Journal: Developmental biology

Article Title: Inducible Intestine-specific Deletion Of Krüppel-Like Factor 5 Is Characterized By A Regenerative Response In Adult Mouse Colon

doi: 10.1016/j.ydbio.2014.01.002

Figure Lengend Snippet: Quantitative PCR was performed on day 3 tamoxifen-treated RNAs as discussed in Methods. The results contained in Panel A show that Klf5, Ki-67, cyclin B1 & Cdk1 were several folds down-regulated when compared to the controls, while Reg3G, Reg1A, Reg3B, Cyp4B1 and Sox9 were up-regulated. Panel B represents immunoblot analyses of Klf5, Sox9, Reg1A and actin. Lane 1 represents control Klf5fl/fl colon lysates and lane 2 represents Klf5ERΔ samples. Actin was used as a loading control.

Article Snippet: Sox9 antibodies (used at 1:100 dilution; Cat# sc-20095) were obtained from Santa Cruz Biotechnologies were used for both immunohistochemistry (IHC) and immunoblots.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Developmental biology

Article Title: Inducible Intestine-specific Deletion Of Krüppel-Like Factor 5 Is Characterized By A Regenerative Response In Adult Mouse Colon

doi: 10.1016/j.ydbio.2014.01.002

Figure Lengend Snippet: Images are stained with DAPI (blue), Sox9 (red) and merged (DAPI + Sox9). Panels A, B and C show colonic tissues from day 5 tamoxifen-treated control Klf5fl/fl and Panels D through O, those from Klf5ERΔ mice. Panels D-F, G-I, J-L and M-O are representative of 3, 5, 7, and 11 day post-tamoxifen treatment respectively. White dashed lines (Panels B, E, H, K and N) mark the zone of epithelial cells staining positive for Sox9. Expansion of Sox9-positive zone was observed in Panels E, H & K compared to control (Panel B). Restoration of Sox9 expansion to the bottom of crypts was observed in Panel N similar to control (Panel B).

Article Snippet: Sox9 antibodies (used at 1:100 dilution; Cat# sc-20095) were obtained from Santa Cruz Biotechnologies were used for both immunohistochemistry (IHC) and immunoblots.

Techniques: Staining, Control

Journal: Developmental biology

Article Title: Inducible Intestine-specific Deletion Of Krüppel-Like Factor 5 Is Characterized By A Regenerative Response In Adult Mouse Colon

doi: 10.1016/j.ydbio.2014.01.002

Figure Lengend Snippet: T84 and HCT116 colon cancer cell lines were used in Panels A and B, respectively. T84 cells were transduced with lentiviruses containing either short hairpin RNA (shRNA) sequence against KLF5 or control shRNA. The lysates were collected from stable T84 knockdown or control cell lines and immunoblots performed for KLF5, Sox9 and actin (loading control) (Panel A). Lane 1 represents lysates from control shRNA transduction and lane 2 from KLF5-specific shRNA transduction. In Panel B, HCT116 cells were either mock-transfected (Lane 1), transfected with empty vector (Lane 2) or with pCI-Neo-KLF5 (Lane 3). Sox9 expression is inversely correlated to KLF5 expression in both T84 and HCT116 experiments (Panels A and B).

Article Snippet: Sox9 antibodies (used at 1:100 dilution; Cat# sc-20095) were obtained from Santa Cruz Biotechnologies were used for both immunohistochemistry (IHC) and immunoblots.

Techniques: Transduction, shRNA, Sequencing, Control, Knockdown, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Developmental biology

Article Title: Inducible Intestine-specific Deletion Of Krüppel-Like Factor 5 Is Characterized By A Regenerative Response In Adult Mouse Colon

doi: 10.1016/j.ydbio.2014.01.002

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) assays were performed as per Methods. HCT116 cells were transfected with KLF5, Sox9 or empty vector before collecting lysates for immunoprecipitations (IP) with Sox9, Reg1Aand cyclin D1 antibodies. DNA purified from the IP samples were then analysed with quantitative PCR using primers against Sox9, Reg1A and cyclin D1 respectively. Promoter binding affinity was plotted in the Y-Axis as Percent Input (%) against different promoter sequences in the X-axis. These results indicate that KLF5 strongly binds to Sox9, Reg1A and cyclin D1 promoters. Sox9 displays strong binding to Reg1A promoter and weak interactions with cyclin D1 and its own promoter.

Article Snippet: Sox9 antibodies (used at 1:100 dilution; Cat# sc-20095) were obtained from Santa Cruz Biotechnologies were used for both immunohistochemistry (IHC) and immunoblots.

Techniques: Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Purification, Real-time Polymerase Chain Reaction, Binding Assay