Table 1.
Oligonucleotide primers and probes used for qRT-PCR to quantify gene expression levels of mx, tnfα, ifnγ, t-bet and nitr9.
Fig 1.
Leukocytes from liver (A), kidney (B) and spleen (C) tissues, based on morphological appearance and FACS forward scatter (FSC) and side scatter (SSC) characteristics.
Table 2.
Size analyses of lymphocyte-like cells from liver, kidney and spleen tissues in rag1-/- mutant zebrafish.
Fig 2.
Leukocytes in rag1-/- mutant zebrafish liver included: (A) monocytes/macrophages, (B) large LLCs, (C) small LLCs.
Cells were stained with Wright Giemsa and examined under oil immersion by light microscopy and viewed at 1000x magnification. The size bar represents 10μm.
Fig 3.
Leukocytes in rag1-/- mutant zebrafish kidney included: (A) megakaryocytes, (B) neutrophils, (C) mast cells, (D) basophils, (E) eosinophils, (F) dendritic cells, (G) monocytes, (H) macrophages, (I) large granular LLCs, (J) small granular LLCs, (K) precursor cell, (L) myeloblasts and (M) megakaryoblast.
Cells were stained with Wright Giemsa and examined under oil immersion by light microscopy and viewed at 1000x magnification. The size bar represents 10μm.
Fig 4.
Leukocytes in rag1-/- mutant zebrafish spleen included: (A) monocytes, (B) macrophages, (C) large granular and small granular and agranular LCCs.
Cells were stained with Wright Giemsa and examined under oil immersion by light microscopy and viewed at 1000x magnification. The size bar represents 10μm.
Fig 5.
Graphs depicting changes in mRNA expression over time of tnfα, ifnγ, t-bet and nitr9 in liver, kidney and spleen after treatment with β glucan.
Only tissues and genes that demonstrated significant changes in expression compared to PBS injected controls are presented. Fold changes in tnfα in liver (A) and kidney (B), ifnγ in liver (C), kidney (D) and spleen (E), t-bet in kidney (F) and nitr9 in kidney (G) are presented as mean fold change relative to the time zero group ± standard deviation as measured by quantitative RT-PCR. Arp was used as a housekeeping gene. hpi = hours post injection; control = PBS (endotoxin-free); Treated = β glucan. *Significant (p<0.05) difference in expression of treated compared to control. No significant changes in expression were observed in tnfα in spleen, in t-bet in liver and spleen and in nitr9 in liver and spleen (S1 Table).
Fig 6.
Graphs depicting changes in mRNA expression over time of mx, tnfα, ifnγ, t-bet and nitr9 in liver, kidney and spleen after treatment with Poly I:C.
Only tissues and genes that demonstrated significant changes in expression compared to PBS injected controls are presented. Fold changes in mx in liver (A), kidney (B) and spleen (C), tnfα in kidney (D) and spleen (E), ifnγ in kidney (F), t-bet in kidney (G) and nitr9 in kidney (H) are presented as mean fold change relative to the time zero group ± standard deviation as measured by quantitative RT-PCR. Arp was used as a housekeeping gene. hpi = hours post injection; control = PBS (endotoxin-free); Treated = Poly I:C. *Significant (p<0.05) difference in expression of treated compared to control. No significant changes in expression were observed in tnfα in liver, in ifnγ, t-bet and in nitr9 in liver and spleen (S1 Table).
Fig 7.
Graphs depicting changes in mRNA expression over time of mx, tnfα, ifnγ, t-bet, and nitr9 in liver, kidney and spleen after treatment with R848.
Only tissues and genes that demonstrated significant changes in expression compared to PBS injected controls are presented. Fold changes in mx in liver (A), kidney (B), and spleen (C), ifnγ in liver (D), kidney (E) and spleen, (F), t-bet in liver (G), and kidney (H) and nitr9 in liver (I) and kidney (J) are presented as mean fold change relative to the time zero group ± standard deviations measured by quantitative RT-PCR. Arp was used as a housekeeping gene. hpi = hours post injection; control = PBS (endotoxin-free); Treated = R848. *Significant (p<0.05) difference in expression of treated compared to control. No significant changes in expression were observed in t-bet and nitr9 in spleen (S1 Table).
Fig 8.
Images of western blots demonstrating NITR9 expression in the liver, kidney and spleen in rag 1-/- zebrafish that were IC injected with R-848 or saline.
The lower images demonstrate GAPDH expression on the same blot. The relative density (bottom) is the pixel density of the NITR9 band divided by the GAPDH.
Table 3.
Summary of expression changes of significantly up-regulated and down-regulated genes at different hours post injection (hpi) following immune stimulation by β glucan, Poly I:C and R848 in liver, kidney and spleen of rag1-/- mutant fish.