Fig 2: Effects of pulmonary and/or renal SP-A on sepsis-induced AKI. (A) Quantitative analysis showed that serum creatinine level was significantly higher in infected KO mice compared to infected WT and hTG mice. Furthermore, when compared to infected WT mice, hTG mice had significantly higher renal injury score. *P< 0.05, **P<0.01, t test (n= 6 mice/group). (B) Renal histological analysis by H&E staining showed normal kidney architecture in all sham groups of mice, suggesting that SP-A deficiency in the kidney did not affect kidney development and formation of normal kidney structure. Sepsis induced AKI was characterized by the presence of vacuolar degeneration of tubular cells (arrows), and brush border loss with tubular lumen dilatation (stars). Magnification 400×. Scale bars= 50μm. (C) Semi-quantitative analysis demonstrated that renal injury score was significantly higher in infected KO mice compared to infected WT and hTG mice. Furthermore, when compared to infected WT mice, hTG mice had significantly higher renal injury score. *P< 0.05, **P<0.01, t test (n= 6 mice/group). (D) Renal NGAL immunofluorescence staining showed sepsis induced AKI was characterized by the presence of NGAL. Magnification 200×. Scale bars= 100 μm. (E) Semi-quantitative analysis demonstrated that renal NGAL fluorescence density was significantly higher in infected KO mice compared to infected WT and hTG mice. Furthermore, when compared to infected WT mice, hTG mice had significantly higher renal injury score. *P< 0.05, **P<0.01, t test (n= 6 mice/group). (F, G) Western blot analysis demonstrated increased cleaved caspase-3 protein level in infected KO and hTG mice compared to infected WT mice 48 hours after infection, *P< 0.05. (H, I) Western blot analysis demonstrated increased cleaved caspase-1 protein level in infected KO and hTG mice compared to infected WT mice 48 hours after infection, *P< 0.05, (n=3 mice/group).

Fig 3: Overexpression of lipocalin 2 in the brain induces anxiety-like behaviors in the light and dark task. Single intracerebroventricular injection of recombinant lipocalin 2 protein (7 μL, 1 μg/mL) or vehicle in the brain of the mouse, and light and dark tasks were assessed after 6 days. A Representative heat maps for the activities of each group during the light–dark test. The frequency during the time spent in the light compartment (B), the latency to the first entry into the light compartment (C), or the cumulative time in the light compartment (D) were determined. The velocity traveled in the light compartment is measured in (E). Quantitative data are presented as mean ± SEM (n = 5 per group). The Student’s t-test was used to examine the significance of the mean. * p < 0.01, vs. the vehicle or control group. LCN2: lipocalin 2, NS: not significant, SEM: standard error of mean

Fig 4: 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.

Fig 5: Overexpression of lipocalin 2 in the brain induces anxiety-like behaviors in the EPM task. Single intracerebroventricular injection of recombinant lipocalin 2 protein (7 μL, 1 μg/mL) or vehicle in the brain of the mice and the EPM task were performed after 6 days. Time spent in the open arms (A), closed arms (B), or center (C) of the EPM apparatus. D Velocity traveled in the EPM apparatus. Quantitative data are presented as mean ± SEM (n = 5 per group). The Student’s t-test was used to examine the significance of the mean. ** p < 0.01 vs. the vehicle group. EPM: elevated plus maze, LCN2: lipocalin 2, NS: not significant, SEM: standard error of mean