Fig. 3: Myosin IIB colocalizes to TDP-25 aggregates and facilitates TDP-25-mediated mitophagy. | Cell Death & Disease

Fig. 3: Myosin IIB colocalizes to TDP-25 aggregates and facilitates TDP-25-mediated mitophagy.

From: Nonmuscle myosin IIB regulates Parkin-mediated mitophagy associated with amyotrophic lateral sclerosis-linked TDP-43

Fig. 3

a Confocal images showing cellular localization of GFP-Myosin IIB together with either Myc-TDP-43 or Myc-TDP-25 in mouse cortical neurons. Scale bar; 10 μm. b The bar graphs show the fluorescence intensity profile across the arrow for green (GFP-Myosin IIB) and red (Myc-TDP-43 or Myc-TDP-25) channels. c Coimmunoprecipitation with anti-Myc using HEK293T cell lysates expressing Myc-TDP-43 or Myc-TDP-25. Western blotting was performed using anti-Myc, anti-Myosin IIB antibody. Number sign (#) indicates the nonspecific band. d Confocal images showing cellular localization of Tom20, or GFP-Myosin IIB with Myc-TDP-25 in mouse cortical neurons treated with either DMSO or Blebbistatin (10 μM, 24 h). Scale bar; 10 μm. e The graphs show the fluorescence intensity profile across the arrow for green (GFP-Myosin IIB), red (Myc-TDP-25) and blue (Tom20). f The bar graph represents % of colocalization of GFP-Myosin IIB with TDP-25 per cell. **p < 0.01. Values represent mean + SEM (n ≥ 10). g Western blotting was performed using the mitochondrial fraction from HEK293T cell lysates expressing Myc-TDP-25 and either Myosin IIB or Myosin VI siRNAs with anti-Myosin IIB, anti-Myosin VI, anti-Myc, anti-Tom20, or anti-β-actin antibody in the presence or absence of NH4Cl. h The bar graphs indicate the percentage of normalized protein levels of Myosin IIB, Myosin VI, Myc-TDP-25, or Tom20 with β-actin. i Cellular images showing mitotracker-positive mitochondria expressing Myc-TDP-25 in the presence or absence of blebbistatin in cultured cortical neurons. Scale bar; 20 μm. j The bar graph indicates the ratio of mitotracker-positive mitochondrial area per cell expressing Myc-TDP-25 in the presence or absence of blebbistatin. *p < 0.05. Values represent mean + SEM (n ≥ 13). k The bar graph indicates the percentage of DAPI-positive dead cells with DNA fragmentation in postmitotic neurons expressing Myc-TDP-25 in the presence or absence of blebbistatin. *p < 0.05. Values represent mean + SEM (n ≥ 6).

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