FIG 4.

Supernatants from B. abortus-infected macrophages induce apoptosis of osteocytes. MLO-Y4 cells were treated with supernatants (added at a 1:2 proportion) from noninfected (NI) or B. abortus-infected macrophages (J774.A1 cell line), and 24 h after stimulation apoptosis was evaluated by annexin V/PI, TUNEL, and Hoechst 33342 assays. (A) Flow cytometry analysis of apoptotic cells by annexin V/PI. (B) Hoechst 33342 staining and TUNEL assays using fluorescence microscopy. (C and D) Quantitative analysis of experiments presented in panel B. H₂O₂ (200 μM) was used as a positive control. NT, nontreated cells. Data are given as the means ± SD from at least three individual experiments. *, P < 0.1; **, P < 0.01; ***, P < 0.001, versus results in noninfected cells.