Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca2+ signaling of differentiated C2C12 myotubes

Figures

• 

  Fig. 1. ANO5 knockdown (ANO5-KD) impairs nuclear positioning without affecting myoblast differentiation. (A) The change of mRNA expression of ANO5 in control and ANO5-KD (shANO5) myoblasts during 3 days of differentiation period (#p < 0.01, 3 independent experiments). Inset: Knockdown efficiency of 4 shANO5 constructs (OriGene) tested were confirmed by Western blotting. The ‘construct #1’ shANO5 was chosen to generate stable ANO5-KD cells. (B) Representative immunostaining images of control and ANO5-KD myotubes after 3 days of differentiation. Shown are DAPI targeted myonuclei and anti-MF-20 targeted MyHC+ myotubes. (C) Bar graphs show the quantification of nuclei from (C) (*p < 0.05). (D) The expression of ANO5 and myogenic markers (MyHC and myogenin) in control and ANO5-KD cells at 3 days of differentiation (DM-3) was evaluated by Western blotting. (E) Bar graphs show the quantitative density of the myogenic marker protein bands (n = 5). GFP, green fluorescence protein; Sc, scrambled shRNA construct for control; MyHC, myosin heavy chain; NS, not significant.

• 

  Fig. 2. ANO5 knockdown (ANO5-KD) causes nuclear positioning defect and reduced expression of Kif5b nuclear motor proteins. (A) Representative DAPI and anti-Kif5b immunostaining images were taken from control and ANO5-KD myotubes after 3 days of differentiation. ANO5-KD myotubes showed aggregated or clustered nuclei at the center of the cell body. (B) The degree of nuclear positioning defect was evaluated by counting the percentage of myotubes with nuclei of ‘aligned’, ‘aggregated’, and ‘other’ mixed type (n = 5, #p < 0.01). (C, D) The changes in Kif5b mRNA expression (#p < 0.01, n = 3) and Kif5b protein expression during 3 days of myogenesis were illustrated. (E) Kif5b protein expression levels at DM-3 were quantified and compared between two groups (*p < 0.05, n = 3). MyHC, myosin heavy chain; DM, differentiation medium.

• 

  Fig. 3. ANO5 knockdown (ANO5-KD) compromises Ca2+ signaling. (A) Representative [Ca2+]i transients that evoked by 100 mM KCl-induced membrane depolarization (high-K solution). Control (n = 24) and ANO5-KD myotubes (n = 17) were repeatedly depolarized at every ~2 min. (B) The line graph shows the quantitated and normalized Ca2+ transients (*p < 0.05). (C) Representative sarcoplasmic reticulum (SR) Ca2+ releases in response to the perfusion of 40 mM caffeine to deplete SR Ca2+ stores. (D) The areas under the Ca2+ release curves were calculated, normalized, and illustrated as bar graphs (#p < 0.01).

• 

  Fig. 4. ANO5 knockdown (ANO5-KD) reduces expression of DHPR and SERCA and impairs co-localization of dihydropyridine receptor (DHPR) and ryanodine receptor subtype 1 (RyR1). (A) The expression of E-C coupling regulatory proteins was compared between control and ANO5-KD myotubes. Western blotting was performed with myotubes harvested at differentiation medium (DM)-3. (B) The quantified protein expression levels were illustrated as bar graphs, showing that DHPR and SERCA expression is reduced by ANO5-KD (*p < 0.05). (C) The myotubes at DM-3 were immunostained against RyR1 and DHPR, and the co-localization of the two proteins was analyzed by calculating overlap coefficient. (D) The calculated overlap coefficient was compared with the bar graph (***p < 0.005). CSQ, calsequestrin-1; SERCA, SR Ca2+-ATPase; JP2, junctophilin-2.