Fig 2: Dominant-negative p38aMAPK prevents dormancy induction in vitro for DKK3, VASN and NEO1, but not BMP1. (A) C4-2B4 cells stably-expressing empty vector or p38aDN mutant were treated with the indicated factors for 72 h and analyzed by live-cell imaging. Quantification of % quiescent cells that did not divide over 72 h relative to total cells examined (mean ± s.e.m.). n, number of cells monitored. N, number of independent experiments for C4-2B4-Vector cells: control (N = 4), DKK3 (N = 4), BMP1 (N = 3), vasorin (N = 3), neogenin (N = 3); and for C4-2B4-p38aDN cells: control (N = 5), DKK3 (N = 6), BMP1 (N = 3), vasorin (N = 3), neogenin (N = 3). P values were by t test. (B) Cells in (A) were treated with the indicated factors for 3 h and immunostained for p-p38 (upper and middle). All bars, 20 µm. Relative p-p38 signals in the nucleus of factor-treated cells compared to those in control cells (lower). n, number of nuclei analyzed. P values were by t test. #p < 0.0001; ns, not significant. (C) C4-2b-Vector or C4-2b-p38aDN cells were treated as in (A), and % cellular quiescence was analyzed by live-cell imaging. n, number of cells monitored. N, number of independent experiments for C4-2b-Vector cells: control (N = 3), DKK3 (N = 3), BMP1 (N = 3), vasorin (N = 3), neogenin (N = 2); and for C4-2b-p38aDN cells: control (N = 6), DKK3 (N = 5), BMP1 (N = 3), vasorin (N = 3), neogenin (N = 3). P values were by t test. (D) Cells in (C) were treated as in (B) and relative p-p38 signals in the nucleus were analyzed as in (B).

Fig 3: Bone-secreted factors induce cellular quiescence in PCa cells. (A) Dose response. C4-2B4 PCa cells were treated without or with various recombinant human bone-secreted factors at different concentrations and analyzed by live-cell imaging as in Fig. 1. About 100 cells were monitored for each factor at each concentration. Using this approach, we obtained an empirically-derived optimal concentration to be used for each factor (arrowheads): DKK3 (10 µg/mL), BMP1 (0.4 µg/mL), VASN (0.25 µg/mL), NEO1 (0.5 µg/mL), MIA (0.1 µg/mL), and NGAL (0.25 µg/mL). (B) (Left) Phase contrast brightfield images. Cells were plated in a Q4 glass-bottom dish, treated with and without various recombinant proteins, and analyzed by live-cell imaging. Representative images are shown for control cells and cells treated with recombinant human DKK3 (cell a), vasorin (cell b), neogenin (cell c), and BMP1 (cell d). Asterisks (*) follow a control cell through 3 cell divisions. Round cells are cells undergoing mitosis. (Right) Immunofluorescence images. At the end of live-cell imaging, cells were immediately fixed and co-immnostained for Ki67 (proliferation marker) and p27 (dormancy marker), and merged with phase contrast images. Cell outlines are traced for ease of view. All bars, 20 µm. (C) C4-2B4 cells were treated with various bone-secreted factors, using concentrations as determined in (A), and analyzed by live-cell imaging. PEDF was used at 0.25 µg/ml47. The dormancy factor BMP7 (0.4 µg/ml)50 was used as a positive control. % quiescent cells that did not divide relative to total cells counted were quantified (mean ± s.e.m), except for PEDF and BMP7 (mean ± s.d.). n, number of cells monitored. N, number of independent experiments for control (N = 24), DKK3 (N = 10), BMP1 (N = 10), vasorin (N = 9), neogenin (N = 4), MIA (N = 4), NGAL (N = 6), PEDF (N = 2), BMP7 (N = 2). P values were by t test. ns, not significant. (D) C4-2b cells were treated with various bone-secreted factors as indicated, monitored by live-cell imaging, and analyzed as in (C). n, number of cells monitored. N, number of independent experiments for control (N = 3), DKK3 (N = 3), BMP1 (N = 3), vasorin (N = 2), neogenin (N = 2). P values were by t test.