ZFIN Image: Sofou et al., 2021, Figure 6

Image

Figure Caption

Figure 6 Analysis of autophagosome formation and turnover in fibroblasts

A

Analysis of p62/SQSTM1 levels in patient and control fibroblasts transduced with control or VPS16‐expressing lentivirus under basal conditions and after 2 h of serum starvation.

B

Summary quantification of (A), normalized to actin (ACTB) and expressed as % of controls (n = 3 biological replicates).

C

Analysis of LC3 isoforms in serum‐starved fibroblasts transduced with control or VPS16‐expressing lentivirus and treated with Bafilomycin A1 (Baf A1), as indicated.

D

Quantification of LC3‐II, normalized to levels of LC3‐I, actin and expressed as % of controls (n = 6 biological replicates).

E

Autophagic flux, calculated as the relative increase in LC3‐II ratios upon addition of Baf A1 (data from D; n = 6 biological replicates).

F–I

Analysis of autophagosome numbers in fibroblasts transfected to express mRFP‐GFP‐LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum‐starved conditions. Scale bar, 20 μm. (G and H) Quantification of autophagosomes, defined as green + red puncta (G) and autophago‐lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n = 50–113 cells from a total of three independent experiments; for exact values see Appendix Table S1). (I) Cell of patient A transfected with mRFP‐GFP‐LC3, labeled with LysoTracker, and imaged live by confocal microscopy under serum‐starved conditions. Insert shows red+blue autolysosomes (arrowheads) and a green+red autophagosome devoid of LysoTracker signal (arrow). Scale bars 10 μm or 2 μm (inserts).

Data information: Bar graphs represent data as mean ±SEM. Statistical comparisons between the indicated groups by one‐way ANOVA with Holm–Sidak’s multiple comparisons tests. *P < 0.05; ***P < 0.001.

Acknowledgments

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