De Palma et al., Suppl Inf., p.1
SUPPLEMENTARY INFORMATION
Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for
myogenic differentiation
Clara De Palma, Sestina Falcone, Serena Pisoni, Sara Cipolat, Chris Panzeri,
Sarah Pambianco, Addolorata Pisconti, Raffaele Allevi, Maria Teresa Bassi,
Giulio Cossu, Tullio Pozzan, Luca Scorrano, Salvador Moncada, Silvia Brunelli
and Emilio Clementi.
SUPPLEMENTARY FIGURES
Supplementary Figure 1 NO regulates myogenesis and mitochondrial network
creation via cGMP.
Myogenic precursor cells transfected with the red fluorescent mitochondrial protein
mitoDsRed were treated with L-NAME, ODQ or vehicle (C, control) as indicated,
and differentiated by serum withdrawal for up to 12 h.
(a) Quantification of the changes in mitochondrial morphology.
(b) Densitometric analyses of the expression of the differentiation markers Mef 2A,
MyoD, myogenin and sarcomeric myosin (MyHC) and of the mitochondrial proteins
Mfn1, Mfn2, Opa1, Drp1, CytC and COX IV, detected by western blot analyses.
Graphs represent the values  SEM (n = 4). Double and triple asterisks represent
statistical probability vs. C (P < 0.01 and P < 0.001, respectively).
Supplementary Figure 2 NO regulates myogenesis and mitochondrial network
creation via cGMP.
De Palma et al., Suppl Inf., p.2
Myogenic precursor cells transfected with the red fluorescent mitochondrial protein
mitoDsRed were treated with L-NAME, ODQ or vehicle (C, control) as indicated,
and differentiated by serum withdrawal for up to 12 h.
Mitochondrial morphology detected by transient transfection with mitoDsRed was
analysed as in Fig 1 and 3D reconstructions on 0.2 um Z stacks for each image were
performed using a VolumeJ plugin of ImageJ software. Mitochondria that were round
in shape in proliferating cells were found to become progressively elongated, forming
an extensive branched network at 12 h of differentiation. Formation of such a network
was inhibited in the presence of L-NAME and ODQ. Results representative of four
reproducible experiments.
Supplementary Figure 3 Effects of nNOS silencing on mitochondrial morphology
and myogenic differentiation.
Myogenic precursor cells were transfected with the red fluorescent mitochondrial
protein mitoDsRed in the presence of the nNOS silencing siRNA or its scrambled
control (SCR) and differentiated by serum withdrawal for up to 12 h.
(a) Efficiency of nNOS silencing verified by western blotting with a nNOS-specific
Ab. Shown is a representative western blot image and the densitometric analysis of
nNOS expression after cell treatment with the nNOS siRNA, the SCR or an unrelated
siRNA GAPDH. (n = 3).
(b) Mitochondrial morphology detected by transient transfection with mitoDsRed.
Bar: 10 µm.
(c) Expression of the myogenic differentiation markers myogenin and sarcomeric
myosin (MyHC), determined by western blotting, at various times of differentiation.
De Palma et al., Suppl Inf., p.3
A representative western blot image and the densitometric analyses are shown (n = 3).
Single and double asterisks represent significant differences from the respective SCR
(P < 0.05 and P < 0.01, respectively).
Supplementary Figure 4 Electron microscopy assessment of mitochondrial
morphology.
Myogenic precursor cells were differentiated for 6 h and then treated with L-NAME,
ODQ or vehicle (C) for 45 min at 37°C. Samples were then pelleted and processed for
conventional transmission electron microscopy. Insets magnified on the right end side
of each panel show the difference in shape of the mitochondria in differentiating cells,
elongated in control cells and short in cells treated with L-NAME or ODQ. Bar = 1
µm.
Supplementary Figure 5 NO and cGMP regulate mitochondrial dynamics.
Myogenic precursors were transfected with the mitoDsRed coding vector and
differentiated. 6 h later cells were treated without (Ctr.) or with L-NAME or ODQ as
indicated. Films show the recording for 50 min.
Supplementary Figure 6 NO and cGMP stimulate myogenesis through inhibition of
mitochondrial fission.
Expression of the myogenic differentiation markers Mef 2A, MyoD, myogenin and
sarcomeric myosin (MyHC) was determined by Western blotting in myogenic
precursor cells transfected with either the myc-tagged pCDNA3 vector empty
(pcDNA3) or containing the dominant negative Drp1 (pcDNA3-Drp1 K38A) at the
De Palma et al., Suppl Inf., p.4
indicated time-points. Densitometric analyses of the expression of the differentiation
markers MEF 2A, Myo D, myogenin and MyHC detected by western blot analyses
are shown. Graphs represent the values  SEM (n = 4). Double and triple asterisks or
crosses represent statistical probability vs. pcDNA3 and vs. pcDNA3 + ODQ (P <
0.01 and P< 0.001, respectively).