Fig. 6

Crotonylation enhanced cell invasive capability by mediating SEPT2-K74Cr-P85α-Akt pathway activity (A, B) P85α interacts with SEPT2. Whole-cell lysates were immunoprecipitated with an anti-SEPT2 antibody (A) and GST-tagged SEPT2-WT or GST-tagged SEPT2-K74R were purified with anti-GST antibody-conjugated beads B. C SEPT2-K74R overexpression decreased the expression of downstream P85α. D SEPT2-K74R overexpression decreased P85α stability. Transient overexpression vectors carrying SEPT2 WT and SEPT2-K74R in SNU449 cells were treated with cycloheximide (CHX), and the P85α protein level was determined by WB (upper panel). The lower panel shows the relative protein levels in the different groups. Error bars represent ± SD on the basis of triplicate experiments. E, F P85α rescued SEPT2 function after sodium crotonate (NaCr) treatment. Knocking down P85α expression inhibited cell migration (E) and invasion (F) after NaCr treatment; overexpression of P85α in SEPT2-K74R-expressing cells rescued the cell migration (E) and invasion (F) abilities after NaCr treatment. G P85α rescued SEPT2 function after NaCr treatment. Quantitative comparison of wound-healing assays performed with Huh7 cells (upper panel) and SNU449 cells (lower panel). H Western blot analysis of proteins expressed in the P85α rescue assays.The data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA, two-way ANOVA). NC, negative control; WT, wild-type SEPT2; K74R, SEPT2-K74R mutant; Cr, NaCr treatment at25 mM; sh, short interfering RNA downregulation of p85α expression; oe, p85α overexpression