Figure 7

Man1b1 ablation enhances ZAAT secretion without affecting retention in hepatocytes. (A) Two gRNA targeting exon 2 of man1b1a or exon 3 of man1b1b were co-injected into outcross embryos from the Tg(lfabp: eGFP-T2A-ZAAT) line at the one-cell stage. (B) RT-PCR of man1b1a (top panel) and man1b1b (bottom panel) using cDNA made from 1 µg RNA extracted from pooled whole embryos at 7 dpf. The housekeeping gene rpl13a was used as a loading control. (C) The ratio of antitrypsin to IgM (left panel) and eGFP to total protein (right panel) in zebrafish blood was detected by immunoblot (Supplementary Figure S17) and quantitated by densitometry. Shown are the pooled data from three independent experiments, with each point representing an individual fish. Statistical significance was determined by Unpaired Student’s t-test. * p < 0.05. (D) Periodic acid-Schiff (PAS) staining with and without diastase was performed on 3 µm thick paraffin zebrafish liver sections. The scale bar represents 50 µm. (E) qPCR analysis of representative cholesterol biosynthesis DEGs in adult non-transgenic zebrafish livers (top panel), as well as livers expressing ZAAT (bottom panel), using data pooled from two independent experiments. Expression values were normalised to the housekeeping genes rpl13a and lsm12b. One-way ANOVA was performed and indicated no significant difference between levels from the four genotypes for each gene.