Figure 6

Synoviocytes apoptosis induced by PBP. A-C: Apoptosis detected using cofocol fluorescence microscope. Synoviocytes were incubated for 24 h with 5 μM PBP (B) and 40 μM celecoxib (C), and PBS as a negative control (A). They were stained by Hoechst33342 and PI. We had observed that viable cells, apoptotic, and necrotic cells. On the one hand, the Hoechst33342 dye stained the nuclei of all cells; therefore apoptosis might show nuclear changes such as chromatin condensation and nuclear fragmentation. So in PBP treatment groups, we could see these changes (see what was directed by arrow in B and C). On the other hand, PI uptake indicated the loss of membrane integrity characteristic of necrotic and late apoptotic cells. D-F: Apoptosis detected using FACS. Synoviocytes were incubated for 24 h with 5 μM PBP (E) and 40 μM celecoxib (F), and PBS as a negative control (D). They were stained by Annexin⊠-FITC and PI. Then, the viability was determined using the FCM. Annexin⊠-FITC (-) and PI (-), living cells; Annexin⊠-FITC (+) and PI (-), early apoptotic cells; Annexin⊠-FITC (+) and PI (+), late apoptotic cells and necrotic cells. The numbers showed the percentages of each fraction.