ED Fig. 1: 20HE feeding promotes sexually dimorphic ISC mitotic activity.

(a) Male ISCs do not divide strongly in response to infection elicited by pathogenic bacteria, but divide to a similar extent as mated female ISCs in response to 20HE feeding, quantified by counting the number of dividing ISCs per midgut using phospho-histone-3 staining (also termed mitotic index) in males and mated females after 16–18 hrs treatment with 5mM 20HE or pathogenic P.e. infection. Males are fully and equally competent to respond to 20HE treatment as mated females.

(b) Mating boosts the mitotic divisions of ISCs. Feeding 0.1% SDS for 16 hrs to virgin females induces ISCs mitoses and this is inhibited by masculinizing ISC clones using sxl or tra RNAi. Mating increases the ISC mitotic responses to SDS feeding and somewhat restores the ability to masculinized ISCs to divide to stress.

(c) Mating induces basal ISC mitoses in both female (control) ISCs and in masculinized ISC clones with tra or sxl depletion.

(d) 20HE feeding leads to the proliferation and expansion of both control ISCs and ISCs of tra^RNAi masculinized progenitors. Representative images are shown 16 hrs after 5mM 20HE feeding. This experiment was repeated 3 times with similar results. Quantification of this experiment is in main Fig 1a.

(e) Quantification of ISC division at different time points (6, 9 and 12h) after feeding 0.1% SDS to mated females.

(f-j) Males or mated females of the genotypes Gal4.DBD-Usp.LBD>GFP (Gal4-Usp>GFP) (panel f) or Gal4.DBD-EcR.LBD>GFP (Gal4-EcR>GFP) (panels g-j) were heat shocked for 30 min to induce expression of the ligand sensor system, and then either infected with P.e., or fed with 5mM 20HE or vehicle and dissected 18–20hrs later. These GFP ligand traps express GFP under the control of heat-inducible promoter and mark cells with active 20HE signaling. When fed with vehicle, both Gal4-EcR>GFP and Gal4-Usp>GFP were expressed in a few cells in the R4 region posterior midgut (image shown) and in many more in the anterior midgut (image not shown). White arrows where applicable indicate cells that are double positive for delta or Su(H) lacZ markers. 5mM 20HE feeding caused a strong increase in GFP expression in the posterior midgut indicating an up-regulation in the activity of both reporters. GFP was expressed in many delta⁺ cells (panels g,h) and much fewer Su(H)⁺ (panels i,j) of both males and females upon 5mM 20HE feeding. The majority of the remaining positive cells are enterocytes. Upon 20 hrs of P.e. infection, GFP signal disappears from males and females guts indicating that EcR does not play a role in infection-induced stress response (panels g,h). However, Usp reporter was still active in many gut cells as a consequence of P.e. infection (panel f). Usp reporter was also positive in many cell doublets and bigger cells of the midgut. These reporter data suggest that EcR and Usp are both activated by exogenous 20HE feeding yet, they act differently in response to infection. Representative images are shown. This experiment was repeated 5 times with similar results.

For all panels, control flies express UAS-GFP instead of the transgene. The period of RNAi induction is indicated above every panel. Results in dot plots are from 3 or more independent biological replicates. Center is the mean and error bars represent ± s.d.. N≥10 are plotted for each genotype in each scatter plot. Statistical analysis was performed using Mann-Whitney test with two-tailed distribution. (**P≤0.01,*** P ≤0.001, **** P <0.0001). Exact n numbers and P values >0.0001 can be found in the online source data. Representative images are shown. GFP, in green; DAPI, in blue; delta lacZ, in red (g-h); Su(H) lacZ, in red (i-j). Scale bars, panel f=50 μm; panels d,g-j=100 μm.

The overnight standard period of feeding the flies was 16–20 hours. ♂ refers to males, ☿ refers to virgins and ♀ refers to mated females.