Fig. 5

PTP1B inhibition attenuates astrocytic TDP-43-induced toxicity and mitochondrial dysfunction in differentiated NSC-34 motor neuron-like cells. a–f NSC-34 cells were treated with 1 μM RA for 8 days and then were analysed. a CMFDA staining of undifferentiated or differentiated NSC-34 cells at 8 days. Scale bars, 100 μm. b Real-time PCR results of motor neuronal marker genes in undifferentiated or differentiated NSC-34 cells at the indicated time points. Data are presented as the mean ± SD. Gapdh was used for normalization. c Differentiated NSC-34 cells were stimulated with GFP ACM, TDP-43 ACM or TDP-43+ PTP1Bi ACM for 4 days. Cell viability was analysed by CCK-8 assay. ***p < 0.005 (one-way ANOVA). d Differentiated NSC-34 cells stimulated with TDP-43 ACM were treated with an IL-1β antibody (50 ng/ml), an IL-6 antibody (50 ng/ml) and a TNF-α antibody (100 ng/ml) for 4 days and then were subjected to CCK-8 assays. TDP-43 ACM-induced toxicity was rescued by IL-1β, IL-6 and TNF-α antibody treatment. Data are presented as the mean ± SD. *p < 0.05; **p < 0.005 (one-way ANOVA). e, f Differentiated NSC-34 cells in XF24-well culture plates were stimulated with GFP ACM, TDP-43 ACM or TDP-43 + PTP1Bi ACM for 4 days. e Mitochondrial dysfunction analysis of ACM-treated cells was performed with a Seahorse XF analyser to detect the basal OCR, ATP production, maximum reserve and respiratory capacity. The OCR was normalized to total protein concentration. f 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. *p < 0.05; **p < 0.005; and N.S. not significant (one-way ANOVA)