Supplementary Figure 8: The RCK Fungal Homolog Vad1 Plays a Role in Decapping and Degradation of Autophagy-related Transcripts

(a) Northern blots of RNA from the indicated strains were hybridized with fragments of the indicated genes. (b) Wild-type fungal cells overexpressing VAD1 from an ACTIN promoter (OE-1, 2) or containing empty vector alone (EV-1, 2) were induced for autophagy by starvation for 1 h followed by northern blot analysis. (c) The indicated strains were observed by DIC for the presence of autophagic bodies (ABs) and quantified in n = 3 independent experiments of 200 cells each by DIC + / − SD. Student’s t-test, ^∗∗∗p < 0.001. (d) RNA from WT (left panel) or vad1D mutant mid-log phase strains (right panel) was subjected to either northern blot (hybridized with a fragment of ATG8) and ratios of signal by densitometry to rRNA plotted over time (top panels) or ATG8 was assayed by quantitative RT–PCR normalized to actin (bottom panels) slope: WT versus vad1D p < 0.001. (e) The indicated strains expressing a GFP-Atg8 fusion protein containing either the native or heterologous 3’UTR (EF1a) under the indicated conditions were incubated with phenylmethanesulfonylfluoride for 30 min and observed by DIC microscopy for the presence of autophagic bodies (arrows). Scale bar = 2 μm. Representative image from n = 50 cells.