Figure 3. Pharmacological inhibition of in vivo SFK signaling elicits reduced Src expression and induction of dFOXO-dependent transcription.

(A, B) Quantitative real-time PCR from total RNA extracts from 2^nd instar larvae are shown. Src42A expression levels are reduced in Src64B mutants and vice versa. (C) Quantitative real-time PCR from total RNA extracts from larvae are shown. Animals were fed on yeast for 48 h and treated with the Src inhibitor SU6656 for 24 h. Bar pairs 1–3: wildtype treated with DMSO as a control or 0.5 mM or 1 mM of SU6656, respectively. SU6656 treatment strongly upregulates the expression of 4E-BP in a dose-dependent manner. Bar pairs 4–6: Heterozygous dFOXO²¹ mutants were treated with DMSO or Src inhibitor. Only high concentrations of SU6656 show a weak and non-significant effect on 4E-BP expression. Bar pairs 7–9: Heterozygous dFOXO²⁵ mutants were treated with DMSO or Src inhibitor. SU6656 has no significant effect on 4E-BP expression levels. (D, E) Fatbody tissue from early 3rd instar wildtypic larvae treated with DMSO (D) or 1 mM SU6656 (E) immunostained with a dFOXO antibody. Insets show fluorescence intensity. The inset false colors correspond to increasing fluorescence intensity in the order blue-green-yellow-red (weak to strong). dFOXO translocates to the nucleus in fatbody cells from SU6656-treated animals. (F) Quantification of the nuclear dFOXO levels using the CTCF value. Cells from animals treated with SU6656 show a significantly stronger nuclear dFOXO signal compared to controls. Statistical significance was tested using ANOVA with Tukey-Kramer post test, except for (B) where data was analyzed with a Kruskal-Wallis test and Dunn post test. Asterisks represent * = p < 0.05, ** = p < 0.01, *** = p < 0.001, or ns for non-significant differences. Significance was tested against the respective wild type control.