Additional file 3: Figure S3. of Coronin 2A (CRN5) expression is associated with colorectal adenoma-adenocarcinoma sequence and oncogenic signalling

CRN5 is methylated by PRMT5, increases cell proliferation, and sensitises cells to TRAIL-induced cell death. (A) Co-immunoprecipitation experiment using lysates from U373 cells with stable over-expression of GFP-CRN5 and anti-GFP magnetic microbeads. Precipitates were subjected to SDS-PAGE, stained with Coomassie brilliant blue, and proteins were identified by mass spectrometry analysis. Arrows indicate the positions of GFP-CRN5 and PRMT5. (B) Pull-down assay using GST-CRN5 or GST for control coupled to glutathione-coated sepharose beads and soluble His6-PRMT5 confirm a direct interaction of CRN5 and PRMT5. Proteins were expressed and purified from E. coli strain BL21. Asterisk, GST signal from partial degradation of GST-CRN5. For illustration purposes the order of lanes from the original immunoblots were digitally re-arranged to omit dispensable lanes. (C) In vitro methylation experiments based on the incubation of recombinant PRMT5/MEP50 complex, GFP-CRN5, and 3H-labelled S-adenosylmethionine showed methylation of CRN5. Controls lacked the substrate GFP-CRN5. Columns indicate mean values and standard deviations derived from three independent experiments. (D) Immunoblot verifications of PRMT5 knock-down and FLAG-CRN5 over-expression in HeLa cells. (E) Left panel: HeLa cell viability after double-transfections as indicated in absence (dark columns) and presence (light columns) of TRAIL ligand. Viability of control cells transfected with both empty vectors is set to 100 %. One representative out of ten independent experiments with similar patterns of cell viability is shown; note, that the level of absolute absorbance values varied from experiment to experiment and did not allow calculation of a mean value. Right panel: For better illustration of the results the three light columns indicate the decrease of cell viability, i.e. the rate of TRAIL-induced cell death. This was calculated by the differences of the values from the three right pairs of dark and light bars from the left panel minus the TRAIL-induced decrease of cell viability of the control cells (first left pair of bars). (TIFF 521 kb)