Fig 1: miR-425 suppresses transcription factor ZNF24 in astrocytes. (A) Predicted miR-425 binding sites within the CREB1, BCOR, and ZNF24 3′-UTRs from TargetScan. (B) ZNF24 mRNA expression is significantly decreased in astrocytes overexpressing miR-425. CREB1 and BCOR mRNA expression is unchanged as measured by RT-qPCR. (C) ZNF24 protein expression is decreased in astrocytes transfected with miR-425 mimic as indicated by Western blot analysis. (D) miR-425 suppresses ZNF24 3′-UTR activity as measured by dual luciferase reporter assay. (E) Ectopic expression of ZNF24 significantly decreases CCL8 and VEGFA mRNA expression, while KITLG is unchanged. mRNA expression measured by RT-qPCR. (F) Ectopic expression of ZNF24 suppresses CCL8 protein expression, but not SCF as indicated by Western blot analysis. (G) ZNF24 knockdown significantly increases CCL8 and VEGFA mRNA expression, but not KITLG. mRNA expression as indicated by RT-qPCR. (H) ZNF24 knockdown increases CCL8 and SCF protein expression as measured by Western blot analysis. Fold change was calculated in panels (B,D,E,G). Student’s t-test was used in panels (B,D,E,G). N = 3 experimental replicates unless otherwise indicated.
Fig 2: miR-425-activated astrocytes secrete CCL8 and SCF to further activate astrocytes and promote mammospheres. (A) Top four highly secreted cytokines in CM from astrocytes overexpressing miR-425 as measured by cytokine arrays. (B) Cytokine stimulation increases GFAP expression (red) in human astrocytes as indicated by GFAP IF. All slides were imaged at 4× or 20× using the Keyence BZ-X810 microscopes (cat. # BZ-X810 Keyence). At least 300 cells per slide were evaluated to identify GFAP+ cells. Nuclei were stained with DAPI (blue). Representative 20× images. Scale bar indicates 100 µm. (C) CCL8, SCF, and MIF stimulation promotes SKBR3 mammospheres. Scale bar indicates 100 µm. (D) CCL8 and SCF promote mammosphere formation of CN34 cells. Scale bar indicates 100 µm. (E) CCL8 and KITLG are upregulated in astrocytes overexpressing miR-425 as measured by RT-qPCR. (F) Human astrocytes stimulated by CM from SKBR3 cells overexpressing miR-425 secrete significantly higher levels of CCL8 compared to astrocytes stimulated with CM from control SKBR3 cells. Secreted CCL8 expression measured by ELISA. (G) Astrocytes stimulated by CM from CN34 overexpressing miR-425 secrete significantly increased levels of CCL8 compared to astrocytes stimulated with CM from control CN34 cells. Secreted CCL8 expression measured by ELISA. (H,I) Astrocytes stimulated by CM from SKBR3 or CN34 cells overexpressing miR-425 secrete high levels of SCF. Secreted SCF expression measured by ELISA. Fold change was calculated in panel (E). Student’s t-test was used in panels (B–I). N = 3 experimental replicates unless otherwise indicated.
Supplier Page from Abcam for Mouse SCF ELISA Kit