Fig 2: Senescent cells induce JNK activation via secreted GMCSF and bFGF(A-B) Western blotting analysis of the phosphorylation/activation status of Stat3, AKT, ERK and JNK in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cell lines co-cultured with medium (None) or young (PD31) or senescent (PD56) BJ cells. (C-D) Western blotting analysis of phosphorylated/activated JNK in MDA-MB-231 and MDA-MB-468 breast cancer cell lines co-cultured with medium (None) or young (PD31) or senescent (PD59) BJ cells, which were incubated with neutralizing antibodies against IgG, GM-CSF (αG, 5 ng/ml), bFGF (αF, 1 ng/ml), or both bFGF and GM-CSF (αG + αF). (E-F) Western blotting analysis of phosphorylated/activated JNK in MDA-MB-231 and MDA-MB-468 breast cancer cell lines treated with GM-CSF, bFGF or both. (B, D, F) Quantification of the Western blot results presented in A, C and E, respectively, showing expression levels of indicated proteins relative to None (B), None + IgG (D) or Ctrl (F). Densitometric quantification of bands in the Western blots was performed by ImageJ. The signals were normalized to that of actin

Fig 3: JNK mediates the stimulation of breast cancer migration by senescent cell-secreted GM-CSF and bFGF(A) Western blotting analysis showing knockdown specificity and efficiency of JNK1 and JNK2 shRNAs in MDA-MB-231 and MDA-MB-468 cells. (B-C) Representative crystal violet-stained images of transwell migration of MDA-MB-231 and MDA-MB-468 cells co-cultured with young (PD31) or senescent (PD59) BJ cells transduced with shRNA control (SC) or shRNAs for JNK1 or JNK2 for 12 h (C), and quantification of number of migrated cells per field (mean ± SD, n = 3) (D). At least 5 randomly chosen 20X fields were counted for each of the triplicates. (D-E) Representative crystal violet-stained images of transwell migration of MDA-MB-231 cells transduced with shRNAs for JNK1 or JNK2 or shRNA control (SC) and treated with vehicle control, 20 ng/ml of GM-CSF, 10 ng/ml of bFGF or both for 12 h (D), and quantification of number per field (mean ± SD, n = 3) of migrated MDA-MB-231 and MDA-MB-468 cells transduced with shRNAs for JNK1 or JNK2 or shRNA control (SC) and treated with vehicle control, 20 ng/ml of GM-CSF, 10 ng/ml of bFGF or both for 13 h (E). At least 5 randomly chosen 20X fields were counted for each of the triplicates. (C, E) **p < 0.01 and ***p < 0.001 between indicated groups in unpaired, 2-sample t tests (dotted lines) or One-way ANOVA corrected for multiple comparisons using Tukey’s multiple comparison correction adjustment (solid lines)

Fig 4: Senescent cells secrete bFGF and GM-CSF that promote the motility of breast cancer cells(A) Relative mRNA levels (mean ± SD, n = 3) of GM-CSF, bFGF and CXCL1 in young (PD31) and senescent (PD62) BJ cells as determined by quantitative real time PCR analysis. (B) Relative protein levels (mean ± SD, n = 3) of GM-CSF, bFGF and CXCL1 in the conditioned medium from young (PD31) and senescent (PD62) BJ cells as determined by ELISA. (C-D) Representative crystal violet-stained images of transwell migration of MDA-MB-231, MDA-MB-468 and MCF-7 cells co-cultured with medium containing DMSO, 10 ng/ml of bFGF, 20 ng/ml of GM-CSF or both for 12 h (C), and quantification of number of migrated cells per field (mean ± SD, n = 3) (D). At least 5 randomly chosen 20X fields were counted for each of the triplicates. (E-F) Representative images of MDA-MB-231, MDA-MB-468 and MCF-7 cells immediately (0 h), 24 h (24 h, for MDA-MB-231 and MDA-MB-468) or 48 h (48 h, for MCF-7) after a scratch wound was made and co-cultured with medium containing DMSO, 10 ng/ml of bFGF, 20 ng/ml of GM-CSF or both (E), and quantification of the distance of the wound edges by ImageJ at 24 h (for MDA-MB-231 and MDA-MB-468) or 48 h (for MCF-7) (mean ± SD, n = 3) (F). (A-B, D, F) * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. BJ-PD31 in unpaired, 2-sample t-tests (A-B) or between indicated groups in One-way ANOVA corrected for multiple comparisons using Tukey’s multiple comparison correction adjustment (D, F)