Fig. 3
From: Reversible induction of mitophagy by an optogenetic bimodular system
AMBRA1-RFP-sspB-mediated mitophagy is reversible. a AMBRA1-RFP-sspB shuttling from mitochondria was assessed by live cell imaging in single Venus-iLID-ActA/AMBRA1-RFP-sspB overexpressing HeLa cells. Upon blue light irradiation AMBRA1-RFP-sspB was found at mitochondria. Subsequently, AMBRA1-RFP-sspB subcellular distribution was recorded every minute without blue light administration. After 3 min, one spike of blue light was reapplied. Images are the sum of a three frames Z-stack. PCC and MOC of the red over the green signal were quantified in ten random fields of five independent experiments. For a ×4 magnification of the inset see Supplementary Figure 13. Scale bar: 10 μm. Data shown mean ± S.E.M. Hypothesis test: ANOVA test. *p < 5 × 10−2. **p < 10−2. ***p < 10−3. b Transfected HeLa cells were treated as follows: 48 h of dark (lane Dark or D), 24 h of dark + 24 h pulsed blue light (1 s light + 1 min dark, lane 24 h), 24 h of pulsed blue light + 24 h of dark state (lane Rescue or R), and 48 h of pulsed blue light only. Cell lysates were loaded on a polyacrylamide gel and immuno-blotted. Levels of the mitochondrial marker SOD2 were investigated; AMBRA1-RFP-sspB was detected to verify the rate of overexpression. Actin and HSP90 were used as loading controls of the two gels, respectively. The graph shows the normalized densitometric SOD2 over actin ratio in three independent experiments. The experiments were repeated in Venus-iLID-ActA/RFP-sspB overexpressing HeLa cells as a control (right panel). Data shown: mean ± S.E.M. Hypothesis test: ANOVA test. *p < 5 × 10−2. Mr (kDa): Relative molecular mass expressed in kilodalton. Source data are provided as a Source Data file
