Figures and data
Caspases fully proteolyze 5 peripheral Nups during myogenesis.
(A) Immunoblots showing the degradation Nups, PARP, and αII-spectrin, the activation of caspase-3/12 and the upregulation of myogenin in differentiating C2C12 ls. (B) Caspase-dependent proteolysis of Nups in differentiating C2C12 cells assessed by immunoblotting. Q-VD-OPh ed at 30 μM. (C) Nuclear-to-total mRNA ratio in C2C12 cells undergoing myogenesis. The average value of three replicates own for each time point. (D) A stable C2C12 cell line that expresses NESRev-GFP. In dashed boxes are cells undergoing -to-MB or MB-to-MT fusion. (E) Cytoplasmic-to-nuclear ratio of NESRev-GFP was quantified for 22 donor myoblast / closest onuclei pairs. Grey lines link each pair. (F) Localization of lamin B1 and α-tubulin in differentiating C2C12 cells assessed immunoblotting. Nuclear α-tubulin marked with an arrow. 30 μg of protein loaded per lane. (G) Cytoplasmic and nuclear els of focal adhesion proteins in differentiating C2C12 cells. 25 μg of protein loaded per lane. MT: myotubes, MB: myoblasts.
Tpr and Pom121 C-terminal fragments dissociate from the NPC during myogenesis.
(A and B) C2C12 cells munostained using two Nup153 and two Tpr antibodies. In dashed boxes are apoptotic nuclei. (C) C2C12 cells undergoing ogenesis immunostained for Crm1, Nup153, and Tpr. In dashed boxes are TprC− cells. (D) Tpr C-term immunoreactivity rmalized against Nup153 internal domain immunoreactivity) determined for 50+ nuclei. (E) Nup153, Tpr, and caspase-3 gments in subcellular fractions of differentiating C2C12 cells visualized by immunoblotting. Q-VD-OPh used at 30 μM. (F) C12 cells undergoing myogenesis immunostained for mAb414, Nup153, and Pom121. (G) Pom121 C-term immunoreac-ty (normalized against Nup153 internal domain immunoreactivity) determined for 50+ nuclei.
Quantitative proteomics reveals how NPC trimming impacts the nuclear proteome of differentiating myo-sts.
(A) Preparation of subcellular fractions for quantitative mass spectrometry. (B) Subcellular fractions were validated ng multiple protein markers. (C) Volcano plot showing how protein levels in the “nuclear soluble” fraction change during the t 24 hours of myogenic differentiation. Each data point colored to represent their transcriptional change during the same e frame. (D) Zoomed-in view of the dotted square area in (C).
NPC trimming accompanies nuclear accumulation of cytoplasmic, plasma membrane, and mitochondrial teins.
(A) Protein groups detected in the “nuclear soluble” fraction. Red dotted line: y = 2. (B) Protein level change in the clear soluble” fraction when C2C12 cells are differentiated with or without 30 μM Q-VD-OPh. Red dotted line: y = 1. Data nts for proteins labeled in (A) are shown in the right box. (C) Visual summary of multiple events that take place during myo-st-to-myotube transition.
Caspase-mediated NPC trimming occurs during neurogenesis and ER stress.
(A) Schematic representation caspase-mediated NPC trimming. (B) Down-/up-regulation of survivin, caspase-3, and myogenin in differentiating C2C12 ls. (C) Relative timing of key events in myogenesis. (D) Expression levels of Nup153, PARP, active caspase-3, survivin, d neuronal differentiation markers (Sox2 and βIII-tubulin) were determined in differentiating neurons. (E) Acute ER stress uced using 1 μg/mL tunicamycin in C2C12 myotubes and reserve cells. The levels of Nups, BiP, XIAP, myosin heavy chain HC), myogenin, and active caspase-3 levels were monitored by immunoblotting. (F) Chronic ER stress was induced in erentiated C2C12 cells for 7 days using low doses of tunicamycin. Nups, BiP, and active caspase-3 levels were assessed.
