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Fig. 9 DALDA reverses glafenine-induced organelle stress and restores proper UPR responses. (A-E) All zebrafish studies were performed after of 12 hours glafenine exposure. (A) TEM with and without DALDA treatment demonstrates a lack of ER and organelle stress and resolved UPR. White arrowheads show pitting from ER stress. White arrow demarks apoptotic IEC. Black arrowheads indicate the brush border. Scale bars: 2 μm. (B) Same conditions as for A, showing granularity in the cytoplasm of segment 1 IECs, indicative of rough ER. Scale bar: 0.5 μm. (C) Glafenine-induced injury causes BiP protein accumulation, an early signal in the UPR cascade. Western blot representative of four independent experiments, each consisting of protein lysate from e10 pooled fish per condition. β-actin was used as loading control. (D) Real-time PCR for UPR genes: u-xbp1 (unspliced), s-xbp1 (spliced) and atf6. Each replicate for each condition was generated from 10 pooled micro-dissected fish gastrointestinal tracts, n≥4/group. (E) Fish were injected with 5 pmol of control or atf6 MO in 4.6 nl of water and subjected to the AO tube assay. Values were pooled from two independent experiments, n≥29/group. veh, vehicle-only group; glaf, glafenine-treated group; G+D, group treated with glafenine plus DALDA. Right: RT-PCR for atf6 in morpholino-injected fish, at 6 dpf. Wild-type band is at 180 bp, the morphant with an intron-1 inclusion is at 314 bp. Representative of triplicate groups of two independent experiments. *P<0.05, ***P<0.001.