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Fluorescence microscopy For imaging, yeast strains tagged with fluorescent markers were cultured in nutrient-rich medium (YPD) and were treated with 300 M IAA for 8 h for conditional Vma2 degradation or shifted to nitrogen starvation (SD-N) medium for 2 h

This allows researchers to isolate metabolic signaling effects without confounding endocrine responses, making AOD 9604 a useful model for studying localized metabolic regulation