Fig 1: 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 2: CSF2 expression in gliomas.a Boxplots represent CSF2 FPKM values in normal brain samples (NB), low-grade gliomas (WHO grade II and III) and GBM (glioblastoma, WHO grade IV) TCGA datasets. b Boxplots represent CSF2 FPKM values in GBM samples with division into molecular subtypes.31 c Genes overexpressed in CSF2 expressing tumours (FDR p-value < 0.05 and FC > 3) were an input for GO enrichment. Dotplot shows the most enriched GO groups in CSF2 expressing GBMs, i.e. related to leukocyte activation, immune and defence responses. d Quantification of CSF2 expression using qPCR in healthy brain samples, Jurkat cells and glioma cell lines. e CSF2 protein levels in cells culture supernatants from normal human astrocytes and human glioma cell lines were determined using ELISA. Data are presented as means± from three independent experiments, performed in triplicates.
Fig 3: Development and characterisation of CSF2-depleted glioma cells.a–d Quantification of CSF2 mRNA (a, c) and protein (b, d) levels in parental LN18 glioma cells (a, b) and U87 glioma cells (c, d), and two clones of each shNeg and shCSF2 cells; normal human astrocytes and normal brain samples were used as a reference. Bars show means ± s.d., n = 3. e–h CSF2 knockdown did not affect the proliferation (e, g) and viability (f, h) of CSF2-depleted LN18 and U87 glioma cells, when compared to control shNeg cells. All results are expressed as the values relative to those obtained for control cells (shNeg1 = 100%) and are presented as the means ± s.d., n = 3.
Fig 4: Recruitment of microglia, but not astrocytes, is impaired in CSF2-depleted gliomas.a CSF2 depleted or shNeg LN18 glioma cells encapsulated in hollow fibres were transplanted to hemispheres of murine brain. After 14 days, mice were sacrificed and brain sections were stained by immunofluorescence using anti-Iba1 (green) and anti-GFAP (red) antibodies. Cell nuclei were counterstained with DAPI (blue). b Quantification of Iba1+ cells (microglia/macrophages) and GFAP+ cells (astrocytes) was performed by counting positive cells in 5 random fields. Data is presented as a relative cell density versus the hemisphere with shNeg-containing hollow fiber. The statistical significance was determined using Student’s t test, *p<0.05, **p<0.01; (mean ± s.d., n = 4 mice).
Fig 5: Apoptotic microglia/macrophages in CSF2-depleted gliomas.a Microglia/macrophages were visualized using Iba1 staining (green); cell nuclei were counterstained with DAPI (blue). DNA fragmentation was detected by TUNEL staining. Scale bar 100 µm. A right panel shows confocal microscopy images with z-stack projections and orthogonal views along the x and y axes, which demonstrate TUNEL+ nuclei in Iba1+ cells. b Quantification of TUNEL+Iba1+ cells in experimental gliomas (8 mice/shCSF2 group, 6 mice/shNeg group). The statistical significance was determined using Student’s t test, *p<0.05, **p<0.01.
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