Research Paper Volume 16, Issue 3 pp 2005—2025

Figure 3. EC-specific VDR knockdown increases age-related ISC phenotypes. (A) EC-specific VDR knockdown induces DNA damage accumulation in ISCs and their progenitors. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotype were incubated at 29° C for 7 days. The guts of flies were dissected and labeled with anti-γH2AvD (red), anti-Pros (white), and anti-GFP (green) antibodies and DAPI (blue). (B) EC-specific VDR knockdown causes centrosome amplification in mitotic ISCs. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotype were incubated at 29° C for 7 days. (a) The entire guts of the flies were dissected, fixed, and labeled with anti-γ-tubulin (red), anti-PH3 (white), and anti-GFP (green) antibodies and DAPI (blue). The original magnification is 400×. (b–d) The number of mitotic ISCs with supernumerary centrosomes (>2) in the midguts of Myo^ts>GFP or Myo^ts>GFP+VDRRi flies increased. (b) EC-specific VDR knockdown increases mitotic ISCs in the guts. (c) Frequency of cells with supernumerary centrosomes per mitotic ISC. (d) Number of cells with supernumerary centrosomes per midgut. Three-day-old female flies were cultured at 29° C for 7 days, and then their dissected guts were fixed and immunostained with anti-γ-tubulin (red), anti-PH3 (white), and anti-GFP (green) antibodies and DAPI (blue). In these guts, the number of abnormal centrosomes in the PH3⁺ cells were determined. Data (mean ± SD) in Myo^ts>GFP or Myo^ts>GFP+VDRRi flies were collated from 154 and 2281 mitotic cells of 21 and 20 guts, respectively. P-values were calculated using Student’s t-test. P < 0.0001 compared with Myo^ts>GFP flies.