FIG. 6.

Mutations in the CXXC motif and the addition of H₂O₂ abolish PAMM ability to inhibit osteoclast formation. (A, top panel) Stably transfected RAW 264.7 clones expressing wild-type (WT) or mutants of PAMM (C85G, C88G, C85G–C88G) were incubated for 5 days on 48-well plates in the presence of RANKL (50 ng/ml) and then stained for TRAP. Cells stably transfected with an empty vector (V) and mock-transfected cells (−) were used as controls. Cells expressing C85G, C88G, and C85G–C88G mutants generated numerous osteoclasts in response to RANKL, similar to mock-transfected cells or cells transfected with an empty vector. Cells transfected with a wild-type PAMM, conversely, did not form osteoclasts. (A, bottom panel) Western blot analysis of protein extracts from: mock-transfected RAW264.7 cells (−), wild-type PAMM (WT), C85G PAMM (85), C88G PAMM (88), C85G–C88G PAMM (85/88), and empty vector (V) by using an anti-PAMM and β-actin antibody shows that all transfected cells overexpress PAMM (with the exception of mock-transfected cells or cells transfected with an empty vector). (B) TRAP staining of: control RAW 264.7 cells (V), PAMM 1 and PAMM 2 stimulated with RANKL for 5 days without (−, left column) or with 50 μm H₂O₂ (+, right column) shows that the addition of H₂O₂ can reverse the effect of PAMM expression on osteoclast formation.