Figure 4. axn mutants are hypersensitive to fasting and energy deficiency.

(A) Compared to the FRT 82 controls, fewer axn¹²⁷ mutant larvae survived after starvation in PBS for 48 hours, and addition of glutamine to PBS largely rescued the lethality of the axn¹²⁷ mutant larvae (p = 0.0002 for PBS vs 25 mM Gln and p<0.0001 for PBS vs 100 mM Gln). (B) With 28 h starvation, survival rates of rbf¹²⁰ larvae or axn¹²⁷ larvae were lower than WT larvae, and rbf¹²⁰ axn¹²⁷ double mutant larvae were much more sensitive to fasting than single mutant larvae (P<0.001 for axn¹²⁷ VS rbf¹²⁰ axn¹²⁷). (C) Quantification of cell death in eye discs fasted for 3 hours. In developing eye discs with mosaic mutant clones, discs without fasting showed very low level of apoptosis in both wild type tissues and axn¹²⁷ or lkb1 axn¹²⁷ mutant clones (D and F). Discs fasted for 3 hours had increased apoptosis in both wild type tissues and mutant mosaic clones (E and G), which are quantified in (C). The level of apoptosis in axn¹²⁷ or lkb1 axn¹²⁷ clones with fasting was significantly higher than surrounding wild type cells (p = 0.0015 for WT vs axn¹²⁷ and p<0.0001 for WT vs lkb1 axn¹²⁷), and lkb1 axn¹²⁷ mutant cells have more apoptosis than axn¹²⁷ mutant cells (P<0.002) (C). lkb1 single mutant did not show significant level of apoptosis (H), lkb1 rbf or lkb1 axn^S mutations induced synergistic apoptosis (I–J), and lkb1 axn^S rbf triple mutant cells had very high levels of apoptosis (K). Quantification of C3 levels in (H–K) is shown in panel (M). The ATP/ADP ratio of eye discs with axn^S, axn^S rbf, or axn^S rbf rheb mutant clones induced in Minute background were determined (L). Comparing to wild type cells (FRT 82B), axn^S mutant cells had slightly lower ATP/ADP ratio (p<0.01); the ATP/ADP ratio of axn^S rbf mutant cells was significantly lower than that of the axn^S or rbf mutant cells (p<0.0001); the ATP/ADP ratio of axn^S rbf rheb mutant cells was similar to that of the axn^S mutants (p = 0.4, between axn^S and axn^S rbf rheb) (L). * and ** indicate p<0.0001 and p<0.002, respectively.