Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Cell Physiol Biochem 2016;38:859-870 - DOI:10.1159/000443040 Fig. 1. Expression and release of CCL2 after exposure of astrocytes to TNF-α. (a) Schematic of experimental protocol describing preparation of astrocytes. (b) CCL2 expression and release in astrocytes after a brief stimulation with TNF-α (10 ng/ml, 15 min). TNF-α evokes CCL2 expression and release even 3 h after TNF-α withdrawal. **P < 0.01, n = 3. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Cell Physiol Biochem 2016;38:859-870 - DOI:10.1159/000443040 Fig. 2. TNF-α-induced microglial activation and CCL2 expression and release after CCL2 siRNA treatment in astrocytes. (a and b) TNF-α-induced CCL2 expression (a) and release (b) after pre-treatment with CCL2 siRNA in cultured astrocytes. (c) The microglial cells were stained with an Iba1 antibody. Expression of Iba1 expression (green) in activated microglia as visualized by confocal microscopy. The blue staining represents DAPI. Scale bar = 50 µm. (d) Graph showing the mean fluorescence intensity (MFI) for Iba1. (e) Levels of Iba1 detected by Western blotting, quantified and normalized to GAPDH levels. Each value was then expressed relative to the control, which was set to 1. ##P < 0.01 versus CM from TNF-α-stimulated astrocytes, **P < 0.01, n = 3 separate cultures from different mice. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Cell Physiol Biochem 2016;38:859-870 - DOI:10.1159/000443040 Fig. 3. Migration of astrocyte-activated microglial cells in transwell assays, (a) Crystal violet-staining of primary microglia that migrated into the lower surface of the polycarbonate membrane inserts (8-µm pore size) at 24 h after seeding. Scale bar = 50 µm. (b) Graph demonstrating the average number of migrating cells per visual field in six random fields. #P < 0.05 versus CM from TNF-α-stimulated astrocytes, **P < 0.01, n = 3 separate cultures from different mice. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Cell Physiol Biochem 2016;38:859-870 - DOI:10.1159/000443040 Fig. 4. Expression of the M1 marker, CD86, after exposure to astrocyte-derived CCL2. (a) Microglial cells stained with CD86 antibody. CD86 expression (green) in activated microglia as observed using confocal microscopy. The blue staining represents DAPI. Scale bar = 50 µm. (b) Graph showing the mean fluorescence intensity (MFI) for CD86. (c and d) For flow cytometric analysis, the cells were incubated with FITC-conjugated CD86 antibody at 37°C for 1 hour. #P < 0.05 versus CM from TNF-α-stimulated astrocytes, **P < 0.01, n = 3 separate cultures from different mice. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Cell Physiol Biochem 2016;38:859-870 - DOI:10.1159/000443040 Fig. 5. Expression of M1 markers after stimulation with astrocyte-derived CCL2. The expression of M1 (TNF-α (a), IL-1β (b), CD86 (c), iNOS (d)) and M2 (IL-4 (e), IL-10 (f), arginase1 (g), CD206 (h)) were examined by quantitative RT-PCT. ##P < 0.01 versus CM from TNF-α-stimulated astrocytes, **P < 0.01 versus control group, n = 3 separate cultures from different mice. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Cell Physiol Biochem 2016;38:859-870 - DOI:10.1159/000443040 Fig. 6. The effect of astrocyte-derived CCL2 on microglia after inhibition of CCR2 with RS102895. Primary microglial cells were pre-treated with RS102895 (5 µM) for 1 h, then incubated with the CM from TNF-α-stimulated astrocytes. (a) Microglial cells were stained with Iba1 and CD86 antibodies. Iba1 and CD86 expression (green) in activated microglia as observed using confocal microscopy. The blue staining represents DAPI. Scale bar = 50 µm. (b and e) Levels of Iba1 detected by Western blotting, quantified and normalized to GAPDH levels. Values are expressed relative to the control, which was set to 1. (c) Crystal violet-staining of primary microglia that migrated into the lower surface of the polycarbonate membrane inserts (8-µm pore size) at 24 h after seeding. Scale bar = 50 µm. (d) Graph showing the mean fluorescence intensity (MFI) for Iba1 and CD86. (f) Graph illustrating the average number of migrating cells per visual field in six random fields. ##P < 0.01 versus CM from TNF-α-stimulated astrocytes, **P < 0.01, n = 3 separate cultures from different mice. (g and h) The expression of Ml (TNF-α, IL-1β, CD86, iNOS) and M2 (IL-4, IL-10, arginase1, CD206) were examined by quantitative RT-PCT. *P < 0.05, **P < 0.01 versus CM from TNF-α-stimulated astrocytes, n = 3 separate cultures from different mice. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0