Journal: Toxics

Article Title: Effects of Short-Term Exposure to Polystyrene Nanoplastics on the Nervous System: Calcium Homeostasis, BDNF and Synaptic Plasticity

doi: 10.3390/toxics14020178

Figure Lengend Snippet: Role of BDNF in the mechanism of synaptic plasticity impairment. All indicators were detected in neurons from the control and PS-NPs intervention groups. ( A ) Western blot analysis of CREB, BDNF, Cav3.1, and CaMKIIα in neurons, with GAPDH as the loading control. ( B ) Protein expression levels of CREB in the neurons. n = 3. ( C ) Protein expression levels of BDNF in the neurons. n = 3. ( D ) Calcium ion content in the neurons. n = 6. ( E ) Protein expression levels of the calcium channel Cav3.1 in neurons. ( n = 3). ( F ) Protein expression levels of CaMKIIα in neurons. n = 3. (○) represents the individual samples within each group. Quantitative results were normalized, and the data in the bar charts are presented as mean ± SD. ** p < 0.01 or *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skim milk (Beyotime, Shanghai, China, P0216-1500 g) and incubated overnight at 4 °C with specific primary antibodies, including Cav3.1 (1:2000, NBP2-59322, Novus, Lone Tree, CO, USA), CaMKIIα (1:2000, 3357S, CST, Danvers, MA, USA), CREB (1:2000, 12208-1-AP, Proteintech, Rosemont, IL, USA), BDNF (1:1000, ab226843, abcam, Cambridge, UK), PSD95 (1:1000, 2507S, CST), SYN (1:2000, 17785-1-AP, Proteintech), ProBDNF (1:1000, 28205-1-AP, Proteintech), and GAPDH (1:2000, UM4002, YouKang, Tianjin, China).

Techniques: Control, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Downregulation of microRNA-27b-3p enhances tamoxifen resistance in breast cancer by increasing NR5A2 and CREB1 expression.

doi: 10.1038/cddis.2016.361

Figure Lengend Snippet: Figure 4 NR5A2 and CREB1 are direct targets of miR-27b-3p. The mRNA expression (a–d) and protein levels (e–h) of NR5A2 and CREB1 were downregulated by miR-27b- 3p in breast cancer cells. MCF-7 (a and e), MCF-7/TAM-1 (b and f), T47D (c and g) and T47D/TAM-1 (d and h) cells were transfected with miR-27b-3p mimics or inhibitors and the negative control (NC), respectively. RT-PCR was performed to detect the mRNA expression of NR5A2 and CREB1. Western blot was performed to detect the protein expression of NR5A2 and CREB1. Actin was used as a loading control. Data were from three independent experiments. (e–h) (B) and (C) Relative protein levels of NR5A2/Actin and CREB1/ Actin were quantified using Image J software. Data are mean ± S.D. from three independent experiments. **Po0.01; ***Po0.001, compared with the control group. (i) The predicted miR-27b-3p target sites in the 3′UTR of NR5A2 and CREB1 mRNA and their mutated version. (j–m) Luciferase activity assays in MCF-7 and 293T cells showed that miR-27b-3p inhibited the expression of NR5A2 and CREB1. MCF-7 and 293T cells were cotransfected with pGL3 vector containing the wild type or mutated 3′UTR of NR5A2 and CREB1, pGL3-Control vector, along with miR-27b-3p mimics and inhibitors or NC. After 48 h, luciferase activity was detected. Data were normalized to luciferase activity in the corresponding cells transfected with NC and are represented as the mean ± S.D. of three replicates

Article Snippet: The NR5A2 and CREB1 expression vectors were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Software, Luciferase, Activity Assay, Plasmid Preparation

Journal: Cell death & disease

Article Title: Downregulation of microRNA-27b-3p enhances tamoxifen resistance in breast cancer by increasing NR5A2 and CREB1 expression.

doi: 10.1038/cddis.2016.361

Figure Lengend Snippet: Figure 5 Overexpression of NR5A2 and CREB1 reverses reduction of cell viability and induction of apoptosis by miR-27b-3p mimics, and depletion of NR5A2 and CREB1 reverses induction of cell viability and reduction of apoptosis by miR-509-5p inhibitors in tamoxifen-treated cells. (a–h) MCF-7/TAM-1 (a) and T47D/TAM-1 (b) cells were cotransfected with negative control (NC) or miR-27b-3p mimics along with control (Ctr) or NR5A2 or CREB1 vectors. MCF-7 (c) and T47D (d) cells were cotransfected with NC or miR-27b-3p inhibitors along with NC or NR5A2 or CREB1 siRNA. After 8 h, cells were treated with indicated dose of 4-hydroxytamoxifen (TAM) for additional 48 h. (a–d) MTT assay was performed to examine cell viability. (e–h) Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, S.D.; **Po0.01; ***Po0.001, compared with NC-treated cells

Article Snippet: The NR5A2 and CREB1 expression vectors were purchased from Origene (Rockville, MD, USA).

Techniques: Over Expression, Negative Control, Control, MTT Assay, Staining, Flow Cytometry

Journal: Cell death & disease

Article Title: Downregulation of microRNA-27b-3p enhances tamoxifen resistance in breast cancer by increasing NR5A2 and CREB1 expression.

doi: 10.1038/cddis.2016.361

Figure Lengend Snippet: Figure 7 MiR-27b-3p is negatively correlated with NR5A2 and CREB1 mRNA levels in breast cancer. (a and b) Relative expression of NR5A2 (a) and CREB1 (b) along with miR-27b-3p was determined by RT-PCR in 32 breast cancer tissues from patients with untreated-tamoxifen. (c and d) Relative expression of NR5A2 (c) and CREB1 (d) along with miR-27b-3p was determined by RT-PCR in 20 breast cancer tissues from tamoxifen-resistant patients. For NR5A2 and CREB1, β-Actin was used as an internal control; for miR-27b-3p, U6 was used as an internal control. Their expression correlation was analyzed by correlation coefficient and t-test

Article Snippet: The NR5A2 and CREB1 expression vectors were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cell death & disease

Article Title: Downregulation of microRNA-27b-3p enhances tamoxifen resistance in breast cancer by increasing NR5A2 and CREB1 expression.

doi: 10.1038/cddis.2016.361

Figure Lengend Snippet: Figure 8 A schematic model depicting miR-27b-3p downregulation promotes tamoxifen resistance in breast cancer cells. Estrogen receptor (ER) inhibitor tamoxifen (TAM) represses miR-27b-3p levels, and estrogen (EST) increases miR-27b-3p levels in breast cancer cells. TAM promotes cell apoptosis by inhibiting ER. On the other hand, miR-27b-3p downregulation by TAM blocks cell apoptosis by upregulating CREB1 and NR5A2. NR5A2 increases ER mRNA levels. CREB1 induces the expression of aromatase, and aromatase promotes the biosynthesis of EST and enhances breast cancer cells resistance to tamoxifen

Article Snippet: The NR5A2 and CREB1 expression vectors were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing