Depletion of CNBP leads to an increased susceptibility to bacterial infection in vivo. (A) The mRNA levels of il-6, but not tnf-α, decreased in the cnbp morphants. Control and cnbp morpholino oligos (MOs) were injected into zebrafish embryos along with LPS (1 mg ml⁻¹) at 24 hpf. Three hours after the infection, il-6 or tnf-α mRNA was measured by RT-qPCR analysis. *P < 0.01 and **P < 0.001 (Student's t-test). (B) Macrophages in cnbp-depleted zebrafish larvae fail to migrate to the inflammatory foci during LPS infection. To compare the infiltration of macrophages between control and cnbp morphants after LPS infection, larvae were collected at 3 h post-infection (hpi) and stained with 2.5 μg ml⁻¹ Neutral Red (NR) solution. Migration of NR-stained macrophages was observed under a dissecting microscope. Images were captured and analyzed using a mono-camera and the NIS-Elements software, respectively. Scale bars, 200 μm, *P < 0.001 (Student's t-test). (C) Infiltration of neutrophils was defective in cnbp morphants during Shigella infection. The Shigella M90T was injected into the otic vesicle (circle) of the control and cnbp morphants at 3 dpf. The zebrafish larvae were fixed with 4% formaldehyde and permeabilized with ethanol for staining with 0.03% SB at 4 hpi. The migration of SB-stained neutrophils observed by a dissecting microscope was photographed and analyzed using NIS-Elements software. Scale bars, 500 μm. *P < 0.001 (Student's t-test). (D) Survival rates of zebrafish in control or cnbp morphants after Shigella M90T or BS176 infection. The larvae were injected intravenously with 1 × 10³ CFU of Shigella at 72 hpf and incubated at 28°C for 24 h. The survival rates of zebrafish larvae were measured during 24 h after the bacteria infection. *P < 0.01 (Mantel-Cox log-rank test). (E) Bacterial burden in control and cnbp morphants after Shigella infection with 1× 10³ CFU at 72 hpf and incubation for 24 h. *P < 0.001 (Student's t-test). (F and G) The delivery of wild-type cnbp mRNA to cnbp morphants rescued il-6 mRNA production and survival during infection. *P < 0.01 and **P < 0.001 (Student's t-test) in F or *P < 0.001 (Mantel-Cox log-rank test) in G. Data are representative of three independent experiments and are presented as mean ± s.d. in A and F.

Generation of cnbp morphants. (A) Schematic of antisense morpholino oligonucleotides for cnbp in Zebrafish. (B) RT-PCR analysis of cnbp in control or cnbp morpholino-treated zebrafish embryos at 24 hpf to assess depletion efficiency. (C) Images highlighting the morphological defects of cnbp morphants during development. Scale bars, 500 μm. (D) Infiltration of neutrophils in control and cnbp morphants after LPS treatment. In cnbp-depleted zebrafish, neutrophils fail to migrate to the inflammatory foci in the yolk of LPS-injected larvae. LPS- or PBS-injected zebrafish larvae were collected at 5 h post-injection. For Sudan Black (SB) staining, the fixed larvae were permeabilized with ethanol, after which the yolk regions were stained with 0.03% SB. Migration of SB-stained neutrophils was observed under a dissecting microscope. Images were captured and analyzed using a mono-camera and NIS-Elements software, respectively. Scale bars, 500 µm, *P<0.01 (Student’s t-test). Data are representative of three independent experiments.