Supplementary Fig. 1 Paternal restraint stress reduces w gene expression in both sexes of w m4 offspring. a To induce restraint stress, a soft sponge plug was inserted into a plastic vial and CO2-anesthetized males (20 flies) were placed on the surface of the sponge and fixed very gently using another soft sponge plug inserted from the top of the vial (right). Flies were also placed into vials not containing fly media as controls (left). b An enlarged photo of a fly 'sandwich'. c Viability of flies at 12 h after restraint stress treatment. Averages with standard deviation (s.d.) are shown (n = 3; ***p < 0.001; N.S., no significant difference, Student's unpaired t-test). d-e Red-eye pigment levels were measured in F1 male (d) and female (e) progeny derived from three paternal conditions (no stress, control, and restraint stress). (d, e) The values of red-eye pigment represent relative to each control. Averages with s.d. are shown (***p < 0.001; **p < 0.01; *p < 0.05; N.S., no significant difference, Student's unpaired t-test). 
Number of flies analyzed indicated in parentheses on each graph. f, g Eye phenotypes of F1 w m4 males derived from dATF-2 KO (f) and Mekk 1Ur36 (g) father with/without restraint stress. Note that F1 offsprings from dATF-2 KO mutant father exhibit high eye-pigment level as like same as dATF-2 PB previously reported 1 , and the eye-pigment level did not increase further in offsprings from restraint stress-exposed fathers. 

Gene name

Forward primer (5'-3') Reverse primer (5'-3') 
rp49 GATGACCATCCGCCCAGCATAC AGTAAACGCGGTTCTGCATGAGC upd1 TCTGGTTGCAGTTGCATTCG GGGTTACTGGGTGGGATTGG upd2 CCCTGGAGTACGGCAATCTG CATGTGGCGGTACCAAGTCT upd3 CTACAGATTCCTGCCCCGTC GTTGCGCATGTACGTGAAGG TotA CCCTGAGGAACGGGAGAGTA CTTTCCAACGATCCTCGCCT spz ACGTACGAGGCCAAGGAGTA GGGTATGGGCATGAAGGGTG spz3 GGTGCCGCTCTTCTCCTATC CACATCCAGTCCCAAAGCCA spz4 ACTGCAGACGATTTGCGATTG CAGATTTCACACTCGCACGC spz5 AGGAAAGACGTACTGCGAGC TTCGTCCACCAACAGAGTGG spz6 AAGAAGCAAACGAGCGGAGA CCTCACCGTAGTCAGATGGC eiger ACTTCCAAAGAGAGCCCTGC AGTCCTCGGATCTGGCTGAA dATF-2-1 CCGGAGACATGGATCACCTGGCC GCAGGATACGTCGCCCTCTGGGG dATF-2-2 CCGCCAGCAACGCAAGCGCGGCC GAAGGAATTTAACTTAACTGAGG