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Additional file 1: Supplementary Figure 1. A The pH of the medium was adjusted by HCl or NaOH and stabilized in a 5% CO2 incubator. the medium pH was measured using a pH meter. B The pH of the medium was adjusted by HCl or NaOH, and MDA-MB-231 cells were incubated for 18 h. mRNA levels were analyzed by qPCR. Supplementary Fig. 2. A–C MDA-MB-231 cells were treated with various conditioned media for 18 h. Protein levels were analyzed by western blotting (A), mRNA levels were analyzed by qPCR (B), and the culture medium pH was measured using a pH meter (C). D, E MDA-MB-231 cells were treated with lactic acid, with/without, NaOH for 24 h. Protein levels were analyzed by western blotting (D) and the culture medium pH was measured using a pH meter (E). Supplementary Fig. 3. A MDA-MB-231 cells were treated with lactic acid or sodium lactate, with/without, NaOH for 24 h. Protein levels were analyzed by western blotting. B MDA-MB-231 cells were treated with various conditioned media with/without NaOH for 18 h. Protein levels were analyzed by western blotting. C The summarization of the process by which various conditioned media are prepared. CON: control; fresh media, NCM: normoxic conditioned media, HCM: hypoxic conditioned media, Oxamate pre-treated HCM). D-F The various conditioned media were treated in MDA-MB-231 breast cancer cells for 18 h. Cells were analyzed by westernblotting (D) and qPCR (E), and cultured media pH was measured using a pH meter (F). G MDA-MB-231 cells were treated with a nifuroxazide under acidic conditions for 18 h. Protein levels were analyzed by western blotting. Supplementary Fig. 4. A Acidic media were treated in MDA-MB-231 breast cancer cells for 18 h. Cell lysates were analyzed by westernblotting. Supplementary Fig. 5. Densitometric and statistical analysis of all western blot data in main figures. Supplementary Fig. 6. Densitometric and statistical analysis of all western blot data in supplementary figures. Supplementary Fig. 7. Full-length blots in Figure 1. The red dotted lines are the main figures. Supplementary Fig. 8. Full-length blots in Figure 2. The red dotted lines are the main figures. Supplementary Fig. 9. Full-length blots in Figure 3. The red dotted lines are the main figures. Supplementary Fig. 10. Full-length blots in Figure 4. The red dotted lines are the main figures. Supplementary Fig. 11. Full-length blots in Supplementary Figure 2. The red dotted lines are the main figures. Supplementary Fig. 12. Full-length blots in Supplementary Figure 3.The red dotted lines are the main figures. Supplementary Fig. 13. Full-length blots in Supplementary Fig. 4.The red dotted lines are the main figures.

Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures.

The naive T-cell pool in peripheral lymphoid tissues is fairly stable in terms of number, diversity and functional capabilities in spite of the absence of prominent stimuli. This stability is attributed to continuous tuning of the composition of the T-cell pool by various homeostatic signals. Despite extensive research into the link between signal transducer and activator of transcription 3 (Stat3) and T-cell survival, little is known about how Stat3 regulates homeostasis by maintaining the required naive T-cell population in peripheral lymphoid organs. We assessed whether the elimination of Stat3 in T cells limits T-cell survival. We demonstrated that the proportion and number of single-positive thymocytes as well as T cells in the spleen and lymph nodes were significantly decreased in the Stat3-deficient group as a result of the enhanced susceptibility of Stat3-deleted T lymphocytes to apoptosis. Importantly, expression of the anti-apoptotic Bcl-2 and Bcl-xL was markedly decreased in Stat3-deleted single-positive thymocytes and T lymphocytes, suggesting that Stat3 helps to maintain the T-cell pool in the resting condition by promoting the expression of Bcl-2 family genes. These findings suggest the importance of Stat3 in the integration of homeostatic cues for the maintenance and functional tuning of the T-cell pool.

IL-7 receptor (IL-7R) plays critical roles in lymphocyte development by promoting survival and proliferation and by inducing V(D)J recombination in TCR and Ig loci. Here, we demonstrate that IL-7R-activated Stat5 binds to consensus motifs in the 5′ regions of Jγ segments and induces germline transcripts. We also show that a constitutively active form of Stat5 restores V-J recombination of TCRγ genes and partially rescues T cell development from IL-7R−/− T cell precursors, especially in favor of γδ T cells. Therefore, this study reveals a potential role of Stat5 in T cell development and also implies that IL-7R may control the accessibility of the TCRγ locus through Stat5-induced germline transcription.