Fig 1. Graphical abstract illustrating the role of ISRAA in mouse brain astrocytes growth regulation through the cytokine network involving IFN-γ. ISRAA acted on embryonic brain astrocytes early at E7 and late at E21. It stimulated them to produce IFN-γ. E7 astrocytes did not express IFN-γR; therefore, they could not respond to the anti-proliferative effects of IFN-γ and continued to proliferate. However, E21 cells did express IFN-γR, and, accordingly, they ceased proliferation and began to differentiate.

Fig 2. Embryonic brain astrocyte proliferation and differentiation and illustration of ISRAA activity on these events. Spontaneous and ISRAA induced cell proliferation and differentiation were assessed by MTT and by morphological observation and determination. Astrocytes were prepared and cultured as described in the Materials and Methods section. Non-stimulated astrocytes from E7 showed high proliferation (Fig 2A; black bar) and no clear differentiation (represented in Fig 2E). E21 astrocytes showed lower proliferation (Fig 2A; black bar) with clear cell differentiation (represented in Fig 2F). Stimulation of E7 with recombinant ISRAA resulted in increased cellular proliferation (Fig 2A; grey bar) and also represented in Fig 2G. In response to ISRAA stimulation, E21 showed less proliferation (Fig 2A; grey bar) and well differentiated cells (represented in Fig 2H). Pre-incubation with anti-ISRAA antibody abrogated the proliferative response in E7 and induced E21 to proliferate (Fig 2B). Intrinsic ISRAA expression in embryonic mouse brain astrocytes was illustrated by immunohistochemistry. As exemplified in Fig 2C, E7 demonstrates few faint cells stained for ISRAA, while E21 showed high numbers of positively and strongly stained cells for ISRAA (exemplified in Fig 2D). Experiments were conducted twice with alike findings. Pictures were gathered via bright field microscopy (x630). Bars denote the means ±SD of eight cultures (***p<0.05).

Fig 3. Spontaneous and ISRAA-induced IFN-γ production by embryonic astrocytes. Cell Released Capturing ELISA (CRC-ELISA) was used assess spontaneous and ISRAA-induced IFN-γ production by embryonic astrocytes. E7 and E21 astrocytes spontaneously produced this cytokine. Stimulation with recombinant ISRAA significantly increased IFN-γ production by E7 and E21 (***p<0.05). However. the induction of IFN-γ was significantly higher in E21 than in E7 astrocyte cultures (***p<0.05). Experiments were conducted twice with alike findings. Bars represent the means ± SDs of 12 cultures.

Fig 4. Expression of IFN-γR by embryonic brain astrocytes. IFN-γR was detected by immunohistochemistry. Counter staining was conducted with haematoxylin. As depicted in the micrograph, E7 astrocytes did not express receptors for IFN-γ (Fig 4A), while E21 astrocytes showed strong expression of IFN-γR (Fig 4B). Pictures were acquired with light microscopy (x630). The micrograph is a representation of several fields scanned in each chamber. All fields showed expression of IFN-γR on E21, but not E7.

Fig 5. Blocking effects of anti-IFN-γ antibody on ISRAA-induced proliferation suppression of E-21 astrocytes. The rationale behind the IFN-γ blocking experiment was to find if the ISRAA-induced proliferation suppression of E21 was achieved by IFN-γ produced in response to ISRAA or not. Incubation with anti-IFN-γ antibody in cultures of E21 astrocytes 15 min prior to ISRAA stimulation significantly inverted the inhibitory effects of ISRAA on proliferation (***p<0.05), but had no effect on E7 (bars with horizontal lines). The IFN-γ blocking effect was obvious on E21 as they express the IFN-γR, but not E7. Experiments were conducted twice with alike results. Bars represent the means ± SDs of 12 cultures.

Fig 6. Illustration of the IFN-γ signaling pathway activated on embryonic brain astrocytes upon ISRAA stimulation. To demonstrate the IFN-γ signalling pathway activation on embryonic brain astrocytes after ISRAA stimulation, tyrosine kinase activity and nuclear localisation of the transcription factor STAT1 in E21 astrocytes were examined using Western blot analyses and immunohistochemistry respectively. A. Phosphorylation of tyrosine-specific protein. Cell lysates were prepared 5min, 15min and 60min after ISRAA stimulation. Note the slight amounts of tyrosine phosphorylated proteins in the non-stimulated control lysates (Ctrl) on lane 1. ISRAA stimulation resulted in a strong and fast rise in tyrosine phosphorylation, which reached its highest point by 15min and declined to the control level at 60min (ISRAA; lane 2, 3, 4). An irrelevant antibody (Ctrl Ab; lane 5, 6, 7) was used to control the specificity of the phosphorylated protein. The band representing activated TPKs (lane 3, 15 min on ISRAA) was not detected. Tyrphostin A47 (a tyrosine kinase specific inhibitor) blocked the ISRAA induced protein kinase as demonstrated by the inhibition of the at the various detection times (A47; lane 8, 9, 10). B. Nuclear localisation of the STAT1. Nuclear localisation of the STAT1 protein was detected in E21 astrocytes 5min, 15min and 60min after ISRAA stimulation. The shown pictures were obtained by a digital camera using a fluorescence microscope at x630 magnification after 15min incubation. B.1 represents non-stimulated cells (note the generalised cytoplasmic antigen distribution before ISRAA treatment; red stained cytoplasm and black non-stained nuclei). B.2 depicts the nuclear translocation of STAT1 15 min after ISRAA stimulation (heavily stained nuclei in red). B.3 demonstrates the blocking effects of tyrphostin A47 on the ISRAA promoted nuclear translocation of STAT1.