Double positive thymocytes survive without LKB1.

<p>(A) CD4^pos thymocytes purified from LKB1^fl/fl CD4Cre^neg or CD4Cre^pos thymi using MACS were lysed at 3×10⁷ cells mL⁻¹ in lysis buffer. Proteins extracted from cells and denatured were resolved on NuPAGE Bis-Tris 4–12% gels under reducing conditions and subsequent immunoblots were probed for indicated proteins, showing that LKB1 protein was efficiently deleted. GSK3 and β-catenin were used as loading controls for equal loading. Data are representative of two independent experiments. (B) Freshly isolated thymi from LKB1^fl/fl CD4Cre^neg or LKB1^fl/fl CD4Cre^pos mice were mashed to single cell suspensions. Cell number of double positive (DP) thymocytes was determined from a given volume using calibrated counting beads and from the frequency of cells co-stained for the MHC-receptors CD4 and CD8 of total number of thymocytes. Data are summary of four to six mice, where each symbol represents one mouse. (C) Single cell suspensions of freshly isolated thymi from LKB1^fl/fl CD4Cre^pos or littermate controls were seeded at a cell density of 5−10×10⁶ cells mL⁻¹ in complete culture medium for 24 h. Frequency of live DP thymocytes was determined by staining for surface co-expression of CD4 and CD8 and exclusion of cells positive for the DNA binding dye DAPI. Statistical analysis using the Mann-Whitney test showed comparable frequencies of DAPI^neg DP cells between LKB1^fl/fl CD4Cre^pos and controls. Data summarise three independent experiments. (D) Thymocytes were isolated and 1×10⁶ cells placed into the fibronectin-coated upper chamber of the transwell plate. Cells were left to migrate into the lower chamber containing medium only or 500 ng mL⁻¹ CXCL12 for three hours. Cells from the lower chamber were collected and counted using counting beads using flow cytometry and the frequency of cells migrated was determined against the input that was used as putatively maximal migration capacity. Data summarise three independent experiments showing mean ± SEM.