Effects of methotrexate on heterochromatin and cell proliferation. (A,C,G) Flies were raised on food with or without methotrexate at the indicated concentrations (10 µM) in 33%DMSO. Salivary glands from 3rd instar larvae were immunostained with anti-H3K9me3 (A) or anti-HP1 (C) and photographed with a compound microscope. Heterochromatin levels were quantified as the ratio of H3K9me3-positive area to the area of the nucleus. Representative images are shown. (G) Total protein was extracted from 3rd instar larvae without or with methotrexate treatment at the indicated concentrations, and was subjected to SDS-PAGE, following by blotting with antibodies specific for H3K9me3, H3, or α-Tubulin. (B,D) Quantification of H3K9me3 (B) or HP1 (D) immunostaining as the ratio of antibody-positive area (dark brown) to the area of the nucleus (light brown). Error bars are standard deviations. *Indicates p < 0.05 by Student’s t-Test. (E) Flies carrying the GMR-Gal4 and UAS-Upd transgenes were raised at 25 °C on food with or without 10 µM methotrexate. Adult male flies were photographed to show the eyes. (F) Eye volumes were quantified by measuring at O.D. 480 nm the amount of red pigments in fly heads.