Fig. 6. Depletion of MLN51 reduces cell viability after stress release. (A) Upper panel: HeLa cells were transfected twice with a MLN51-specific siRNA, treated with arsenite before fixation and double stained with anti-MLN51 (green) and anti-FMRP (red) antibodies. Cells showing undetectable level of MLN51 and no FMRP-positive foci are indicated by arrows and cells showing no depletion of MLN51 and FMRP-positive foci are indicated by asterisks. Bottom panel, MLN51-silenced HeLa cells were transfected using a siRNA-insensitive plasmid pEYFP-MLN51ins and treated with arsenite and stained with anti-MLN51 (purple) and anti-FMRP (red) antibodies. A cell showing undetectable level of MLN51 and no FMRP-positive foci is indicated by an arrow and a cell showing expression of EYPF-MLN51 and FMRP-positive foci is indicated by an asterisk. (B) Silencing of MLN51 alters SG formation. SGs were traced using an anti-FMRP antibody in arsenite-treated HeLa cells transfected with either synthetic control siRNA (siRNA/control, black bar) or MLN51-specific siRNA (siRNA/MLN51, white bar). The numbers of cells showing clear FMRP-positive foci were counted. Values presented are mean percentages ± s.e.m. of n=2 independent experiments done in triplicate, with ^*** indicating a P value of less than 0.001. (C) Depletion of MLN51 results in reduced cell viability following stress. HeLa cells were transfected with either control synthetic siRNA (black circles) or MLN51-specific siRNA (white circles). 48 hours after transfection, cells were stressed with sorbitol for 2.5 hours, followed by incubation in normal medium. Cellular viability was measured at each time point. Values presented are mean percentages ± s.e.m. of a typical experiment made in triplicate with ^** and ^*** indicating a P value of less than 0.01 and 0.001, respectively. Top right: western blot analysis showing the level of MLN51 depletion in the experiment presented.