Figure 4.

Figure 4.

Temporal control of transgene expression to regulate autophagic flux. (a-c) Larvae from crosses of elavl3:ERT2-Gal4 and UAS:EGFP were used to optimize the timing and concentration of tamoxifen treatment for temporal control of transgene expression. (a) Schematic diagram of different treatment conditions. (b) Treatment of larvae from 8 h.p.f. to 96 h.p.f. with 1 µM tamoxifen (condition ii) resulted in transgene expression which was evident from 24 h.p.f. and strongly expressed in the CNS from 48 h.p.f. onwards. Representative images taken using GFP filter (excitation 395–455 nm; emission 480 nm). (b and c) Treatment with 2 µM tamoxifen was required to induce GFP expression at later time points, from 24 or 48 h.p.f. onwards (conditions iii and iv, respectively). (c) Western blot and quantification of EGFP protein levels (samples run from 3 independent experiments on single gel). (d) Temporal induction of Atg5 expression results in upregulation of autophagy. All larvae from crosses of ubb:ERT2-Gal4 and UAS:atg5 were treated with 0.1 µM tamoxifen from 8 h.p.f. to 72 h.p.f. to prime transgene expression and then with 3 µM tamoxifen from 72 h.p.f. to 96 h.p.f. Atg5 expression was induced, although not as strongly as when treatment was initiated early (quantified in Fig. S1B). Although no increase in Lc3-II was observed in basal conditions, the strong increase in Lc3-II observed in Atg5 expressing larvae with NH₄Cl treatment, which is much greater than that observed in non-transgenic siblings with NH₄Cl treatment, demonstrates that late induction was able to induce autophagic flux. (e) Temporal induction of Atg4b^C74A expression results in a block in autophagic flux. All larvae from crosses of ubb:ERT2-Gal4 and UAS:FLAG-atg4b_C74A were treated with 0.1 µM tamoxifen from 8 h.p.f. to 72 h.p.f. to prime transgene expression and then with 2 µM tamoxifen from 72 h.p.f. to 96 h.p.f. Strong induction of FLAG-tagged Atg4b^C74A was observed in double transgenic larvae with this late induction protocol which correlated with an increase Lc3-II expression. Lc3-II levels did not increase in controls versus Atg4b^C47A expressing siblings in NH₄Cl treatment conditions indicating that Atg4b^C74A expression causes a block in autophagic flux. Nonspecific bands (blue arrowheads) were observed in all treatment groups and genotypes. (d and e). Graphs show mean values (± SEM) of densitometry of Lc3-II normalized to Actb (loading control) from at least 3 independent experiments. All graphs are normalized to the control (no treatment) condition. Statistical analysis was performed using paired t-tests: ns – not significant; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.