Research Paper Volume 10, Issue 3 pp 322—338

Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila

Figure 2. Effects of the knockdown of EC-specific DNA damage response (DDR)-related factors on ISC proliferation. (A-B) EC-specific knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, or Chk2 induce ISC division. Flies carrying Myots>GFP, Myots>GFP+Mre11i, Myots>GFP+Rad50i, Myots>GFP+Nbs1i, Myots>GFP+ATMi, Myots>GFP+ATRi, Myots>GFP+Chk1i, or Myots>GFP+Chk2i genotypes were cultured at 29 °C for 4 days. The guts of flies were dissected and labeled with anti-GFP (green) and anti-PH3 (red) antibodies and DAPI (blue). Original magnification is 400×. (B) A graph showing the PH3+ cell number in the midgut with an EC-specific knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, or Chk2. The gut specimens of Myots>GFP, Myots>GFP+Mre11i, Myots>GFP+Rad50i, Myots>GFP+Nbs1i, Myots>GFP+ATMi, Myots>GFP+ATRi, Myots>GFP+Chk1i, or Myots>GFP+Chk2i flies (kept at 29 °C for 4 days) were labeled with anti-GFP (green) and anti-PH3 (red) antibodies and DAPI (blue). The numbers of PH3+ cells were counted in the whole gut under a microscope. Data (mean±SE) in Myots>GFP, Myots>GFP+Mre11i, Myots>GFP+Rad50i, Myots>GFP+Nbs1i, Myots>GFP+ATMi, Myots>GFP+ATRi, Myots>GFP+Chk1i, or Myots>GFP+Chk2i flies were collated from 21, 22, 13, 20, 9, 9, 26, and 10 guts, respectively. p-values were calculated using student’s t-test. *p < 0.01, ***p < 0.0001. (C) EC-specific knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, or Chk2 increased the number of Delta-positive cells. Flies carrying Myots>GFP, Myots>GFP+Mre11i, Myots>GFP+Rad50i, Myots>GFP+Nbs1i, Myots>GFP+ATMi, Myots>GFP+ATRi, Myots>GFP+Chk1i, or Myots>GFP+Chk2i genotypes were cultured at 29 °C for 4 days. The guts of flies were dissected and labeled with anti-GFP (green) and anti-Delta (red) antibodies and DAPI (blue). Original magnification is 400×.