APOPT1/COA8 ASSISTS COX ASSEMBLY AND IS OPPOSITELY REGULATED BY UPS AND ROS (2018)
Figure 5

Figure 5 - Defective COX assembly and accumulation of assembly intermediates in APOPT1-less human cells

A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The expression levels were tested at different days after transduction. The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line. Data are presented as mean ± SEM (n = 3). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0030 (S6 naïve vs S6 APOPT1HA), *P = 0.0165 (S6 EV vs S6 APOPT1HA).

B APOPT1GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line. Data are presented as mean ± SEM (n = 4). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0066 (S6 naïve vs S6 APOPT1GFP), ***P = 0.0006 (S6 EV vs S6 APOPT1GFP), *P = 0.0169 (S2 naïve vs S2 APOPT1GFP), ***P = 0.0008 (S2 EV vs S2 APOPT1GFP).

C Western blot analysis of 1D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows).

D Western blot analysis of 2D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows). Anti-GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein.

E L-[35S]-Methionine pulse-chase labeling of mtDNA-encoded proteins. After a two-hour exposure with the radioactive label (pulse), cells were cultured in cold medium for the indicated chase times. The graphs show the densitometric quantification of the bands corresponding to MT-CO1 (left graph) and MT-CO2+MT-CO3 (right graph) normalized to the ATP6 band over the indicated time points. Graphs represent the values of three biological replicas for each cell line. Data are presented as mean ± SEM (n = 4 for controls, n = 2 for S2/S6/S2 APOPT1GFP/S6 APOPT1GFP). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: MT-CO1 - **P = 0.0039 ( 3.5 hours, controls vs S6), *P = 0.0202 (3.5 hours, controls vs S2), *P = 0.0163 (5 hours, S6vs S6 APOPT1GFP), *P = 0.0118 (5 hours, controls vs S2), **P = 0.0011 (20 hours, controls vs S6), *P = 0.0343 (20 hours, S2 vs S2 APOPT1GFP), ***P = 0.0007 (20 hours, S6 vs S6 APOPT1GFP), ***P = 0.0002 (20 hours, controls vs S2), *****P < 0.0001 (20 hours, controls vs S6), MT-CO2/MT-CO3 - *P = 0.0194 (3.5 hours, S6 vs S6 APOPTGFP), ***P = 0.0003 (3.5 hours, controls vs S6), *P = 0.0375 (5 hours, S6 vs S6 APOPT1GFP), *P = 0.0234 (20 hours, S2 vs S2 APOPT1GFP), ****P < 0.0001 (20 hours, S6 vs S6 APOPT1GFP), **P = 0.0013 (20 hours, controls vs S2), ****P < 0.0001 (20 hours, controls vs S6).

Data information: C1 and C2: control immortalized fibroblasts. EV: Cells transduced with the empty vector. Naïve: Non-transduced immortalized fibroblasts. GFP: Cells transduced with a recombinant GFP expression vector. Tubulin, SDHB and SDHA were used as loading controls in the different experiments as indicated.