Fig. 4

NP treatment inhibits protein synthesis in osteosarcoma cells. a, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) for 24 h, and STAT3 mRNA expression was determined by RT-PCR. b, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) with or without protein synthesis inhibitor cycloheximide (CHX) for 0, 12, and 24 h. Cytoplasmic extracts were prepared and analyzed by Western blot using anti-STAT3 antibodies. c and d, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) in the presence and absence of proteasome inhibitor MG132 (5.0 μM) for 24 h. Cytoplasmic extracts were prepared and analyzed by Western blot using anti-STAT3 antibodies. e and f, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) with or without lysosome protein degradation inhibitor Baf-A1 (200 nM) for 24 h, and cytoplasmic extracts were prepared and analyzed by Western blot using anti-STAT3 antibodies. g, ¹⁴C-valine–labeled MG63 cells were treated with vehicle and NP (3.0 μM) in the presence and absence of MG132 (5.0 μM) for 4, 12, and 16 h, and protein degradation was analyzed as described in the Methods. *P < 0.05 versus vehicle control; ** P < 0.01 versus vehicle control