OPA1 loss of function affects in vitro neuronal maturation
Bertholet Ambre M. , Millet Aurélie , Guillermin Oriane , Daloyau Marlène , Davezac Noélie
,
Miquel Marie-Christine and Belenguer Pascale.
Supplementary data
Figure S1 :
Histograms representing the viability (Trypan blue exclusion assay) of
untransfected cells from 2 days in vitro to 12 days in vitro (A) and of control small interfering
RNA- and small interfering RNA against OPA1-transfected cells at days in vitro 2 (B) and
days in vitro6, days in vitro9 and 12 days in vitro (C) (mean ± s.e.m; 300 cells per condition;
n = 3). (D) Histogram representing the percentage of neurons transfected by control small
interfering RNA or small interfering RNA against OPA1 containing Hoechst-labeled
apoptotic nuclei with condensed and fragmented chromatin at days in vitro6, days in vitro9
and 12 days in vitro. (mean ± s.e.m; 250 cells per condition; n = 4).
Figure S2 : (A) MAP2 staining (red) showed neurites extension from 2 days in vitro to 12
days in vitro. (B) Micrographs showing, at days in vitro6, days in vitro9 and 12 days in vitro,
immuno-stained pre-synaptic (synaptophysin, red) and post-synaptic (PSD95, green) puncta
in control small interfering RNA- or small interfering RNA against OPA1-transfected
neurons. In control (control small interfering RNA) both pre- and post-synaptic puncta
increased, as expected, upon time. Down-regulation of OPA1 (small interfering RNA against
OPA1) led to a decreased of the pre- and post-synaptic puncta at all times. (scale bars 10 m)
Figure S3 : Representative immunoblot showing the five OPA1 isoforms named a to e. Note
the higher expression of the b and d isoforms.
Figure S4 : Histograms representing protein levels analyses by Western blot during neuronal
maturation (days in vitro0-12 days in vitro) of (A) MFF1 relative to HSP60 (B) actin relative
to HSP60 or GAPDH. (AU : Arbitrary Units; mean +/- s.e.m; n = 4). (C) Histogram
representing the protein levels estimated by Western blot of actin relative to HSP60 in control
small interfering RNA- and siOPA-treated neurons at days in vitro6 (AU : Arbitrary Units;
mean +/- s.e.m,; n = 8).
Statistical significance was determined by the Kruskal-Wallis non parametric test (A, B) and
(C) Student’s t-test.
Figure S5 : Confocal micrographs of NRF2 immunolabeling (green) and Hoechst labeling
(blue) in days in vitro6 neurons showing NRF2 nuclear translocation (*).
Figure S6 : Histograms represent the numbers of Map2-immunostained primary and
secondary dendrites at days in vitro6, days in vitro9 and 12 days in vitro after downregulation of OPA1 (mean +/- s.e.m;, average of 30 neurons per conditions; n = 5).
Statistical significance between small interfering RNA against OPA1- and control small
interfering RNA-treated neurons was determined by Student’s t-test.
Figure S7 : Histograms representing the quantity of OPA1 protein estimated by Western blot
relative to HSP60 levels after RNA interference at days in vitro9 and 12 days in vitro,
normalized to control values (AU: Arbitrary Units; mean ± s.e.m., n =
6). Statistical
significance between small interfering RNA against OPA1 and control small interfering
RNA-treated neurons was determined by Student’s t-test: *P < 0.05, **P < 0.01.
Figure S8 : Histograms representing the mean mitochondrial area (A and B, left panels) and
the number of mitochondria (A and B, right panels) in each region of interest in eight
dendritic regions of interest (region of interest, 21-m-long and 5m-large segments) starting
from the most proximal region. Analysis was performed at days in vitro6 in control small
interfering RNA- (A) and small interfering RNA against OPA1- (B) transfected neurons
(mean ± s.e.m., average of 7 neurons, n = 4).
Statistical significance was determined by ANOVA. *P < 0.05, **P < 0.01.