Fig. 3: Proline activates cGMP-PKG signaling to promote breast cancer stem-like traits.

A, B The enriched KEGG pathways of downregulated genes following shP1-1 (A) and shP1-2 MDA-MB-231 cells (B). C Overlapping the enriched KEGG pathways from downregulated genes of MDA-MB-231 shP1-1 cells and shP1-2 cells. The common pathways were listed. D The cGMP levels were measured in MDA-MB-231 tumor and PY8119 tumor following PYCR1 knockdown (n = 3). E Relative mRNA levels of GUCY1A2, PRKG1 and PRKG2 were determined following PYCR1 knockdown in MDA-MB-231 tumor (n = 6). F Relative protein levels of sGC, PRKG1 and PRKG2 were determined following PYCR1 knockdown in MDA-MB-231 tumor. G The cGMP levels were determined following PYCR1 knockdown and supplemental addition of proline in PY8119 tumor (n = 3). H Relative mRNA levels of Gucy1a2, Prkg1 and Prkg2 were determined following PYCR1 knockdown and supplemental addition of proline in PY8119 tumor (n = 6). I The protein levels of sGC, PRKG1 and PRKG2 were determined following PYCR1 knockdown and supplemental addition of proline in PY8119 tumor. J ALDH-positive populations were analyzed following treatment proline and PKGi in MDA-MB-231 cells. Differences of ALDH-positive cells among groups were analyzed (n = 3). K Sphere formation ability was analyzed following treatment proline and PKGi in MDA-MB-231 cells. The representative images were presented (Upper, scale bar = 100 μm) and the number (Bottom Left) and diameter (Bottom Right) of spheroids were measured and counted (n = 4). PKGi, PKG inhibitor. Graph data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values were calculated with two-tailed, unpaired Student’s t-test (D, E) or one-way ANOVA (G, H, J, K).