(A) Schematic of MLF2 ProSRSA. (B) Top 10 candidate genes of MAGeCK analysis. (C and D) Western blotting analysis and quantification of MLF2 and PIM3 protein levels in U2OS and 143B stable cells. (E) 143B cells were subjected to co-IP using anti-PIM3 or anti-IgG to detect endogenous MLF2. (F, I, and K) HEK293T cells were cotransfected with the indicated plasmids for 48 hours and then subjected to IP assay. (G and H) HEK293T cells transfected with MLF2-SFB or S65A-SFB for 36 hours were incubated with 40 μg/mL cycloheximide (CHX) for the indicated periods and then subjected to Western blotting. Quantification of MLF2 protein levels was based on the Western blotting results. (J) Purified MLF2-SFB WT or S65A mutant protein were incubated with or without purified V5-PIM3 protein in vitro as described in the methods and then analyzed by Western blotting. (L) HEK293T cells cotransfected with MLF2-SFB and V5-PIM3 for 24 hours were incubated with PIM447 (10 μM) for another 24 hours and then subjected to IP assay. (M) Quantification analyses of migration and invasion assays using MLF2 WT or S65A knock-in 143B cells. (N) The procedure for in vivo orthotopic model of osteosarcoma metastasis and representative bioluminescence images of mice. (O) Quantification analyses of lung metastasis (n = 6). (P and Q) Western blotting analysis and quantification of MLF2 and PIM3 protein levels in human osteosarcoma tissues. N, normal; T, tumor. (R) The correlation between MLF2 and PIM3 protein levels is shown. The coefficient of correlation and P value were calculated using nonparametric Spearman’s test. Data in C, E–G, and I–L are representative of 3 independent experiments. Data in D, H, and M are presented as mean ± SD; n = 3 biologically independent experiments. P values were calculated using 1-way ANOVA with Dunnett’s test (D) and 2-tailed Student’s t test (H, M, O, and Q). NC, negative control; sh, short hairpin; sr, steradian; RRA, robust rank aggregation.