From 80b04fbf0e0bcfdc748acfe3e4af5466d21fda9e Mon Sep 17 00:00:00 2001 From: Dominik Brilhaus <brilhaus@nfdi4plants.org> Date: Mon, 21 Oct 2024 06:58:09 +0000 Subject: [PATCH] link images / movie to README --- assays/AutophagicActivityAssay/README.md | 43 ++++++++++++++++++++++++ 1 file changed, 43 insertions(+) diff --git a/assays/AutophagicActivityAssay/README.md b/assays/AutophagicActivityAssay/README.md index e69de29..664a934 100644 --- a/assays/AutophagicActivityAssay/README.md +++ b/assays/AutophagicActivityAssay/README.md @@ -0,0 +1,43 @@ + +# 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. + + \ No newline at end of file -- GitLab