Fig 1: Short-term hypoxia-inducible factor 1 (HIF-1) stabilization in primary mouse trophoblasts leads to decreased mitochondrial abundance and cellular senescence.HIF-1 is detected in cultured trophoblasts exposed to CoCl2 (A). After 48 hr of CoCl2 exposure, trophoblasts exhibit decreased mitochondrial abundance reflected by Cox2 mRNA expression levels (B; p=0.014) and COX IV protein levels (C; p=0.0047). Senescence marker Glb1 is increased (D; p=0.038) and senescence-associated beta galactosidase (SA-βGal) accumulation is noted by X-gal assay (E; p=0.012). Each data point represents a technical replicate (e.g. protein, RNA, or β-Gal measured from an individual well of cells grown in treated or control condition). Data normalized to mean of control treatment group. See Figure 4—source data 1 for uncropped blots. Figure 4—source data 1.Uncropped, unedited blots from 4a (left) and 4c (right).
Fig 2: Senescence, hypoxia-inducible factor 1 (HIF-1) signaling, and decreased mitochondrial abundance characterize late-gestation human placenta.mRNA expression of senescence marker GLB1 and HIF-1 targets HK2 and SLC2A1 trends higher in placentas from >39-week cohort vs <35-week cohort (A; two-way ANOVA gestational age factor p=0.057). Mitochondrial abundance, reflected by mitochondrial genes ATP6 and COX2 (B; two-way ANOVA gestational age factor p=0.042) and COX IV protein (C; p=0.0036) decreases with advancing gestational age. Each data point represents a biological replicate (RNA or protein isolated from an individual placenta). Data normalized to mean in <35-week group. See Figure 3—source data 1 for uncropped blots. Figure 3—source data 1.Uncropped, unedited blots from Figure 3.
Fig 3: Mouse placental aging is characterized by cellular senescence, hypoxia-inducible factor 1 (HIF-1) signaling, and mitochondrial dysregulation.Weighted gene correlation network analysis (WGCNA) yielded 20 gene clusters. Functional pathways overrepresented in clusters found to increase (blue) and decrease (turquoise) across gestation highlight enhanced cellular senescence, increased HIF-1 signaling, and decreased mitochondrial synthesis and respiration late in pregnancy (A). mRNA expression of senescence marker Glb1 peaks at e17.5 (B; one-way ANOVA p=0.0048). HIF-1 protein abundance is higher at e17.5 versus e13.5 and e15.5 (C; one-way ANOVA p=0.019), as is expression of HIF-1 targets Hk2 and Slc2a1 (D; two-way ANOVA p<0.0001 for gestational age factor). (See Figure 2—figure supplement 1 for analysis of gene expression changes across timepoints by placental sex.) Mitochondrial abundance, reflected by COX IV protein, decreases with gestational age (E, one-way ANOVA p=0.0064), and mitochondrial DNA lesion rate peaks at e17.5 in the regions of the D-loop (one-way ANOVA p=0.0001), COII/ATPase6 (p=0.0027), and ND5 (p=0.036) (F). (B–F) Each data point represents a biological replicate (e.g. RNA, protein, or DNA extracted from an individual placenta, in turn collected from one of 2–4 pregnant dams per group). Data normalized to mean at e13.5. See Figure 2—source data 1 for uncropped blots. Figure 2—source data 1.Uncropped, unedited blots from 2c (left) and 2e (right).
Supplier Page from Abcam for Beta Galactosidase Staining Kit (Senescence)