Figure 4
From: Neuronal hemoglobin affects dopaminergic cells’ response to stress
Hb induces nucleolar stress and impairs autophagy in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with rotenone at the indicated concentrations for 16 h. (a) qRT-PCR of pre-rRNA levels. Pre-rRNA levels were normalized to β-actin. Untreated control cells was used as reference and set to 1. (n=5) (b) Western blotting analysis of phosphorylated 4E-BP1 (P-4E-BP1) status and LC3 expression. For normalization, the total levels of 4E-BP1 were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. β-Actin was used as a loading control. (n=5) (c) LysoTracker Red staining. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 10 μm. (n=3) (d) Quantification of autophagosome. Ninety randomly chosen cells were counted for quantification of each condition. Values are expressed as a percentage relative to the total. Autophagy activity was defined as follows: high: >10 LysoTracker Red foci/cell; medium: 6–10 LysoTracker Red foci/cell; low: <6 LysoTracker Red foci/cell. Values are mean±S.D. Data were evaluated statistically by Student’s t-test. The P-values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P-values are indicated (NS=not significant)
