Fig. 6

Characterization of DTT and tunicamycin induced ER stress in Tg(ef1α:xbp1δ-gfp) transgenic zebrafish embryos. Transgenic embryos with the paternally derived Tg(ef1α:xbp1δ-gfp) transgene were treated with 0.5 mM of DTT, 1 µg/ml or 2 µg/ml of tunicamycin between 24 and 48 hpf. XBP1Δ-GFP expression and mRNA splicing were analyzed at 48 hpf after the DTT or tunicamycin treatment. (A, E) Side and dorsal views of non-transgenic fish embryos at 48 hpf. (B, F) Side and dorsal views of Tg(ef1α:xbp1δ-gfp) transgenic embryos at 48 hpf without DTT treatment. (C, G) XBP1Δ-GFP expression in transgenic embryos at 48 hpf with 24 h of 0.5 mM DTT treatment. (D, H) XBP1Δ-GFP expression in transgenic embryos at 48 hpf with 24 h of 1 µg/ml tunicamycin treatment (D) or without the treatment (H). (I) RT-PCR analysis shows the expression and splicing of the Tg(ef1α:xbp1δ-gfp) transgene and the endogenous xbp1 gene in DTT treated or control paternal transgenic (P-) and wild type (WT-) embryos. P-Endo represents xbp1 expression from the endogenous gene. P-Exo represents xbp1 expression from the exogenous Tg(ef1α:xbp1δ-gfp) transgene. WT-Endo represents xbp1 expression from the endogenous gene in wild type embryos. Arrows indicate spliced xbp1 transcripts. (J). Western blot analysis shows the expression of the myc-tagged XBP1Δ-GFP fusion protein derived from the paternal transgenic line at 48 hpf in control and DTT treated embryos. (K). RT-PCR analysis shows the splicing of endogenous and exogenous xbp1 in paternal transgenic zebrafish embryos treated with 1 µg/ml (TUN-1) and 2 µg/ml (TUN-2) tunicamycin for 24 hpf between 24 and 48 hpf. EF1α expression was analyzed as an internal control. Arrows indicate spliced xbp1 transcripts. (L). RT-PCR analysis shows the expression of ER stress markers, xbp1 and BIP, in zebrafish embryos treated with 0.5 mM DTT for 24 hpf between 24 and 48 hpf.