 nM) or 27-OHC (0.1 and 1 μM) for 15 min, followed by incubation in phenol red-free SFM. Corresponding vehicle controls included phenol red-free SFM alone and ethanol at concentrations of 0.01% and 0.1%, matching the solvent concentrations used for 27-OHC. All experiments were performed in three independent biological replicates, each in technical triplicate ( n = 3). Proliferation data were normalised to the phenol red-free SFM control and presented as fold change. Statistical analysis was conducted using one-way ANOVA with Dunnett's post hoc test. Data are presented as mean ± SD. ( B ) Differential gene expression in SW620 cells treated with 10 nM E2 for 24 h based on mRNA sequencing data ( GSE112568 ). The graphs depict up-regulation of GPER1 and down-regulation of ZEB1 following E2 treatment. Each group was analysed in duplicate ( n = 2). ( C ) Intracellular Ca 2+ concentrations and cAMP levels measured in HCT-116 and HT-29 cells following 15-min treatment with E2 (10 nM) or 27-OHC (0.1 and 1 μM). Control groups received phenol red-free SFM or 0.1% ethanol, respectively. All data were normalised to the SFM control and presented as fold change. Experiments were conducted in three independent biological replicates ( n = 3), each in technical triplicate. Statistical comparisons were made using one-way ANOVA with Dunnett's post hoc test. Data are shown as mean ± SD. ( D ) Anti-proliferative effects of G1, a selective GPER1 agonist, following a 15-min exposure in HCT-116 and HT-29 cells. Cells were subsequently incubated in phenol red-free SFM for 48 h. Additionally, mRNA expression of GPER1 following short-term exposure to E2 and G1 was assessed. All experiments were performed in triplicate ( n = 3), and data were normalised to vehicle controls. Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Data are expressed as mean ± SD. ( E ) GPER1 expression in CRC based on TCGA and GTEx datasets. E(i) Differential expression of GPER1 in colorectal tumour and normal tissues analysed via GEPIA2. For COAD, 275 tumour and 349 normal samples were included; for READ, 92 tumour and 318 normal samples were analysed. Gene expression is shown in log 2 (TPM + 1) units. Purple boxes represent normal tissues, and pink boxes represent tumour tissues. Statistical comparisons were performed using Student's t -test. E(ii) GPER1 expression levels in 438 TCGA colon cancer patients (204 females and 234 males), highlighting sex-specific expression differences. Data are shown as mean ± SD and analysed by Student's t -test. E(iii) Kaplan–Meier survival analysis of GPER1 and ERβ expression in 438 colon adenocarcinoma patients. Patients were stratified into high and low expression groups. The orange line denotes high expression and the blue line denotes low expression. Censored observations are indicated by plus signs (+). A steeper curve corresponds to lower survival probability. Statistical significance was P = 0.16 and P = 0.039.

Article Snippet: HCT-116 and HT-29 cells (3 × 10 5 cells/well) were seeded into six-well plates and transfected with 50 nM siRNA (Dharmacon, ON-TARGET plus Human ESR2 (2100) siRNA-SMART pool, 20 nmol (L-003402-00-0020)) as performed previously [ ] using RNAiMAX Lipofectamine (Invitrogen) in phenol red-free Opti-MEM medium.

Techniques: Incubation, Solvent, Control, Gene Expression, Sequencing, Expressing, Quantitative Proteomics