Figure 4.
Deregulation of Jagn1b results in UPR activation, but UPR induction alone is not sufficient to induce neutropenia in zebrafish. (A-E) Relative expression levels (fold change) of UPR-associated genes measured with quantitative RT-PCR after interference with jagn1b by overexpressing jagn1bΔT12-G14 mRNA or inhibiting jagn1b with morpholino. Relative expression levels of (A) ddit3, coding the UPR marker Chop, (B) atf4, (C) ero1a and ero1b, (D) ppp1r15a, and (E) xbp1 were measured, corresponding groups are indicated. Expression levels were normalized to the housekeeping gene b-actin and fold change was calculated with respect to the expression levels of wild-type embryos. Each dot represents the value of the relative expression of 1 sample. Data are presented as mean ± standard deviation. The data represent combined results from 2 to 3 independent experiments. N represents the total number of analyzed complimentary DNA samples. (F-G) Relative expression (fold change) quantified by quantitative RT-PCR, normalized to the housekeeping gene b-actin and fold changes were calculated respect the expression levels of wild-type embryos of (F) UPR-marker ddit3 (G) and spliced xbp1 after UPR induction with tunicamycin (1 μg/mL), DTT (0.7 mM) or DMSO control (0.7%) alone or in combination with jagn1b morpholino. Data are presented as mean ± standard deviation. Each dot represents the value of relative expression of one sample. The data represent combined results from 2 to 3 independent experiments. N represents the total number of analyzed complimentary DNA samples. (H-I) Effect of chemically induced UPR on neutrophil counts. (H) Quantification of lyz+ neutrophils stained with WISH after UPR induction with tunicamycin (1 μg/mL), DTT (0.7 mM) or DMSO control (0.7%) alone or in combination with jagn1b morpholino. Each dot represents the number of cells in the hematopoietic tissue of a 2 dpf individual embryo after 24 hours of treatment. Data are presented as mean ± standard deviation. The data represent the combined results from 2 to 3 independent experiments. N represents the total number of analyzed embryos. (I) Representative images were taken with 10× magnification. Scale bars represents 100 μm. ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Deregulation of Jagn1b results in UPR activation, but UPR induction alone is not sufficient to induce neutropenia in zebrafish. (A-E) Relative expression levels (fold change) of UPR-associated genes measured with quantitative RT-PCR after interference with jagn1b by overexpressing jagn1bΔT12-G14 mRNA or inhibiting jagn1b with morpholino. Relative expression levels of (A) ddit3, coding the UPR marker Chop, (B) atf4, (C) ero1a and ero1b, (D) ppp1r15a, and (E) xbp1 were measured, corresponding groups are indicated. Expression levels were normalized to the housekeeping gene b-actin and fold change was calculated with respect to the expression levels of wild-type embryos. Each dot represents the value of the relative expression of 1 sample. Data are presented as mean ± standard deviation. The data represent combined results from 2 to 3 independent experiments. N represents the total number of analyzed complimentary DNA samples. (F-G) Relative expression (fold change) quantified by quantitative RT-PCR, normalized to the housekeeping gene b-actin and fold changes were calculated respect the expression levels of wild-type embryos of (F) UPR-marker ddit3 (G) and spliced xbp1 after UPR induction with tunicamycin (1 μg/mL), DTT (0.7 mM) or DMSO control (0.7%) alone or in combination with jagn1b morpholino. Data are presented as mean ± standard deviation. Each dot represents the value of relative expression of one sample. The data represent combined results from 2 to 3 independent experiments. N represents the total number of analyzed complimentary DNA samples. (H-I) Effect of chemically induced UPR on neutrophil counts. (H) Quantification of lyz+ neutrophils stained with WISH after UPR induction with tunicamycin (1 μg/mL), DTT (0.7 mM) or DMSO control (0.7%) alone or in combination with jagn1b morpholino. Each dot represents the number of cells in the hematopoietic tissue of a 2 dpf individual embryo after 24 hours of treatment. Data are presented as mean ± standard deviation. The data represent the combined results from 2 to 3 independent experiments. N represents the total number of analyzed embryos. (I) Representative images were taken with 10× magnification. Scale bars represents 100 μm. ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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