Skip to main content
. 2018 Apr 18;12:92. doi: 10.3389/fncel.2018.00092

Figure 3.

Figure 3

AMBRA1ActA improves vitality of SH-SY5Y cells treated with rotenone. (A) SH-SY5Y cells were transfected with vectors coding for PcDNA3 or for Myc-AMBRA1ActA and then treated with rotenone (10 μM). Viable cells were estimated using the MTS assay. Results are expressed as arbitrary unit (A.U.). Each point represents the mean (± SD) of triplicate wells from three independent experiments. Statistical analysis was performed using One-way ANOVA. **P < 0.01. *P < 0.05. (B) SH-SY5Y cells were transfected with vectors encoding Venus-ActA or Myc-AMBRA1ActA or Myc- AMBRA1ActALIRAA for 24 h. Six hours after transfection, cells were treated with rotenone (10 μM). After DAPI staining, cells with condensed or fragmented nuclei were scored as pyknotic (arrows indicate pyknotic nuclei). The graph shows the percentage of pyknotic nuclei in transfected cells (± S.D). For each condition, transfected cells were counted in random fields from three independent experiments. Statistical analysis was performed using One-way ANOVA. ****P < 0.0001; ***P < 0.001; *P < 0.05. (C) SH-SY5Y cells were transfected with vectors encoding PcDNA3 or Myc-AMBRA1ActA for 24 h. Six hours after transfection, cells were treated with rotenone (10 μM). After extraction of proteins, we performed a western blot analysis using antibodies directed against AMBRA1 (Myc), PARP/Cleaved-PARP and ACTIN. The graph shows the cleaved PARP/full-length PARP ratio, resulting as the mean of three independent experiments (± S.D). n = 3. One-way ANOVA, *P < 0.05. (D) SHSY5Y cells transfected with a siRNA-Ctr or siRNA-AMBRA1 were treated with 6-OHDA for 18 h. Total lysates were subjected to immunoblotting for AMBRA1, PARP and ACTIN antibodies. The graph represents the cleaved PARP/Full-length PARP ratio (± S.D). Statistical analysis was performed using One-way ANOVA. *P < 0.05. n = 3 independent experiments.