Fig 1: Senescent cells promote the migration of breast cancer cells via secreted bFGF and GM-CSF. (A-B) Representative crystal violet-stained images of transwell migration of MDA-MB-231, MDA-MB-468 and MCF-7 cells co-cultured with conditioned medium from young (PD29) or senescent (PD61) BJ cells, which were left untreated (None) or incubated with neutralizing antibodies against β-actin (1 ng/ml), bFGF (1 ng/ml), GM-CSF (5 ng/ml) or both bFGF and GM-CSF for 12 h (A), and quantification of number of migrated cells per field (mean ± SD, n = 3) (B). At least 5 randomly chosen 20X fields were counted for each of the triplicates (C-D) Representative crystal violet-stained images of transwell migration of MDA-MB-231, MDA-MB-468 and MCF-7 cells co-cultured with conditioned medium from young (PD29) or senescent (PD61) BJ cells transduced with shRNA control (SC) or shRNAs for bFGF or GM-CSF for 20 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. (B, D) ns, not significant; * p < 0.05; ** 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 2: 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)
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 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
from Cell Signaling Technology for Basic FGF (E9S5A) Rabbit mAb