Figure 4.

Upregulation of the TGF-β signaling axis by progranulin depletion. a) Kasumi-1 cells were cultured with anti-progranulin antibody (200 μg/ml) or control antibody for 8 h. RNA extraction and mRNA microarray were performed, and significantly upregulated pathways were identified. b) Progranulin specific siRNA or control siRNA transfected Kasumi-1 cells were cultured for 48 h. Whole cell lysate was collected and expression of TGF-β, P-Smad2 were analyzed by western blotting. c) Expression level of TGF-β was normalized to that of β-actin, expression level of P-Smad2 was normalized to that of t-Smad2. Error bars indicate the SD from mean; n = 4. (∗P < 0.05; two-tailed student's t test) d) The supernatant was collected 48 h after transfection and extracellular TGF-β level was determined by Ella Simple Plex. Error bars indicate the SD from mean; n = 4. (∗P < 0.05; two-tailed Student's t test) e) Expression level of P-Smad3 and t-Smad3 in Kasumi-1 cells transfected progranulin-specific siRNA or control siRNA were analyzed by Western blotting. f) Total RNA was extracted from untreated cell lines to perform RT-qPCR. Expression level of TGF-β receptor type II was normalized to that of GAPDH. Error bars indicate the SD from mean; n = 4. (∗∗∗P < 0.001 compared with Kasumi-1; Dunnett's test).