Additional file 1 of The overexpression of TDP-43 in astrocytes causes neurodegeneration via a PTP1B-mediated inflammatory response
Creators: name: Shinrye Lee
name: Seyeon Kim
name: Ha-Young Kang
name: Lim, Hye Ryeong
name: Younghwi Kwon
name: Myungjin Jo
name: Jeon, Yu-Mi
name: Kim, Sang Ryong
name: Kiyoung Kim
name: Ha, Chang Man
name: Seongsoo Lee
name: Kim, Hyung-Jun
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology

description: Additional file 1: Figure S1. Purity of cultured primary mouse cortical neurons and astrocytes. (a) Primary cortical neuron (upper) or astrocyte (lower) enriched cultures were stained with antibodies for neuron, astrocyte and microglia markers. Triple immunostaining of MAP2 (neuron; red), GFAP (astrocytes; green), and Iba-1 (microglia; pink) in primary neuronal cells at DIV 7 and astrocytes at DIV 21. DAPI staining was used to determine the number of cells. Scale bar, 200 μm. n=248 cells (primary cortical neuron culture), n=447 cells (primary astrocyte culture). Figure S2. Insoluble TDP-43 protein was significantly increased in TDP-43-overexpressing primary astrocytes. (a) Immunoblot analysis of TDP-43 protein in the insoluble and soluble fractions of TDP-43-Gfp-transfected astrocytes. The immunoblot results from 3 independent experiments were normalized to those of tubulin. Figure S3. Transfection of the control plasmid did not affect the viability of astrocytes. (a) Astrocytes were treated with the Lipofectamine only or with a GFP expression DNA vector + Lipofectamine mixture for 3 days; then, CCK-8 assays were performed. Data are presented as the mean ± SD of 3 independent experiments. N.S., not significant (Student’s t-test). Figure S4. PTP1B and proinflammatory genes are upregulated by TDP-43 overexpression in primary astrocytes. (a-e) TDP-43-Gfp-transfected astrocytes were treated with a PTP1B inhibitor (PTP1Bi, 5 μM) for 1 day, and then real-time PCR was performed. 18S rRNA was used as a normalization gene for real-time PCR data. PTP1B inhibition greatly attenuated TDP-43-induced inflammatory upregulation. Quantification data for Il-1b (a), Il-6 (b), Lcn2 (c), iNos (d), and Nf-κb. (e) Quantification data are presented as the mean ± SD from 3 independent real-time PCR experiments. *p&lt;0.05; **p&lt;0.005; and ***p&lt;0.001 (one-way ANOVA with Bonferroni’s multiple comparison test). (f-j) Astrocytes were cotransfected with TDP-43 expression construct and a control siRNA or mouse Ptp1b siRNA for 3 days, and then FACS of Gfp-transfected live cells was performed. These cells were allowed to acclimate for 1 day and then were subjected to real-time PCR experiments. The TDP-43-induced upregulation of inflammatory gene transcription was attenuated by PTP1B downregulation. 18S rRNA was used as a normalization gene for RT-PCR. Quantification data for Il-1b (f), Il-6 (g), Lcn2 (h), iNos (i), and Nf-κb. (j) All data are presented as the mean ± SD from 3 independent real-time PCR experiments. *p&lt;0.05; **p&lt;0.005 (one-way ANOVA with Bonferroni’s multiple comparison test). Figure S5. ACM from astrocytes treated with a PTP1B inhibitor does not affect the viability of mouse cortical neurons. (a) Primary cortical neurons were treated with DMSO ACM or PTP1Bi ACM for 5 days and then were subjected to CMFDA staining. CMFDA-positive neurons were counted under a fluorescence microscope. The percentage of CMFDA-positive cells was quantified (lower). Data are presented as the mean ± SD of 3 independent experiments. N.S., not significant (Student’s t-test). Scale bars, 20 μm. Figure S6. The secretion of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α mediates astrocytic TDP-43-induced neuronal toxicity and mitochondrial dysfunction. Primary cortical neurons (a) and differentiated NSC-34 motor neurons (b) were stimulated with GFP ACM or GFP + PTP1Bi ACM supplemented with IL-1β protein (50 ng/ml), IL-6 protein (50 ng/ml), and TNF-α protein (10 ng/ml) for 4 days, and then they were subjected to CCK-8 assays. Similar to what was observed with TDP-43 ACM treatment, GFP or GFP + PTP1Bi ACM supplemented with IL-1β, IL-6, and TNF-α caused neurotoxicity. Data are presented as the mean ± SD of 3 independent experiments. **p&lt;0.005 (Student’s t-test). (c) Differentiated NSC-34 motor neurons stimulated with GFP ACM or GFP + PTP1Bi ACM supplemented with IL-1β protein (50 ng/ml), IL-6 protein (50 ng/ml), and TNF-α protein (10 ng/ml) for 4 days and then subjected to mitochondrial dysfunction analysis. Mitochondrial dysfunction analysis of ACM-treated cells was assessed through the detection of basal OCR, ATP production, maximum reserve and respiratory capacity by a Seahorse XF analyser. The oxygen consumption rate (OCR) was normalized to the total protein concentration (OD). Quantification of the OCR, ATP production, maximum reserve and respiratory capacity as a percentage of the basal values. Data are presented as the mean ± SEM of 3 independent experiments. *p&lt;0.05; **p&lt;0.005; and ***p&lt;0.001 (one-way ANOVA with Bonferroni’s multiple comparison test). Figure S7. Knockdown of Il-1b, Il-6, and Tnf-α mitigated astrocytic TDP-43-induced neuronal toxicity. (a-c) Astrocytes were cotransfected with the TDP-43-Gfp expression construct and control siRNA, Il-1b siRNA, Il-6 siRNA, or Tnf-α siRNA, and after 3 days the ACM were harvested. The concentrations of secreted cytokines (IL-1β, IL-6, and TNF-α) in the GFP ACM and TDP-43 ACM groups were measured by ELISA. TDP-43-induced secretion of cytokines (IL-1β, IL-6, and TNF-α) was significantly suppressed by the downregulation of IL-1β (a), IL-6 (b), or TNF-α. (c). Data are presented as the mean ± SD. *p&lt;0.05; **p&lt;0.005; and ***p&lt;0.001 (one-way ANOVA with Bonferroni’s multiple comparison test). (d) Primary cortical neurons stimulated with TDP-43ACM from astrocytes were treated with Il-1b siRNA, Il-6 siRNA, and Tnf-a siRNA for 5 days, and then they were subjected to CCK-8 assays. TDP-43 ACM-induced toxicity was rescued by siRNA knockdown of proinflammatory cytokine genes. Data are presented as the mean ± SD of 3 independent experiments. *p&lt;0.05; **p&lt;0.005; and ***p&lt;0.001 (one-way ANOVA with Bonferroni’s multiple comparison test). Figure S8. Inflammation induced by glial TDP-43 is mitigated by Drosophila Ptp1b downregulation. (a-f) Levels of Dorsal (Nf-κb), iNos, Attacin-C, Diptericin B, TDP-43 and Ptp1b mRNA from fly head lysates of control or TDP-43-expressing glial transgenic flies were analysed by real-time PCR. 18S rRNA was used as a normalization gene for real-time PCR. TDP-43-induced expression of genes involved in inflammation and genes that are downstream of NF-κB was significantly suppressed by the downregulation of PTP1B. Quantification data of Dorsal (Nf-κb) (a), iNos (b), Attacin-C (c), Diptericin B (d), TDP-43 (e), and Ptp1b (f) mRNA transcript levels are presented as the mean ± SD from 3 independent real-time PCR experiments. 18S rRNA was used for normalization. *p&lt;0.05; **p&lt;0.005; and ***p&lt;0.001 (one-way ANOVA with Bonferroni’s multiple comparison test).