Fig. 4. CHCHD10^S59L increases TDP-43 insolubility and mitochondrial translocation.

a HeLa cells were transfected with FLAG-tagged CHCHD10^WT or CHCHD10^S59L. After 24 h, cells were subjected to sequential protein extraction with RIPA and urea buffers. Immunoblotting was conducted with anti-FLAG and anti-actin (loading control) antibodies. Data are shown as mean ±  SD (one-way ANOVA and post hoc Tukey’s test, two-sided, NS, not significant; n = 3 independent experiments). b HeLa cells transfected with FLAG-tagged CHCHD10^WT or CHCHD10^S59L with TARDBP were subjected to sequential protein extraction with RIPA and urea buffers. Immunoblotting was performed with anti-TDP-43 or anti-actin (loading control) antibodies. Data are shown as mean ± SD (one-way ANOVA and post hoc Tukey’s test, two-sided, ***p = 0.000738 for WT, ***p = 0.000533 for S59L + WT; n = 3 independent experiments). c, d HeLa cells were co-transfected with FLAG-tagged TARDBP^WT or disease-causing mutants combined with empty vector (EV), CHCHD10^WT, or CHCHD10^S59L. After 24 h, mitochondria were fractionated. Immunoblotting was conducted with anti-TDP-43 (arrowhead indicates transfected TDP-43), anti-HSP60, and anti-TOM20 (loading controls) antibodies. Data are shown are mean ± SD (one-way ANOVA and post hoc Dunnett test, two-sided, *p = 0.01284 and ***p = 0.00021 in TDP-43 WT, **p = 0.0032 and ***p = 7.9e − 05 in G298S, *p = 0.0475 in A315T, *p = 0.02604 and ***p = 0.00031 in A382T). e, f HeLa cells were co-transfected with FLAG-tagged TARDBP^WT or disease-causing mutants combined with EV or CHCHD10^WT. After 24 h, mitochondria were fractionated. Immunoblotting was conducted with anti-TDP-43 (arrowhead indicates transfected TDP-43) or anti-HSP60 and anti-TOM20 (loading control) antibodies. Data are shown as mean ± SD (two-sided t test, *p = 0.011759, **p = 0.008552, *p = 0.001604, and **p = 0.018798 for TDP-43 WT, G298S, A315T, and A382T, respectively). g HeLa cells expressing CHCHD10^S59L were treated with a control peptide (scPM, 5 μM) or TDP-43 inhibitor (PM1, 5 μM). Representative images of HeLa cells stained with anti-FLAG (green, CHCHD10^S59L) and TOM20 (red, mitochondria) antibodies. Data are shown as mean ± SD (two-sided t test, ***p = 1.26e − 06 and 8.98e − 15 for S59L and Tom20, respectively; n = 20 cells from three independent experiments). Scale bar = 20 μm. h HeLa cells expressing CHCHD10^S59L were treated with a control peptide (scPM, 2 μM) or TDP-43 inhibitor (PM1, 2 μM). Mitochondrial respiration was measured by Seahorse XF Cell Mito Stress tests. A representative graph of a single experiment is shown (mean ± SD). Actual statistical analyses were performed with four independent experiments (one-way ANOVA and post hoc Dunnett test, two-sided, comparison with EV, *p = 0.024685; Detailed information on statistical analyses is available in Supplementary Fig. 9f). i HEK293 cells were transfected with FLAG-tagged CHCHD10 ^WT or CHCHD10^S59L with/without HA-tomato-tagged TARDBP. After 24 h, lysates were subjected to co-immunoprecipitation with anti-FLAG (M2) affinity gels. Immunoblotting was conducted with anti-FLAG (CHCHD10) and HA (TDP-43) antibodies. A representative image from three independent experiments is shown.