onecut2 overexpression plasmid (OriGene)
Structured Review

Onecut2 Overexpression Plasmid, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/onecut2+overexpression+plasmid/pmc08972808-54-1-8?v=OriGene
Average 92 stars, based on 3 article reviews
Images
1) Product Images from "Chemotherapy-induced extracellular vesicle miRNAs promote breast cancer stemness by targeting ONECUT2"
Article Title: Chemotherapy-induced extracellular vesicle miRNAs promote breast cancer stemness by targeting ONECUT2
Journal: Cancer research
doi: 10.1158/0008-5472.CAN-18-4055
Figure Legend Snippet: (A) MDA231, MCF-7, and BT474 cells were transfected with siRNA against ONECUT2 (GeneSolution/GS including equal mixture of 4 preselected siRNAs, or individual siRNA #1 and #2), or a control siRNA, or with PBS. After 48 h, cells were collected for sphere formation assay. (B) ALDEFLUOR assay of MDA231 cells transfected with siRNA as indicated for 72 h. (C) RT-qPCR-determined RNA levels of indicated genes in various BC cells at 48 h following transfection with indicated siRNA. (D) Western blots showing the expression levels of indicated proteins in BC cells at 48 h following transfection of ONECUT2 siRNA-GS or control siRNA. (E) MDA231 cells were transfected with indicated siRNA and seeded at an equal number on day 0. DTX (10 nM) or DOXO (500 nM) was added on day 1, and replenished every 24 h. On day 3, cell viability (left) and cell number (right) were determined by MTS assay and cell counting, respectively, and compared to the PBS treatment group. (F) RT-qPCR of indicated genes in MDA231 cells at 72 h after transfection with indicated siRNA. *P<0.05, **P<0.01, ***P<0.001 compared to control siRNA or as indicated. Numbers below Western images indicate quantification after normalization to GAPDH with the first lane set as 1.
Techniques Used: Transfection, Control, Tube Formation Assay, Quantitative RT-PCR, Western Blot, Expressing, MTS Assay, Cell Counting
Figure Legend Snippet: (A,B) MDA231 cells stably expressing a mammalian expression plasmid of human ONECUT2 cDNA or the empty vector were exposed to PBS or EVs from PBS/DTX/DOXO-treated MDA231 cells for 48 h before being analyzed by sphere formation assay (A) and Western blots (B). (C) RT-qPCR-determined RNA levels of indicated genes in MDA231 cells stably expressing ONECUT2 or vector. (D) MDA231, MCF-7, and BT474 cells transfected with the ONECUT2 expression plasmid or empty vector were analyzed by Western blots. (E) MDA231 cells stably expressing ONECUT2 or vector were seeded at an equal number on day 0. DTX (10 nM) or DOXO (500 nM) was added after 6 h, and replenished every 24 h. Cell viability (left) was measured by MTS assay every 24 h and cell number (right) was determined by cell counting on day 3. Data were normalized to the PBS control group. (F) RT-qPCR of indicated genes in MDA231 cells stably expressing ONECUT2 or vector. *P<0.05, **P<0.01, ***P<0.001. Numbers below Western images indicate quantification after normalization to GAPDH with the first lane set as 1.
Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Tube Formation Assay, Western Blot, Quantitative RT-PCR, Transfection, MTS Assay, Cell Counting, Control
Figure Legend Snippet: (A) A schematic representative showing putative binding sites of miR-9–5p, miR-203a-3p, and miR-195–5p in the 3’UTR of human ONECUT2 and the regions cloned into the luciferase reporter plasmid constructs. (B) MDA231 cells were transfected with psiCHECK2 reporter plasmids containing indicated ONECUT2 3’UTR region or with the psiCHECK2 vector (2 µg DNA per 2×105 cells). After 12 h, transfected cells were exposed to PBS or EVs from PBS/DTX (4 nM)/DOXO (125 nM)-treated MDA231 cells for 48 h before luciferase activities were measured. Ratio between Renilla luciferase and firefly luciferase activities (Rluc/Fluc) is shown. (C) MDA231 cells were co-transfected with indicated psiCHECK2 reporter plasmids (2 µg DNA per 2×105 cells) and miRNA mimics (individually or with a 1:1:1 mixture of miR-9–5p, miR-203a-3p, and miR-195–5p mimics for a total of 25 pmol). Luciferase activities were analyzed at 48 h. *P<0.05, **P<0.01, ***P<0.001.
Techniques Used: Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, Transfection
Figure Legend Snippet: (A) Left: Western blots showing modified expression of Rab27a and Onecut2 in indicated cell lines. Right: nanoparticle tracking analysis of EVs from an equal number of producing cells showing reduced EV secretion by MDA231/Rab27aKD cells. (B-E) Xenograft tumors were established in NSG mice by injecting 2×105 of indicated cells into the #4 mammary fat pad. When tumor size reached ~300 mm3, mice were treated weekly with DTX (15 mg/kg) for 3 weeks. (B) Tumor onset and volume. The time of DTX treatments were indicated by arrows. (C) Western blots of indicated proteins using tumors collected before and after DTX treatment. Numbers below Western images indicate quantification after normalization to GAPDH with the first lane set as 1. (D) RT-qPCR analysis of indicated genes using tumors collected before and after DTX treatment. (E) EVs were prepared from the sera of indicated mice before and after the 3-week DTX treatment. Levels of miRNAs were determined by RT-qPCR using a cel-miR-39–3p spike-in control for normalization. (F) Twelve pairs of pre- and post-NT human breast tumors were analyzed by IHC to determine the ONECUT2 expression levels in tumor cells. Wilcoxon test was performed. *P<0.05, **P<0.01, ***P<0.001.
Techniques Used: Western Blot, Modification, Expressing, Quantitative RT-PCR, Control
