Journal: PloS one

Article Title: The CREB-miR-9 negative feedback minicircuitry coordinates the migration and proliferation of glioma cells.

doi: 10.1371/journal.pone.0049570

Figure Lengend Snippet: Figure 5. CREB regulates NF1 and knocking down CREB inhibits migration. (A) The migratory capacity of AD-shcreb or AD- shNC-infected U87MG, T98G and U251 cells were analyzed in a transwell migration assay (top). The bound crystal violet staining was released and quantified by measuring the OD570-630 (mean 6 SD, n = 3) (bottom). (B) The mRNA and protein levels of NF1 were detected in control T98G and U251 cells or in cells with CREB knocked down by quantitative RT-PCR and western blotting, respectively. *, P,0.05; **, P,0.01; ***, P,0.001, two-tailed unpaired Student’s t test. (C) T98G cells were transfected with miR-9 antagomirs or controls followed by infection of adenovirus-mediated shRNA for CREB (AD-shcreb) or AD- shNC, and the protein level of NF1 was detected by western blotting. doi:10.1371/journal.pone.0049570.g005

Article Snippet: Quantitative real-time PCR The expression levels of pri-miR-9-1, -2, and -3 as well as CREB and NF1 mRNAs were determined by real-time PCR using a SYBR-green-containing PCR kit (Takara) according to the manufacturer’s instructions.

Techniques: Migration, Infection, Transwell Migration Assay, Staining, Control, Quantitative RT-PCR, Western Blot, Two Tailed Test, Transfection, shRNA

Journal: PloS one

Article Title: The CREB-miR-9 negative feedback minicircuitry coordinates the migration and proliferation of glioma cells.

doi: 10.1371/journal.pone.0049570

Figure Lengend Snippet: Figure 6. MiR-9-1 is under CREB’s control. (A) Location of putative CREs within the 59 flanking regions of miR-9-1 and miR-9-2. MiR-9-1 is located in an intron of the gene c1orf61 (chromosome 1), whereas miR-9-2 is located in an exon of linc00461 (chromosome 5). Three pairs of primers (miR-9- 1-a, miR-9-1-b and miR-9-2) were designed to detect the binding capacity of CREB to the predicted CREs of miR-9-1-a, miR-9-1-b and miR-9-2, respectively, by ChIP-qPCR assays. Both the 59 flanking sequences (2 kb) and the pre-miRNA bodies of miR-9-1 and miR-9-2 were inserted upstream of the luciferase reporter (gray box shown by LUC). The arrows denote the positions of primers used for ChIP-qPCR. (B) ChIP-qPCR assays were performed in T98G and U251 cells to detect the binding capacity of CREB to the putative CREs of miR-9-1 and miR-9-2 (mean 6 SD, n = 3). (C) In AD- shNC/AD-shcreb-infected T98G and U251 cells, ChIP-qPCR was performed to detect the binding capacity of CREB on CRE-miR-9-1-a (mean 6 SD, n = 3). (D) CREB enhances the transcription of miR-9-1. The 59 flanking sequences (22 kb+miR-9-1; 22 kb, 2570 bp; 2560 bp+miR-9-1) without mutations or with a mutation of CRE-a (2569+miR-9-1, from TGACGGGC to TGGAGGGC) in miR-9-1 were inserted upstream of the luciferase cassette. The luciferase reporter constructs were co-transfected with CREB expression plasmids or control vectors and the normalized luciferase activity was determined (mean 6 SD, n = 4). (E) The mRNA expression levels of CREB, pri-miR-9-1 and mature miR-9 were detected in T98G and U251 cells infected with AD-shcreb or AD-shNC by quantitative RT-PCR (mean 6 SD, n = 3). *, P,0.05, **, P,0.01, two-tailed unpaired Student’s t test. doi:10.1371/journal.pone.0049570.g006

Article Snippet: Quantitative real-time PCR The expression levels of pri-miR-9-1, -2, and -3 as well as CREB and NF1 mRNAs were determined by real-time PCR using a SYBR-green-containing PCR kit (Takara) according to the manufacturer’s instructions.

Techniques: Control, Binding Assay, ChIP-qPCR, Luciferase, Infection, Mutagenesis, Construct, Transfection, Expressing, Activity Assay, Quantitative RT-PCR, Two Tailed Test

Journal: PloS one

Article Title: The CREB-miR-9 negative feedback minicircuitry coordinates the migration and proliferation of glioma cells.

doi: 10.1371/journal.pone.0049570

Figure Lengend Snippet: Figure 7. A low concentration of glucose induces the expression of miR-9. (A and B) T98G cells were maintained in DMEM/high glucose (4.5 g/L, HG) or DMEM/low glucose (1.0 g/L, LG) DMEM for 24 h. Cells were then harvested for RNA and protein extraction. The mRNA expression levels of miR-9, pri-miR-9-1, pri-miR-9-2, CREB and NF1 were determined by quantitative RT-PCR (mean 6 SD, n = 3), and the protein levels of CREB and NF1 were detected by western blotting. (C) The negative feedback minicircuitry comprised of miR-9 and CREB. MiR-9 is highly expressed in glioma cells with amplification of the miR-9-2 gene copy number. The up arrow denotes genomic amplification. The transcription of miR-9-1 is regulated by CREB and miR-9 can directly target the 39UTR of CREB, forming negative feedback minicircuitry. CREB inhibits the migration of glioma cells by elevating the expression level of NF1, whereas miR-9 promotes migration by directly targeting NF1 and CREB. In addition, miR-9 inhibits the proliferation of glioma cells by directly targeting the proliferation-promoting transcription factor, CREB. (D) The balance between miR-9 and CREB coordinates the migration and proliferation of glioma cells. The glioma cells with low levels of miR-9 and high CREB protein levels prefer to proliferate rather than migrate. As the glioma progresses, certain events, such as glucose reduction and mir-9-2 gene copy number amplification trigger the substantial increase of miR-9. By targeting migration-inhibitory CREB and NF1, miR-9 promotes the migration of glioma cells accompanied by proliferation repression. doi:10.1371/journal.pone.0049570.g007

Article Snippet: Quantitative real-time PCR The expression levels of pri-miR-9-1, -2, and -3 as well as CREB and NF1 mRNAs were determined by real-time PCR using a SYBR-green-containing PCR kit (Takara) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Protein Extraction, Quantitative RT-PCR, Western Blot, Amplification, Migration

Journal: Molecular Cancer

Article Title: Reversal of chemosensitivity and induction of cell malignancy of a non-malignant prostate cancer cell line upon extracellular vesicle exposure

doi: 10.1186/1476-4598-12-118

Figure Lengend Snippet: EV-mediated transfer of proteins via Kinexus phospho-protein microarray analysis in DU145 cells co-cultured with PrEC EVs

Article Snippet: To determine the proteins that are involved in or might be responsible for “phenotypic switching”, we utilized Kinexus phospho-protein microarray analysis (Vancouver, BC) in the DU145 cells co-cultured with PrEC EVs.

Techniques: Microarray