A: Intestines of flies carrying the gstD::lacZ reporter and esg::Gal4, UAS::GFP (ISC/EB marker; green). A’: Intestines are stained for β-galactosidase (β-gal, red) and DNA (DAPI, blue) or armadillo and the enteroendocrine (EE) marker prospero (arm/pros, blue). β-gal channel is shown separately in the lower panels. White arrowheads indicate ISCs, red arrowheads point to EEs. β-Gal is expressed in esgGFP+ cells and EEs in wild-type flies. A”: Intestines are stained for β-gal (red), the ISC marker delta (red), and DNA (DAPI, blue) as indicated. Lower panels show β-gal and delta separately. Note co-localization of β-gal and Dl expression, indicating that CncC activity is present in ISCs.
B: gstD::lacZ reporter activity (β-gal) is absent from CncC^RNAi - expressing ISCs. Note that β-gal expression in EEs is not affected by esg-mediated expression of CncC^RNAi. GFP, green; β-gal, red; DAPI or arm/pros, blue.
C: GFP-marked (MARCM) ISC clones mutant for cnc^VL110; cnc^K6; keap1^EY5; keap1⁰³⁶; and keap1⁰³⁶, cnc^K6 at 7 days after heat shock. Single confocal sections: GFP, green; arm/pros, red; DAPI, blue.
D: Quantification of clone sizes at 7 days after heat shock (averages and standard deviations of numbers of cells per clone; Student's T-test).
E: MARCM clones over-expressing only GFP (left, WT) or GFP and CncC (right). Intestines were dissected 8 days after heat-shock. GFP, green; arm/pros, red.
F: “Flp-out” clones over-expressing CncC at 7 days after heat-shock (GFP, green; DAPI, blue).
G: Inhibition of ISC proliferation by CncC is reversible. CncC over-expressing clones were induced using the Flp-out system in the presence of the temperature-sensitive Gal4 inhibitor Gal80^ts. After heat-shock, flies were maintained at the permissive (18°C) or the restrictive (29°C) temperatures for 14 days (18-18, 29-29, respectively) or switched from one to the other temperature after 7 days (18-29, 29-18). Intestines were dissected at 7d (7d 18) or after 14 days (all others). In wild-type control flies, the restrictive temperature does not affect clone growth (at 7 or 14 days after heat shock), while in CncC over-expressing flies incubation at 29°C for 14 days inhibits growth (only in these flies, CncC is over-expressed throughout the experiment). In CncC over-expressing flies maintained at 29°C for 7 days (in which clones fail to grow, F), but then shifted to 18°C (29-18), clone size recovers, indicating that CncC-mediated repression of ISC proliferation can be reversed. Conversely, clone size decreases in CncC over-expressing flies maintained at 18°C for 7days and then transferred to 29°C (18-29). Averages and standard deviations of clone cell numbers are plotted; *p<0.001, Student's T-test. Numbers of clones assessed (n) is shown as insert in each bar.
See also Figure S1.

A: CncC represses JNK- and InR- induced ISC proliferation. Quantification of pH3⁺ cells in intestines expressing CncC, InR and/or the JNKK Hep in ISCs and EBs using the TARGET system. Intestines were analyzed 5 days after shift to the restrictive temperature (Average and SEM; Student's T-test; N shown in each bar).
B: Expression of CncC or repression of Keap (Keap1^RNAi) limits growth of ISC and ee tumors, while reduced cnc genedose (cnc^VL110 heterozygosity) enhances tumor growth in Notch loss-of-function conditions. Expression of N^RNAi and other transgenes was induced by shifting flies to restrictive temperature (29°C). Intestines were analyzed 5 days after induction. Representative intestines are shown (GFP, green and armadillo/prospero, red). Quantification of tumor sizes is shown below.
See also Figure S3.

A: Increased ISC proliferation in response to loss of mitochondrial electron transport chain components (NADH:ubiquinone reductase 75kD and 42kD subunit precursors; ND75^RNAi and ND42^RNAi). PH3⁺ cells per gut are quantified (Average and SEM, Student's T-test, N in bars).
B, C: Representative images and quantification of DHE fluorescence in intestines over-expressing Gclc or Jafrac1 in ISCs (7 day old flies, Average and SEM, Student's T-test, N in bars; esgGFP, green; DHE, red).
D: Cell numbers in MARCM clones over-expressing Gclc or Jafrac1 quantified at 4 and 14 days, or at 7 days after heat shock (average cell numbers and standard deviations; Student's T-test).
E: Quantification of mitotic figures in intestines of 10 day-old flies over-expressing Gclc or Jafrac1 under the control of esgG4::UASGFP (Average and SEM, Student's T-test).
F: Cnc-mediated repression of ISC proliferation in response to Paraquat exposure requires jafrac1 gene function. CncC was over-expressed under the control of esgGal4 in wild-type or jafrac1^KG05372 heterozygous backgrounds (Average and SEM, Student's T-test).
See also Figure S5.

Nrf2/CncC induces anti-oxidant gene expression, promoting a reducing environment in ISCs that ensures cytoprotection and limits ISC proliferation. Keap1-mediated inhibition of CncC is critical to allow proliferation of ISCs in response to stress and mitogenic signaling. By limiting ISC proliferation, the Keap1/CncC signaling module thus balances the need for ISC-mediated tissue regeneration with the necessity for proliferation control, promoting proliferative homeostasis in the intestinal epithelium.

A: gstD1::lacZ expression is repressed in esgGFP+ cells in response to oxidative stress. As shown in Figure 1, β-Galactosidase is expressed in ISCs and EBs of mock-treated gstD1::lacZ carrying flies (top panels; GFP, green; armadillo/prospero, blue; β-gal, red). After Paraquat exposure (5mM, 24 hours), β-Gal is strongly expressed in ECs, but cannot be detected in esgGFP+ cells (bottom panels, arrowheads). Overviews are shown left, higher magnification on the right. Single confocal sections are shown.
B: Intestines treated as in A, co-stained for the ISC marker delta (red, left panels) and β-gal (red, middle and right panels). Dl+ cells are also β-gal+ in mock treated intestines (top panels), but become β-gal- in response to paraquat (lower panels). See Figure S3 for more examples.
C: Quantification of relative β-gal fluorescence intensity of ISCs compared to EEs in mock and paraquat treated intestines. Note that gstD1::lacZ expression in EEs is not affected by Paraquat (Figure S3A). Only Dl+ cells were analyzed. Averages and standard deviation, Student's T-test.
D: BrdU incorporation in intestines of paraquat - treated flies expressing CncC or Keap1^RNAi in ISCs and EBs using the TARGET system (transgene expression was induced by shifting flies to 29°C for 3 days prior to paraquat exposure). GFP (green), BrdU (red). Mock treated flies incorporate BrdU in very few cells during a 24hour exposure (upper panels, see also Figure S2). In response to Paraquat exposure, BrdU is widely incorporated in wild-type flies (WT), but not in flies expressing CncC or Keap^RNAi (lower panels). A single confocal section is shown in each panel. Arrowheads point to selected BrdU⁺ cells. Frequency of BrdU incorporation (left; calculated as the fraction of GFP+ cells that are also BrdU⁺; averages and standard deviation, Student's T-test) and amounts of pH3⁺ cells per gut (right; averages and SEM; Student's T-test) are shown in the histograms (N, numbers of analyzed intestines, are shown in the bars).
See also Figure S2.

A-C: Di-hydro-ethidium (DHE) staining of live intestines of flies treated with Paraquat (PQ, 5mM in 5% sucrose) or mock (5% sucrose) for 24 hrs. Representative images are shown in A (single confocal section (GFP, green; DHE, red; DHE channel is shown alone in lower panels). B: Quantification of fluorescence intensity of the DHE channel (Average and standard deviation; Student's T-test; N shown in each bar). Note that fluorescence intensities increase in both ISCs and ECs after paraquat treatment. C: relative DHE fluorescence in ISCs, calculated as ratio of fluorescence intensity in ISCs and nearby ECs. N=number of analyzed guts (total analyzed cells in parenthesis) shown in each bar.
D: DHE staining of PQ-treated intestines over-expressing CncC or Keap1^RNAi in ISCs and EBs. DHE channel is separated in grayscale in lower panels. Images are from single confocal sections. Arrowheads point to individual ISCs for orientation (basal location in the epithelium, isolated esg+ cells in CncC or Keap^RNAi expressing guts). Quantification of relative DHE fluorescence of ISCs compared to surrounding ECs is shown in the graph (Average and standard deviation; Student's T-test; N=number of analyzed guts (total analyzed cells in parenthesis) shown in each bar).
E: Loss of CncC results in increased ROS levels in the ISC lineage and increases ISC proliferation. DHE fluorescence in intestines from flies expressing CncC^RNAi and GFP (CncC^RNAi) or only GFP (w¹¹¹⁸) under the control of esg::Gal4 (representative images left, quantification middle). Fluorescence intensity in GFP⁺ cells is normalized to DHE fluorescence in adjacent ECs. (Average and standard deviation; Student's T-test; N=number of analyzed guts (total analyzed cells in parenthesis) shown in each bar).
Quantification of pH3⁺ cells in intestines of 2, 5, and 10 day-old flies is shown in the histogram on the right (average and SEM; Student's T-test; N shown in each bar).
See also Figure S4.

A, B: Elevated ROS concentration in the intestinal epithelium of aging flies. Images (A) and quantification (B) of DHE fluorescence in intestines of young (3 day) and old (30 day) flies (DHE, red). Intestines were co-stained in the same well to ensure equal DHE staining, young and old guts were identified by GFP expression (3d: OreR, 30d: esg::G4;UAS::GFP; Average and standard deviation, Student's T-test, N in bars; GFP, green; DHE, red. Normalized fluorescence (ISC/EC ratio) is shown on the right. See Figure S7 for additional images.
C: Reporter activity in young (3 day) and old (30 day) flies (GFP, green; β-gal, red). gstD1::lacZ expression is reduced in most ISCs of old flies (white arrowheads). Infrequently, single esg-GFP+ cells expressing β-gal are detected (red arrowheads).
D: Reporter activity detected in 30-day-old flies counterstained with Dl antibody to identify ISCs (Dl, red, left panels; β-gal, red, right panels). White arrowheads point to ISCs for orientation, red arrowheads to EEs.
E: quantification of β-gal fluorescence intensity. As in D, ISCs detected as esg-GFP+, Dl+ cells. Average and standard deviation, Student's T-test, N in bars.
F: ISC proliferation in aging wild-type flies (esg::Gal4, UAS::GFP, blue) and in flies expressing CncC (red) or CncC^RNAi (green) in ISCs and EBs. Flies were analyzed by counting PH3⁺ cells in the gut every 5 days. Data points represent averages +/- SEM.
G: Four categories based on the extent of accumulation of esg⁺ cell clusters used for quantification of intestinal degeneration (representative pictures). The four categories correlate with increasing numbers of PH3⁺ cells (graph on right).
H: Quantification of intestinal degeneration in aging flies (5-30 days of age) by blind scoring of individual intestines. In addition to containing fewer proliferating cells, guts with elevated levels of CncC in ISCs/EBs show significantly reduced intestinal degeneration compared to wild-types, whereas CncC knock-down in ISCs/EBs accelerates the progression of intestinal degeneration. P-values from Pearson χ² test (compared to wild-types of same age), N shown in each bar.
See also Figure S6.