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assays/AutophagicActivityAssay/README.md

+
+# 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.
+
+![41598_2024_63226_MOESM3_ESM.mp4](assays/AutophagicActivityAssay/dataset/41598_2024_63226_MOESM3_ESM.mp4)
+
+
+## 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.
+
+![FigS1.png](FigS1.png)
+
+
+
+## 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.
+
+![FigS2.png](assays/AutophagicActivityAssay/dataset/FigS2.png)
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