Fig. 3.

rHR activates a noncanonical UPR^mt pathways. (A and B) rHR triggers noncanonical UPR^mt pathways. BeWo cells were subjected to rHR for 24 and 48 h. Western blot was used for measurement expression of UPR^mt molecular markers CLPP, paraplegin, TID1, HSP60, GRP75, and CS. Data were normalized to CS and are expressed as mean ± SEM, n = 5. *P < 0.05; **P < 0.01 (2-tailed paired t test at either 24 h or 48 h). (C–E) No increase in cellular expression but decreased nuclear translocation of ATF5 under rHR. Cells were exposed to 48 h of rHR. Western blot was used to quantify ATF5 while immunocytochemistry and subcellular fractionation was used to show its cellular localization. Data are presented as mean ± SEM, n = 3 to 4. *P < 0.05; **P < 0.01 (2-way ANOVA with Sidak’s multiple comparisons test). Magnification, 200×. (Scale bar: 200 μm.) (F) Potential conformation change of ETC complexes. Isolated mitochondria were subjected to immunoprecipitation with conformation-sensitive mitoprofile complex II antibody to pull out complex II before resolving in SDS/PAGE gel. Silver staining was used to reveal 4 subunits of complex II. The 20 N indicates cells were incubated under normoxic conditions with 20% O₂ for 24 or 48 h; 1/20 HR indicates cells were exposed to a 6-h cyclic pattern of 1% and 20% O₂ for 24 or 48 h.