Figure 5.
TNFα secreted by B-ALL cells perpetuates the sEV-associated circuit via regulation of PLEKHM1 expression in MSCs. (A) Representative immunoblot of PLEKHM1, syntenin, and syndecan-1 expression in WT MSCs treated with 15 ng/mL TNF-α for 24 hours or cocultured with BCR-ABL1+ BΑ/F3 cells for 24 hours. The data represent the results of 4 independent experiments. (B) Representative immunoblot of PLEKHM1 expression in WT MSCs cocultured with or without BCR-ABL1+ BA/F3 cells and treated with IgG control (IgG) or adalimumab (ada). The data represent the results of 6 independent experiments. (C) Number of BCR-ABL1+ BA/F3 cells, which were exposed for 40 minutes to sEVs derived from untreated MSCs (control = ctrl) or MSCs treated with TNF-α for 24 hours and 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. The data represent the results of 4 independent experiments. (D) Representative immunoblot and its quantification showing expression of pFAK (Tyr397) and FAK in BCR-ABL1+ BA/F3 cells after exposure to sEVs derived from untreated MSCs (control = ctrl), MSCs treated with TNF-α for 30 minutes or 24 hours. The values are normalized to the housekeeping protein GAPDH. The data represent the results of 4 to 5 independent experiments (one-way ANOVA, Dunnett multiple comparisons test). (E) Representative immunoblot and its quantification showing expression of PLEKHM1 in HS5 cells after coculture with vehicle, SUPB15 (BCR-ABL1+), NALM6 (BCR-ABL1−), or K562 cells for 24 hours. The values are normalized to the housekeeping protein vinculin. The data represent the results of 5 independent experiments (one-way ANOVA, Dunnett multiple comparisons test). (F) Representative immunoblot and its quantification showing expression of PLEKHM1, phospho-p65, and p65 in HS5 cells treated with vehicle or 15 ng/mL human TNF-α (hTNF-α) for 24 hours. The results represent 5 independent experiments (t test). (G) Representative immunoblot and its quantification showing expression of PLEKHM1 in primary human MSCs treated with vehicle or 15 ng/mL hTNF-α for 24 hours. The results represent 5 independent experiments (t test). (H) Representative immunoblot and showing expression of syntenin, flotillin-1, and CD81 and quantification of syntenin in sEVs isolated from HS5 cells treated with vehicle or human TNF-α for 24 hours. The results represent 6 independent experiments (t test). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

TNFα secreted by B-ALL cells perpetuates the sEV-associated circuit via regulation of PLEKHM1 expression in MSCs. (A) Representative immunoblot of PLEKHM1, syntenin, and syndecan-1 expression in WT MSCs treated with 15 ng/mL TNF-α for 24 hours or cocultured with BCR-ABL1+ BΑ/F3 cells for 24 hours. The data represent the results of 4 independent experiments. (B) Representative immunoblot of PLEKHM1 expression in WT MSCs cocultured with or without BCR-ABL1+ BA/F3 cells and treated with IgG control (IgG) or adalimumab (ada). The data represent the results of 6 independent experiments. (C) Number of BCR-ABL1+ BA/F3 cells, which were exposed for 40 minutes to sEVs derived from untreated MSCs (control = ctrl) or MSCs treated with TNF-α for 24 hours and 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. The data represent the results of 4 independent experiments. (D) Representative immunoblot and its quantification showing expression of pFAK (Tyr397) and FAK in BCR-ABL1+ BA/F3 cells after exposure to sEVs derived from untreated MSCs (control = ctrl), MSCs treated with TNF-α for 30 minutes or 24 hours. The values are normalized to the housekeeping protein GAPDH. The data represent the results of 4 to 5 independent experiments (one-way ANOVA, Dunnett multiple comparisons test). (E) Representative immunoblot and its quantification showing expression of PLEKHM1 in HS5 cells after coculture with vehicle, SUPB15 (BCR-ABL1+), NALM6 (BCR-ABL1−), or K562 cells for 24 hours. The values are normalized to the housekeeping protein vinculin. The data represent the results of 5 independent experiments (one-way ANOVA, Dunnett multiple comparisons test). (F) Representative immunoblot and its quantification showing expression of PLEKHM1, phospho-p65, and p65 in HS5 cells treated with vehicle or 15 ng/mL human TNF-α (hTNF-α) for 24 hours. The results represent 5 independent experiments (t test). (G) Representative immunoblot and its quantification showing expression of PLEKHM1 in primary human MSCs treated with vehicle or 15 ng/mL hTNF-α for 24 hours. The results represent 5 independent experiments (t test). (H) Representative immunoblot and showing expression of syntenin, flotillin-1, and CD81 and quantification of syntenin in sEVs isolated from HS5 cells treated with vehicle or human TNF-α for 24 hours. The results represent 6 independent experiments (t test). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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