Fig. 2: Validation of hypoxia sensors under environmental hypoxia.
a Schematic representation of the FlHypox- and FlHypoxP850A-expressing transgenes. HβDD (HIF-1β dimerisation domain) was mutated at three residues (Q339E, V413E and Y417T) to prevent dimerisation with HIF-1β; ODD oxygen-dependent degradation domain. mNeonGreen was inserted between amino acid residues S725 and A726. The P850A mutation prevents hydroxylation of HIF-1α, thereby inhibiting oxygen-dependent degradation. b-b’ Anti-V5 immunofluorescence analysis of L3 discs dissected from larvae cultured in either 20.9% (normoxia) or 5.0% environmental oxygen. All discs were imaged under identical conditions. Scale bar = 100 μm. Here, and in subsequent relevant images, the pouch was outlined with a dotted line to mark the area for quantification shown in (b’). n ≥ 8 for each condition. Data normality was confirmed with the Shapiro–Wilk test. Statistical significance was assessed by a two-sided unpaired t test (p ≥ 0.05, not significant). For a detailed description of box plots, see Supplementary Fig. 1e–e’. c-c’ HypoxyprobeTM adduct analysis of L3 wing discs dissected from larvae cultured in different environmental oxygen. All the discs shown in (c) were imaged under identical conditions. Quantification of wing pouch fluorescence intensity is shown in (c’). Scale bar = 100 μm. n ≥ 10 for each condition. After confirming normality of the data, statistical significance was assessed by a two-sided unpaired t test. d Schematic representation of the SyHREns transgene. HRE hypoxia response element. Note that the presence of two copies of SyHREns leads to a mild developmental delay (approximately one hour over the approximately five days to pupariation) but has no other detectable effects on development. e-e’ Fluorescence from SyHREns in L3 discs dissected from larvae cultured in different environmental oxygen for 2.5 h. Representative images are shown in (e) (all discs imaged under identical conditions), and quantification of wing pouch fluorescence intensity is shown in (e’). Scale bar = 100 μm. n ≥ 7 for each time point. After confirming normality of the data, statistical significance was assessed by a two-sided unpaired t test.
