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CEPLAS
Guichard-2024
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[curation] link figures and movies in assay READMEs
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[curation] link figures and movies in assay READMEs
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# Autophagic Activity Assay
## Movie S3. Application of the RoPod for time-lapse imaging of pHusion-ATG8 in roots treated with autophagy modulators.
Representative movies of WT seedlings expressing the autophagosomal marker pHusion-ATG8
grown in a RoPod chamber and treated with vehicle (0.01% DMSO), 500 nM Concanamycin A (ConA)
or 500 nM AZD8055 (AZD). ConA blocks the final step of autophagy, i.e. degradation of the
autophagic bodies in the vacuole, causing massive accumulation of the pHusion-ATG8-positive
puncta in the vacuolar lumen. AZD8055 induces autophagic activity, causing incorporation of the
ATG8a-based reporter into autophagosomes followed by its delivery to the vacuole and
degradation. Note decrease of the fluorescent signal after ca 4h of treatment. The magenta and
green lines indicate respectively hair and non-hair cell files used for quantification. Scale bar, 50
µm.

## Figure S1. Dynamic changes in the basal autophagic activity detected in root hair and non-hair
cells.
Quantification of pHusion-ATG8-positive puncta per area in the epidermal root cells of WT seedlings
grown in RoPods and treated with 500 nM ConA. The data represents 0h, 4h and 12h time points of
the time-lapse assays shown in the Figure 5 and Movie S3. T-test, p-value<0.01.

## Figure S2. Root hair cells accumulate less autophagic bodies than non-hair cells under prolonged ConA treatment.
Arabidopsis seedlings co-expressing autophagy reporter (GFP-ATG8) with vacuolar marker (spL-
mRFP) were grown in a RoPod v23.4 and treated with 0.5uM ConA overnight prior to imaging
using confocal microscope. (a) A representative maximal projection of a tiled z-tack encompassing
complete cells with vacuoles was used to segment hair (yellow outline) and non-hair (blue outline)
cells; scale bar, 100 µm. (b) illustrates segmented non-hair (left) and hair (right) cells outlined in
(a). Scale bars, 20 µm.
The number of GFP-positive puncta was quantified per µm3 (c) and per a cell (d). Charts in c and d
represent data from one out of four independent experiments. Four roots were analyzed,
selecting five cells for each cell type for each root, n= 20. OneWay Anova, p-value <0.01 for both
charts.

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