Fig. 1: Characterization of the deleterious effects of ACM from Tg mutSOD1 mice. | Nature Communications

Fig. 1: Characterization of the deleterious effects of ACM from Tg mutSOD1 mice.

From: Systematic elucidation of neuron-astrocyte interaction in models of amyotrophic lateral sclerosis using multi-modal integrated bioinformatics workflow

Fig. 1

a Representative images of MN cultures exposed to NTg (blue) and mutSOD1 (red) ACM stained with the neuronal marker SMI-32. Scale bar: 50 μm. ACM from NTg (n = 3) or mutSOD1 (n = 3) astrocytes were passed through anion exchange Q column or cation exchange S column. Elutes were collected and then applied to mouse primary MNs. GFP+MNs were counted manually using epifluorescent microscope. Data are means ± SEM of independent experiments (n) analyzed by two-way ANOVA (Interaction F(2,12) = 17.61; P = 0.0003) followed by Sidak post hoc test: ***P = 0.0001 Control NTg vs mutSOD1 (CI 22.61–57.39%; d = 4.73); ****P ≤ 0.0001 Q eluate NTg vs mutSOD1 (CI: 47.61–82.39%; d = 28.47). b ACM from NTg (blue; n = 5) or mutSOD1 (red; n = 5) were passed through different molecular weight cutoff filters. The retentates were resuspended in fresh media and then applied to mouse primary MN culture and counted manually. Data are means ± SEM of independent experiments (n) analyzed by two-way ANOVA (Interaction F(5,24) = 19.14; P < 0.0001) followed by Sidak post hoc test: ****P ≤ 0.0001 Control NTg vs mutSOD1 (CI: 34.26–70.94%; d = 6.35) and 5 K NTg vs mutSOD1 (CI: 31.06–67.74%; d = 5.76); **P = 0.0015 10 K NTg vs mutSOD1 (CI: 9.06–45.74%; d = 1.55). c To examine molecular property of the deleterious effect in MNs, ACM from NTg (blue) or mutSOD1 (red) mice was either untreated (n = 10), heat-inactivated using water bath (n = 8 HI 15 min and n = 7 HI 30 min), treated with pepsin (n = 8), charcoal (n = 6) or chloroform (n = 7) and then applied to mouse primary MNs and counted manually. Data are mean ± SEM of independent experiments (n) analyzed by two-way ANOVA (Interaction F(5,40) = 21.22; P < 0.0001) followed by Sidak post hoc test: ****P ≤ 0.0001 Control NTg vs mutSOD1 (CI: 33.61–53.99%; d = 5.86), HI 15 min NTg vs mutSOD1 (CI: 11.98–34.77%; d = 1.63), charcoal NTg vs mutSOD1 (CI: 20.68–46.99%; d = 3.24), and chloroform NTg vs mutSOD1 (CI: 26.82–51.18%; d = 6.10). In Fig. 1, all primary spinal MN cultures were from Tg HB9::EGFP mice. See also Supplementary Fig. 1. Source data provided as source data file.

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