plko tet on vectors  (Addgene inc)

Journal: Carcinogenesis

Article Title: Targeting RLIP with CRISPR/Cas9 controls tumor growth

doi: 10.1093/carcin/bgaa048

Figure Lengend Snippet: Target gene knockdown by CRISPR/Cas9. Schematic representation of a sensitive and efficient inducible CRISPR/Cas9 system: The pCW-CAS9 and pLX-sgRNA constructs were used for inducible CRISPR/Cas9-dependent RLIP gene knockdown.

Article Snippet: Then, pLX-U6-sgRLIP-Blast-5p, pLX-U6-sgRLIP-Blast-3p, and a Tet-inducible Cas9 vector, pCW-CAS9 (Addgene: #50661), were used for lentiviral production in HEK293T cells.

Techniques: CRISPR, Construct

Journal: Carcinogenesis

Article Title: Targeting RLIP with CRISPR/Cas9 controls tumor growth

doi: 10.1093/carcin/bgaa048

Figure Lengend Snippet: RLIP knockdown activated by doxycycline inhibits cell survival and tumorigenicity. MCF7 and MDA-MB231 Cas9/sg RLIP clones were treated with doxycycline (final concentration 0.5 µg/ml) for 24–96 h, and MTT assays were used to assess cell viability. Values are presented as mean ± SD from three independent experiments with eight replicates each (n = 24). Bars represent the percentage of surviving cells after doxycycline (doxy) treatment at each time point for all five clones from both cell lines (*P < 0.05 compared with control) (upper panels). Colony formation assays were performed and colonies were counted using an Innotech Alpha Imager HP. The bars represent the percent colony formation (*P < 0.03 compared with control) (lower panels).

Article Snippet: Then, pLX-U6-sgRLIP-Blast-5p, pLX-U6-sgRLIP-Blast-3p, and a Tet-inducible Cas9 vector, pCW-CAS9 (Addgene: #50661), were used for lentiviral production in HEK293T cells.

Techniques: Clone Assay, Concentration Assay

Journal: Carcinogenesis

Article Title: Targeting RLIP with CRISPR/Cas9 controls tumor growth

doi: 10.1093/carcin/bgaa048

Figure Lengend Snippet: Effect of Cas/sg RLIP on RLIP gene and protein expression. qPCR using RLIP primers (upper panels), Western blot analysis using antibodies against RLIP (middle panels), and TUNEL assays (lower panels) were performed using MCF7 and MDA-MB231 Cas9/sg RLIP clones (without [control] or with doxycycline treatment, final concentration 0.5 µg/ml) and normalized against β-actin. In qPCR values are means with 95% confidence intervals (n = 4 for each clone); *P < 0.01 and **P < 0.03 compared with respective controls. Data were analyzed using the 2−ΔΔCt method. Effect of Cas9/sg RLIP on apoptosis. MCF7 and MDA-MB231 Cas9/sg RLIP clones without or with doxycycline treatment (0.5 µg/ml, 48 h) were grown on coverslips. TUNEL assays using a Promega fluorescence detection kit were performed using a Zeiss LSM 510 META laser scanning fluorescence microscope with 520 and >620 nm filters. Photographs presented were taken at identical exposure with 400x magnification. Apoptotic cells showed green fluorescence and characteristics of cell shrinkage.

Article Snippet: Then, pLX-U6-sgRLIP-Blast-5p, pLX-U6-sgRLIP-Blast-3p, and a Tet-inducible Cas9 vector, pCW-CAS9 (Addgene: #50661), were used for lentiviral production in HEK293T cells.

Techniques: Expressing, Western Blot, TUNEL Assay, Clone Assay, Concentration Assay, Fluorescence, Microscopy

Journal: Carcinogenesis

Article Title: Targeting RLIP with CRISPR/Cas9 controls tumor growth

doi: 10.1093/carcin/bgaa048

Figure Lengend Snippet: Effect of CRISPR/Cas9-mediated RLIP knockdown on the size of subcutaneously implanted human BC (MDA-MB231) xenograft tumors in nude mice. Thirty 10-week-old mice were divided into five groups: (1) MDA-MB231, (2) MDA-MB231-Cas9/sg control, (3) MDA-MB231-Cas9/sg control + doxycycline, (4) MDA-MB231-Cas9/sg RLIP, and (5) MDA-MB231-Cas9/sg RLIP + doxycycline. Tumors grew more slowly in mice that received MDA-MB231-Cas9/sg RLIP + doxycycline (Group #5) than in mice in the other groups (panel A). The average wet weights of the tumors at day 60 was also lower for the Cas9/sg RLIP + doxycycline group (0.45 g) than for the other groups (Group #1: 2.1 g, Group #2: 2.0 g, Group #3: 1.8 g, and Group #4: 1.5 g, respectively; panel B). The Cas9/sg RLIP + doxycycline-treated mice did not exhibit any signs of overt toxicity, such as impaired movement or posture, indigestion, or areas of redness or swelling. The initial and final body weights of the controls and treated mice did not differ significantly (panel C).

Article Snippet: Then, pLX-U6-sgRLIP-Blast-5p, pLX-U6-sgRLIP-Blast-3p, and a Tet-inducible Cas9 vector, pCW-CAS9 (Addgene: #50661), were used for lentiviral production in HEK293T cells.

Techniques: CRISPR

Journal: Carcinogenesis

Article Title: Targeting RLIP with CRISPR/Cas9 controls tumor growth

doi: 10.1093/carcin/bgaa048

Figure Lengend Snippet: CRISPR/Cas9-mediated RLIP knockdown regulates the levels of signaling proteins in human BC (MDA-MB231) xenograft tumors in nude mice. Western blot analyses of signaling proteins in MDA-MB231 human BC tumor tissue lysates from (1) MDA-MB231, (2) MDA-MB231-Cas9/sg control, (3) MDA-MB231-Cas9/sg control + doxycycline, (4) MDA-MB231-Cas9/sg RLIP, and (5) MDA-MB231-Cas9/sg RLIP +doxycycline groups. β-actin was used as a loading control.

Article Snippet: Then, pLX-U6-sgRLIP-Blast-5p, pLX-U6-sgRLIP-Blast-3p, and a Tet-inducible Cas9 vector, pCW-CAS9 (Addgene: #50661), were used for lentiviral production in HEK293T cells.

Techniques: CRISPR, Western Blot

Journal: Carcinogenesis

Article Title: Targeting RLIP with CRISPR/Cas9 controls tumor growth

doi: 10.1093/carcin/bgaa048

Figure Lengend Snippet: Immunohistochemical analyses revealing that CRISPR/Cas9-mediated RLIP knockdown inhibits proliferation and angiogenesis markers in BC (MDA-MB231) xenograft tumors in mice. Sectioned tumor tissues from nude mice bearing Cas9/sg control and Cas9/sg RLIP MDA-MB231 tumors, without or with doxycycline treatment, were used for histopathologic analyses. Presented are H&E-stained sections and immunohistochemical analyses for the expression of RLIP, Ki67, CD31, E-cadherin, and vimentin. The difference between expression in sections from MDA-MB231 Cas9/sg RLIP tumor-bearing mice treated with doxycycline and sections from mice in the control groups was statistically significant (P < 0.02, determined by a two-tailed Student’s t-test). Photomicrographs at 40x magnification were acquired using an Olympus DP72 microscope. Percent staining was determined by measuring positive immunoreactivity per unit area. The intensity of antigen staining was quantified by digital image analysis using Pro Plus software. Bar represent mean ± SE (n = 5).

Article Snippet: Then, pLX-U6-sgRLIP-Blast-5p, pLX-U6-sgRLIP-Blast-3p, and a Tet-inducible Cas9 vector, pCW-CAS9 (Addgene: #50661), were used for lentiviral production in HEK293T cells.

Techniques: Immunohistochemical staining, CRISPR, Staining, Expressing, Two Tailed Test, Microscopy, Software