sEVs derived from Plekhm1 KO MSCs alter signaling in B-ALL cells and may promote B-ALL progression. (A) Representative immunoblot showing the expression of syntenin and syndecan-1 in BCR-ABL1⁺ BA/F3 cells after a 40-minute exposure to sEVs derived from WT or Plekhm1 KO MSCs (left). The quantification of syntenin (middle) and syndecan-1 (right) protein expression, normalized over the housekeeping protein GAPDH is shown on the right. The data represent the results of 6 (syntenin) or 7 (syndecan-1) independent experiments (t test). (B) Representative immunoblot showing the expression of pAKT (Ser473), AKT, syntenin, and syndecan-1 in BCR-ABL1⁺ BA/F3 cells after exposure to sEVs from WT or Plekhm1 KO MSCs with or without 20-μM dynasore for 6 hours (left) and quantification of pAKT (Ser473) expression (normalized to the +WT sEV control) (right). The data represent the results of 3 independent experiments (one-way ANOVA, Tukey multiple comparisons test). (C) Representative immunoblot showing the expression of pFAK (Tyr397) and FAK in BCR-ABL1⁺ BA/F3 cells after exposure to WT or Plekhm1 KO sEVs. The quantification of pFAK (Tyr397) expression, normalized over the housekeeping protein beta-actin, is shown on the right. The data represent the results of 7 independent experiments (t test). (D) Number of BCR-ABL1⁺ BA/F3 cells after a 40-minute exposure to WT or Plekhm1 KO sEVs, which migrated to the lower chamber of a transwell (5 μm pore size) within 6 hours. The medium in the upper vs the lower chambers of the transwell was substituted with 2% or 20% fetal bovine serum, respectively. (E) Kaplan-Meier style survival curve of mice transplanted with BCR-ABL1–transduced BM, which had previously been incubated for 1 hour with WT (red solid line) or Plekhm1 KO (blue dashed line) sEVs (sEVs from donor MSCs were incubated with BCR-ABL1-transduced BM cells in a 1:5 ratio) and transplanted into WT recipient mice (+WT sEVs n = 6; +Plekhm1 KO sEV n = 6; log-rank test). (F) Immunoblot showing pAKT (Ser473) and AKT expression in sorted primary GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells from the BM of WT recipient mice transplanted with BCR-ABL1–transduced BM cells previously exposed to WT or Plekhm1 KO sEV from panel E. Each lane represents an individual mouse. (G) Kaplan-Meier style survival curve of NOD SCID interleukin-2 (IL-2) receptor γ KO mice transplanted with 1.5 × 10⁶ SUBP15 cells, which had previously been incubated with WT (red line) or Plekhm1 KO (blue line) sEVs (sEVs from donor MSCs were incubated with SUBP15 cells in a 1:5 ratio) for 1 hour (P = .057; +WT sEVs n = 7; +Plekhm1 KO sEVs n = 7; Gehan-Breslow-Wilcoxon test). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.