Fig. 8

Upregulation of hspb9 and hspb15 mRNA expression in DKO larvae and their modulation by MG-H1 and BOR treatments. (A–B) Hspb9 and hspb15 mRNA expression levels were upregulated in DKO larvae compared to WT, GLO1KO and ALDH3A1KO larvae. n = 5. (C–D) The mRNA expression levels of hspb9 and hspb15 were significantly increased in larvae treated with 1 mM MG-H1 compared to the larvae without treatment, and significantly decreased in larvae co-incubated with MG-H1 and CAR. n = 4. (E–F) The mRNA expression levels of hspb9 and hspb15 were significantly increased in larvae treated with 7.5 μM BOR compared to the larvae without treatment, and significantly decreased in larvae co-incubated with BOR and BA. n = 6. mRNA expression level was measured by RT-qPCR and normalized to arnt2. (G) Validation of splice-modifying morpholino of hspb15 targeting exon2-intron2: SB-hspb15-MO. RT-PCR of Control-MO, and SB-hspb15-MO injected larvae showed wild type and generation of morphant hspb15 signals at 120hpf. 2 ng of morpholinos: Control-MO and SB-hspb15-MO were injected into the one-cell stage of zebrafish embryos, respectively. (H) Representative confocal images of the hyaloid vasculature showed alleviated alteration in DKO larvae injected with hspb15 morpholino at 120hpf compared to the control-MO. White scale bar = 20 μm. (I) Quantification of deceased branch points formation in the hyaloid vasculature in DKO larvae injected with hspb15 morpholino. n = 31–35. The bars indicate mean ± SD values. Statistical analysis was applied by Student's t-test and one-way ANOVA, ns = not significant, *p < 0.05, **p < 0.01, ****p < 0.0001. MG-H1, methylglyoxal-derived hydroimidazolone 1; CAR, L-Carnosine; BOR, bortezomib; BA, betulinic acid; arnt2, aryl hydrocarbon receptor nuclear translocator 2; hspb9, heat shock protein, alpha-crystallin-related, 9; hspb15, heat shock protein, alpha-crystallin-related, b15; WT, wild type; MO, morpholino; Morph, morphant; RT-PCR, reverse transcription-polymerase chain reaction.