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. 2020 Nov 9;62:103097. doi: 10.1016/j.ebiom.2020.103097

Fig. 6.

Fig. 6

Silencing of CXCL13 expression heightened MN loss and astrocytosis in non-transgenic and mSOD1 primary co-cultures. (a-c) Confocal micrographs showing the co-localisation of CXCL13 (red) with (a, b) the neuronal marker Smi32 (green) and with (c) the microglia marker Iba1; Scale bar, (a, b): 100 μm; (c): 200 μm. (d) CXCL13 concentration (pg/mL) in the conditional media of shRNA-CXCl13- and shRNA- scramble (SCR)-treated or untreated (UT) mSOD1 and Ntg co-cultured with mSOD1 and Ntg microglia/astrocytes, respectively. Data are expressed as the mean ± SEM from three independent experiments for each experimental group. *P < 0.05; ⁎⁎P < 0.001; ⁎⁎⁎P < 0.001 Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. (e) Representative SMI-32 immunostaining images (white) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg motor neurons co-cultured with mSOD1 and Ntg microglia/astrocytes, respectively; scale bar 200 μm. (f) Cell count analysis showing greater MN loss in both shRNA-CXCl13 treated mSOD1 and Ntg co-cultures than or shRNA-SCR- treated or untreated (UT) co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. *P < 0.05; ⁎⁎P < 0.001; ⁎⁎⁎P < 0.001; ⁎⁎⁎⁎P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. (g) Representative GFAP immunostaining images (green) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg astrocytes co-cultured with mSOD1and Ntg motor neurons/microglia, respectively; scale bar 100 μm. (h) Cell Area Fraction (percentage) analysis showing greater astrocytosis in shRNA-CXCl13 treated mSOD1 co-cultures than shRNA-SCR-treated or UT mSOD1 co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. *P < 0.05; ⁎⁎P < 0.01; ⁎⁎⁎P < 0.001 ⁎⁎⁎⁎P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. (i) Representative Iba1 immunostaining images (red) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg microglia co-cultured respectively with mSOD1and Ntg motor neurons/astrocytes; scale bar 100 μm. (j) Morphometric parameter of Iba1-positive microglia showing higher activation (mean microglial cell area) of shRNA-CXCl13-treated mSOD1 microglia than shRNA-SCR- treated or UT mSOD1 co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. *P < 0.05; ⁎⁎P < 0.001; ⁎⁎⁎P < 0.001; ⁎⁎⁎⁎P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis.