Supplementary Figure 10: STMN2 levels are decreased in the TDP-43 mislocalization model.
From: ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair
a, Experimental strategy used to assess the effect of proteasome inhibition on TDP-43 localization in hMNs. b, Pearson’s correlation analysis for TDP-43 immunostaining and DAPI fluorescence of cells treated with MG-132 (1 μM). Data are displayed as dots representing individual neurons with mean and s.d. (unpaired t-test, two-sided, P value for untreated versus DMSO (0.53), Day 1(< 0.0001), Day 2 (< 0.0001), Day 3 (< 0.0001), Day 4 (0.0018), Day 5 (0.69), Day 6 (0.26) and Day 7 (0.78)). c, Micrographs of HUES3 hMNs untreated or treated with MG-132 and immunostained for TDP-43 (red), β-III tubulin (green) and counterstained with DAPI (blue). Scale bar, 100 μm. Similar results were obtained in n = 2 independent experiments. d, hMNs were treated with DMSO or MG-132 (1 μM) for 24 h and then immunostained for TDP-43 (red), β-III tubulin (green) and counterstained with DAPI (blue). The nuclear TDP-43 intensity and the cytoplasmic TDP-43 intensity was determined to compare the nuclear/cytoplasmic ratio between the MG-132-treated cells and DMSO-treated cells. Data are displayed as dots representing individual neurons with mean and s.d. (unpaired t-test, two-sided, P < 0.05). Similar results were obtained in n = 2 independent experiments. e, Immunoblot analysis of TDP-43 in the detergent-soluble (RIPA) and detergent-insoluble (urea) fractions in neurons treated with MG-132 (unpaired t-test, P < 0.05). Similar results were obtained in n = 4 biologically independent experiments. f, qRT-PCR analysis of STMN2 expression for motor neurons treated with MG-132 at the indicated concentrations and durations relative to DMSO control (unpaired t-test, two-sided, P < 0.05). Similar results were obtained in n = 2 independent experiments. g–h, Diagram of RT-PCR detection stratagey for STMN2 cryptic exon (g) and Sanger sequencing of the PCR product confirmed the splicing of STMN2 Exon 1 with the cryptic exon (h). Similar results were obtained in n = 2 independent experiments.
