MMP9 inhibitors attenuate activated TGF-β signaling in human RPS14-deficient cells and in bone marrow cells from a patient with del(5q) MDS. (A) Human BMCD34⁺ cells were cultured in liquid culture media. After 5 days of culture, cells were treated by rMMP9 for 2 days, and protein was collected for western blot analysis. (B) Human BMCD34⁺ cells were cultured in liquid culture media. After 3 days of culture, cells were treated by rMMP9 for 2 days followed by SB431542 treatment for an additional 2 days. At 7 days after culture, cells were analyzed for the CD71 expression by using flow cytometry. (C) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14. After 5 days of transduction, cells were treated by MMP9 inhibitors for 2 days and were analyzed for pSMAD2/3 or SMAD2/3 expression by using flow cytometry. (D) RNA was isolated from bone marrow CD34⁺ cells from a patient with del(5q) MDS or healthy control subjects and analyzed by using RT-qPCR. (E) BMCD34⁺ cells from a patient with del(5q) MDS or a healthy control subject were cultured in liquid culture media. After 1 day of culture, cells were treated by MMP9 inhibitors for 4 days. Cells were cultured for an additional 4 days without MMP9 inhibitors and then analyzed for CD71 and CD235a expressions by using flow cytometry. (F) BMCD34⁺ cells from a patient with del(5q) MDS or a healthy control subject were cultured in liquid culture media. After 1 day of culture, cells were treated by MMP9 inhibitors for 4 days and then analyzed for pSMAD2/3 or SMAD2/3 expression by using flow cytometry. (G) Model of the defective erythroid development in RPS14 deficiency through increased MMP9 expression. Data are representative of 3 independent transduction experiments (A-C) or 1 independent experiment (D-F). *P < .05, **P < .01, ***P < .001.