(A) Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, and protruding-mouth stages for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). The mean fluorescence intensity (FI) of Nrf2a protein in the (B) body tissue, (C) brain, (D) heart, (E) gut, and (F) pancreas was measured; liver data are presented at higher magnification in Figure 3 below. Representative heatmaps were generated to visualize Nrf2a protein fluorescence differences at the (G) pharyngula, (H) hatching, and (I) protruding-mouth stages. The gut and pancreas are not present (n.p.) at the pharyngula stage. Statistics were performed with a two-way ANOVA and Fisher’s LSD post hoc test. n = 7–12 fish. Letters indicate significant differences (p ≤ 0.05) among groups. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bar = 100 µm.

(A) Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, or protruding-mouth stage for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). Z-stacks of the islet were acquired via confocal microscopy. (A) Islet volume and (B) islet mean fluorescence intensity (FI) of Nrf2a protein were measured with Nikon NIS elements software (available at Light Microscopy Core analysis workstations at the UMass Amherst Institute for Applied Life Sciences; https://www.microscope.healthcare.nikon.com/bioimaging-centers/nic-and-cofe/university-of-massachusetts-amherst). Representative images of each group at the (C) pharyngula, (D) hatching, and (E) protruding-mouth stage are shown. Images show max intensity projections of the islet Z-stack (circled in yellow) where FITC (green) represents β-cells, TRITC (red) represents Nrf2a protein, and DAPI (blue) represents nuclei. Statistics were performed with a two-way ANOVA and Fisher’s LSD post hoc test. n = 5–12 fish. Letters indicate significant differences (p ≤ 0.05) among groups. Scale bar = 25 µm.

Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the protruding-mouth stage for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). Liver images were acquired using confocal microscopy. (A) The mean fluorescence intensity (FI) of labeled Nrf2a protein in the liver was quantified via image analysis. (B) Representative images of the zebrafish liver are shown, where the liver is outlined in yellow, TRITC (red) represents Nrf2a, and DAPI (blue) represents nuclei. Statistics were performed using a one-way ANOVA followed by Fisher’s LSD post hoc test. n = 8–13 fish. Letters indicate significant differences (p ≤ 0.05). Scale bar = 50 µm.

Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, and protruding-mouth stages for 6 h and then fixed, and Nrf2a protein was labeled using immunohistochemistry (IHC). Colocalization analysis was performed on 40× confocal images of the liver, and a representative image of the pancreatic islet was taken from the Z-stack. Pearson’s R coefficients were converted to normally distributed Z-scores, shown as means ± SEMs, where higher Z-scores indicate greater colocalization between Nrf2a protein and nuclear DAPI staining within the region of interest. Significance was assessed with a two-way or three-way ANOVA followed by Fisher’s LSD post hoc test. n = 5–13 fish. Different letters indicate significant differences (p ≤ 0.05) among tissues. **** p ≤ 0.0001 between islet and liver of the DMSO WT groups.

(A) Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the pharyngula, hatching, and protruding-mouth stages for 6 h and incubated with BioGee for 2 h prior to fixation (24 h post-exposure). BioGee protein conjugates were labeled in situ using immunohistochemistry. (B) Mean fluorescence intensity (Fl) of BioGee protein conjugates. Representative heatmaps were generated to visualize BioGee-protein conjugate fluorescence at the (C) pharyngula/hatching, (D) hatching/protruding-mouth, and (E) protruding-mouth/larval stages. Statistics were performed using a two-way ANOVA followed by Fisher’s LSD post hoc test. n = 8–10 fish. Different letters indicate significant differences (p ≤ 0.05) among groups. Scale bar = 100 μm.

Zebrafish were treated with 40 µM SFN or 1 µM tBHQ for 6 h during the pharyngula, hatching, and protruding-mouth stages and incubated with BioGee for 2 h before fixation 24 h after the start of the exposure. BioGee protein conjugates were labeled in situ using IHC. Confocal microscopy was used to acquire Z-stacks of the islet. (A) Islet volume and (B) islet mean fluorescence intensity (FI) of BioGee. Representative images at the (C) pharyngula (start of exposure)/hatching (BioGee labeling and fixation), (D) hatching/protruding-mouth, and (E) protruding-mouth/larval stages are shown. Images are max intensity projections of the Z-stack the islet (outlined in yellow), where FITC (green) represents β-cells, TRITC (purple) represents BioGee protein conjugates, and DAPI (blue) represents nuclei. Statistical significance was assessed via two-way ANOVA followed by Fisher’s LSD post hoc test. n = 7–9 fish. Letters indicate significant differences (p ≤ 0.05) among groups. Scale bar = 25 µm.

Zebrafish were treated with 40 µM SFN or 1 µM tBHQ during the hatching and protruding-mouth stages for 6 h and incubated with BioGee for 2 h before fixation 24 h after treatment. BioGee protein conjugates were labeled in situ using IHC. Confocal microscopy was used to acquire images of the liver using a 40× objective. (A) Liver mean fluorescence intensity (FI) of BioGee protein conjugates was determined via image analysis. Representative images of the zebrafish liver treated during the (B) hatching/protruding-mouth and (C) protruding-mouth/larval stage are shown where the liver is outlined in yellow, TRITC (purple) represents BioGee, and DAPI (blue) represents nuclei. The liver is not present (n.p.) at the stage visualized. Statistics were performed using a two-way ANOVA followed by Fisher’s LSD post hoc test. n = 6–12 fish. Different letters indicate significant differences (p ≤ 0.05) among groups. Scale bar = 50 µm.

Zebrafish were treated with 40 µM SFN or 1 µM tBHQ for 6 h during the pharyngula, hatching, and protruding-mouth stages and then collected for gene expression via quantitative real-time PCR. mRNA expression was measured for glutathione S-transferase Pi (gstp) along with the housekeeping gene β2-Microglobulin (b2m). The ΔΔCT method was used to calculate fold change. Values are mean fold change ± SEM. Statistical significance was assessed via two-way ANOVA followed by Fisher’s LSD post hoc test. n = 2–3 pools of 15–20 (hatching and protruding-mouth stages) or 20–40 (pharyngula) fish. Different letters indicate significant differences (p ≤ 0.05) among groups.