Fig 2: AFMSCs immunosuppress PHA-stimulated PBMCs. Bright field observation and flow cytometry analysis of CFSE, Ki67 and PCNA were used for qualitative and quantitative analysis of PHA-stimulated PBMCs and UCMSCs and AFMSCs immunosuppression effect (A), bar = 100 µm. Statistical analysis of CFSE (B), Ki67 (C) and PCNA (D) positive rate in different groups (n = 7 for CFSE, n = 6 for Ki67 and PCNA, *P < .05). Specific genes expression of mRNA was assayed, IL-6 (E), IFN-? (F) and TNF-ß (G) were for immuno-activity, and Ki67 (H) and PCNA (I) were for proliferation (n = 3, *P < .05). AFC was indicated AFMSCs cultured by AFC1 media

Fig 3: Muscarinic signaling regulates the acinar lineage and SOX2+ cells.(A–B) E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. White dashed line outlines acini. Arrows indicate double positive SOX2+EdU+ cells. Scale bar is 50 µm. The number of SOX2+, EdU+ and SOX2+EdU+ cells was quantified in B. (C–E) E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP (10 µM). The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS, normalized to control and expressed as percentage of total ECAD+ cells (C). In D and E, cultured glands were immunostained for SOX2, nerves (TUBB3), AQP5, KRT19 and Ecadherin (ECAD). Images are 50 µm projections of 5 µm confocal sections. Scale bars are 100 µm. (F–H) E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and immunostained for AQP5 (F) and the number of AQP5+ and KRT19+ cells counted (G). SMG+SLG were also subjected to qPCR analysis (H). Data in B, C, G and H are means ±s.d of three biological replicates and three experiments. Data in B, C and G were subjected a Student’s t-test. *p<0.05 **p<0.01. Data in H were analyzed using a one-way analysis of variance with a post-hoc Dunnett’s test. *p<0.05, **p<0.01. Additional data for this figure in Figure 5—figure supplement 1.DOI: http://dx.doi.org/10.7554/eLife.26620.02210.7554/eLife.26620.023Figure 5—source data 1.Source data relating to Figure 5B.E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. The number of SOX2+, EdU+ and SOX2+EdU+ cells were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.02310.7554/eLife.26620.024Figure 5—source data 2.Source data relating to Figure 5C.E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP (10 µM). The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS, normalized to control and expressed as percentage of total ECAD+ cells. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.02410.7554/eLife.26620.025Figure 5—source data 3.Source data relating to Figure 5G.E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and the number of AQP5+ and KRT19+ cells counted. Counts were normalized to the control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.02510.7554/eLife.26620.026Figure 5—source data 4.Source data relating to Figure 5H.E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and subjected to qPCR analysis. Data were normalized to Rsp29 and control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.026

Fig 4: Axl reduction in PCa cells suppresses the cellular dormancy of PCa cells in bone marrow.(A) Tumor growth monitored by bioluminescence imaging (BLI) in marrow. PCa sh Control and sh Axl cells labeled with luciferase were injected into tibiae of SCID mice (n = 5). (B) Immunohistochemistry showing Ki-67 expression in PCa sh Control and PCa sh Axl cells in BM. PCa cells were recognized by pan cytokeratin with green color, and Ki-67 was shown with red color. Scale bar, 20 µm. Ki-67 negative and positive PCa cells were counted and the percentage of Ki-67 negative PCa cells was shown. *p < 0.05 compared to PC3 sh Control cells. #p < 0.05 compared to DU145 sh Control cells. (C) Immunohistochemistry showing TGFGBR2 expression in PCa sh Control and PCa sh Axl cells in BM. PCa cells were recognized by pan cytokeratin with green color, and TGFBR2 was shown with red color. Scale bar, 10 µm. TGFBR2 positive and negative PCa cells were counted and the percentage of TGFBR2 positive PCa cells was shown. *p < 0.05 compared to PC3 sh Control cells. #p < 0.05 compared to DU145 sh Control cells. (D) Immunohistochemistry showing TGFGBR3 expression in PCa sh Control and PCa sh Axl cells in BM. PCa cells were recognized by pan cytokeratin with green color, and TGFBR3 was shown with red color. Scale bar, 10 µm. TGFBR3 positive and negative PCa cells were counted and the percentage of TGFBR3 positive PCa cells was shown. **p < 0.01 compared to PC3 sh Control cells. N.D. means not detected.

Fig 5: Tumor cell-intrinsic PD-L1 alters in vitro BC cell proliferation. MTT viability assay of MB49 (A) and RT4 (C) control and PD-L1KO cell lines at 72 h. MB49 (B) and RT4 (D) cell counts after control and PD-L1KO cells were uniformly seeded in 12-well plates for 72 h. (E) Comparison of BC cell-intrinsic PD-L1 effects between cell lines. Flow cytometry staining for Ki67 of MB49 (F) and RT4 (G) cells after 72 h. P, unpaired t-test. SSC-A, side scatter area