Additional file 2: of The integrated stress response regulates BMP signalling through effects on translation

Figure S2. crc is Drosophila ATF4. (A) HA-crc-expressing S2 lysates and matched samples incubated with λ phosphatase (λ ppase) were subjected to SDS-PAGE and transferred to nitrocellulose. Immunoblotting was performed using an anti-HA antibody. (B) The 5’UTR of crc transcript E: small upstream open reading frames (uORFs) in orange; coding sequence in red. (C) Luminescence signal of luciferase control (blue bars) or 5’UTR-crcE-luciferase reporter (red bars) expressed in HEK293T cells presented as the ratio of firefly/Renilla luminescence fold change compared to vehicle-treated samples. Cells were treated with the indicated concentrations of ISRIB and/or tunicamycin for 16 h. Mean ± standard error of the mean (SEM). n = 3. P value calculated using ANOVA with Bonferroni post hoc testing. (D) Representative photomicrographs of adult eyes. Gmr (gmrGAL4 driver control), gmr > perk (gmrGAL4 > UAS-perk), gmr > crc RNAi (gmrGAL4 > UAS-crc RNAi) and gmr > perk;crc RNAi (gmrGAL4 > UAS-crc RNAi;UAS-perk). Scale bar = 200 μm. (E) Representative photomicrographs (5× objective) of adult wings of the indicated genotypes. en (enGAL4 driver control), en > dicer2;ppp1r15 RNAi (enGAL4 > UAS-dicer2;UAS-ppp1r15 RNAi), en > dicer2;crc RNAi (enGAL4 > UAS-dicer2;UAS-crc RNAi) and en > dicer2;crc RNAi;ppp1r15 RNAi (enGAL4 > UAS-dicer2;UAS-crc RNAi;UAS-ppp1r15 RNAi). Lower panels are enlargements of the crossvein territories. Scale bars = 250 μm. (F) Quantification of ACV phenotype in (E). (G) Representative photomicrographs of adult wings of the indicated genotypes. nab (nabGAL4 driver control), nab > ppp1r15 RNAi (nabGAL4 > UAS-ppp1r15 RNAi), nab > crc RNAi (nabGAL4 > UAS-crc RNAi) and nab > ppp1r15 RNAi;crc RNAi (nabGAL4 > UAS-crc RNAi;UAS-ppp1r15 RNAi). Lower panels are enlargements of the crossvein territories. Scale bars = 250 μm. (H) In situ hybridisation of w1118 wing imaginal disc with sense or antisense probes to residues 1405–1900 of crc transcript A. (I) Representative photomicrographs of adult wings of the indicated genotypes. nab (nabGAL4 driver control), nab > crc (nabGAL4 > UAS-crcA). Lower panels are enlargements of the crossvein territories. Scale bar = 250 μm. (PDF 3370 kb