Figure 5. Bolus dosing with different LDEs or reactive covalent electrophilic drugs elicits complex AR responses.

Also see Figure 6, Figure 3—figure supplements 1 and 4 and 5E, Figure 4—figure supplement 1, and Figure 5—figure supplement 1. (A) Representative IF images of Tg(gstp1:GFP) fish expressing Halo-•-hKeap1-(2xHA), following bolus exposure to indicated electrophiles. (B) Quantitation of data in (A). Image quantitation was performed on the head/tail-regions as illustrated. No. embryos analyzed: Tail, DMSO (55), Sulforaphane (16), HNE (12), Tecfidera (24), NE (9); Head, DMSO (43), Sulforaphane (16), HNE (15), Tecfidera (24), NE (9). Sulforaphane and HNE elicit non-significant and 1.5-fold AR upregulation, respectively. Tecfidera gives medium (~2-fold) AR response and NE elicits the strongest AR upregulation (~3-fold) in tail. Consistent with data elsewhere (e.g., Figure 3A and C-E), head is not responsive. (C) qRT-PCR analysis of endogenous AR-responsive genes following bolus exposure of native reactive LDEs to whole fish similarly shows mixed responses. Whole-head/-tail separation was performed as indicated in inset (left), prior to RNA isolation selectively from the tails. 2 hr post Z-REX, embryos were euthanized, and RNA was isolated separately from tail (see inset, left). Data are presented as mean ± sem. n>3 independent biological replicates and two technical repeats for each sample. All numerical data present mean ± sem. Numbers above the bars represent analysis by two-tailed t-tests.

Figure 5—figure supplement 1. Uncontrolled bulk exposure of developing larvae with reactive electrophiles results in differential proteome labeling extent and adversely affects development/viability.

(A) Treatment with 25 µM CDDO-Me for 4 hr negatively impacts the morphology of the developing embryos (curved tail; malformed/missing caudal fin), whereas Z-REX photocaged probe treatment shows no negative impacts. See also Figure 3—figure supplement 1. Top: CDDO-Me treated, Bottom: Ht-PreNE treated. (B) Comparison of relative whole-reactive-proteome labeling extent by the respective alkyne-bearing small-molecules, following either bolus LDE treatment (2^nd to 4^th bars) or treatment with Z-REX-photocaged-LDE precursor alone (6^th bar) or full Z-REX execution (7^th bar) in live embryos. Zebrafish embryos at 32 hpf were directly treated with 25 μM of the indicated LDEs for 2 hr. After fixing and permeabilization, Cy5 azide was conjugated to alkyne labeled proteins using copper-assisted Click procedure as described in Methods. Larvae were imaged after washing away unbound dye. The Cy5 fluorescence intensity was analyzed using Image-J(NIH). Electrophiles devoid of 4-OH group such as DE and NE (4^th and 5^th bars) (see also Figure 1A inset) efficiently label the whole-reactive-proteome upon bolus treatment of whole embryos, whereas 4-OH-group containing electrophile such as HNE and HDE (2^nd and 3^rd bars) show a lower degree of whole-reactive-proteome labeling. See Figure 6 for representative images related to the data in 1^st to 5^th bars. All numerical data present mean ± sem. Numbers above the bars represent analysis by two-tailed t-tests.