FIGURE SUMMARY
Title

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Regulates the Production of Acute-Phase Reactants from the Liver

Authors
Jacome Sanz, D., Saralahti, A.K., Pekkarinen, M., Kesseli, J., Nykter, M., Rämet, M., Ojanen, M.J.T., Pesu, M.
Source
Full text @ Liver Int.

Homozygous pcsk9tpu-13/tpu-13 and pcsk9tpu-2,+15/tpu-2,+15 mutant zebrafish show reduced pcsk9 expression but normal morphology. A, A schematic representation of zebrafish pcsk9 (ENSDARG00000074185), and the gRNA target site for CRISPR/Cas9 mutagenesis in the third exon. B, The in vivo mutagenesis efficiency was estimated in the gRNA and Cas9 protein injected F0-generation embryos and in the uninjected controls using a T7 endonuclease I (T7EI) assay and 2.5% agarose TAE gel electrophoresis. The uninjected controls: a 169-bp WT PCR product (lanes 8 and 9); the gRNA and Cas9-injected mutant embryos: three bands of 169 bp (WT), ~100 bp and ~70 bp (lanes 1-7). The mutagenesis efficiency was calculated as described previously.27 C, A schematic representation of the indel mutations (−13 bp deletion, pcsk9tpu-13 and −2 bp deletion, +15 bp insertion, pcsk9tpu-2,+15), leading to truncated protein products of 173 and 159 amino acids (aa) respectively. Red boxes indicate altered amino acids caused by the frameshift. D, The expression of pcsk9 was determined in the brain of pcsk9tpu-13/tpu-13 (n = 5; 2 females, 3 males) and pcsk9tpu-2,+15/tpu-2,+15 zebrafish (n = 5; 2 females, 3 males) and in the WT siblings (n = 5 in both control groups; 2 females, 3 males) with qPCR. Gene expression levels were normalized to eef1a1l1 and a 2-tailed Mann-Whitney test was used for statistics. E, Anesthetized WT and homozygous pcsk9 mutant zebrafish were imaged using a Canon EOS 7D Mark II camera with an exposure time of 17 ms. Fish were kept submerged in water during image acquisition. Images in (B) and (E) were cropped to exclude empty background from the figure

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Adult
PHENOTYPE:
Fish:
Observed In:
Stage: Adult

Nonsense mutations in pcsk9 do not affect zebrafish survival upon an S pneumoniae infection. S pneumoniae (4 700 000 CFU; SD 990 000 CFU) was injected into the abdominal cavity of (A) pcsk9tpu-13/tpu-13 (n = 21; 8 females, 13 males; survived: n = 16; 8 females, 8 males), pcsk9tpu-13/+ (n = 24; 7 females, 17 males; survived: n = 17; 6 females, 11 males) and WT (pcsk9tpu-13) (n = 26; 5 females, 21 males; survived: n = 15; 4 females, 11 males) as well as (B) pcsk9tpu-2,+15/tpu-2,+15 (n = 20; 4 females, 16 males; survived: n = 16; 3 females, 13 males), pcsk9tpu-2,+15/+ (n = 54; 10 females, 44 males; survived: n = 49; 9 females, 40 males) and WT (pcsk9tpu-2,+15) (n = 23; 12 females, 11 males; survived: n = 19; 10 females, 9 males) adult zebrafish, and their survival was followed for 7 days. Pneumococcus infected (240 CFU; SD 76 CFU) zebrafish larvae from both the (C) pcsk9tpu-13 and (D) pcsk9tpu-2,+15 background were followed until 5 dpi and their survival was recorded. Group sizes; pcsk9tpu-13/tpu-13 (n = 24; survived: n = 5), pcsk9tpu-13/+ (n = 48; survived n = 19), WT (pcsk9tpu-13) (n = 24; survived n = 4) and pcsk9tpu-2,+15/tpu-2,+15 (n = 17; survived n = 10), pcsk9tpu-2,+15/+ (n = 24; survived n = 21), WT (pcsk9tpu-2,+15) (n = 15; survived n = 13). The data were collected from single experiments. A log-rank (Mantel-Cox) test was used for statistics. dpi, days post infection

pcsk9 is up-regulated on a pneumococcal infection and associated with the expression of acute-phase reactants. A, The relative expression of pcsk9 was determined in the liver of PBS injected (n = 5; all males) and S pneumoniae infected (635 000 CFU; SD 276 000 CFU, n = 5; all males) WT AB zebrafish at 7 dpi using qPCR. Gene expression levels were normalized to eef1a1l1 expression and target genes were run once as technical duplicates. A 2-tailed Mann-Whitney test was used for statistics. B-C, RNA was isolated from the liver of unchallenged (n = 2 in both groups; 2 females/group), PBS injected (n = 3 in both groups; 3 females/group) and S pneumoniae infected (3 370 000 CFU; SD 840 000 CFU, n = 3 in both groups; 3 females/group) adult pcsk9tpu-13/tpu-13 and WT (pcsk9tpu-13) zebrafish at 1 dpi and the transcriptome was analysed using RNA sequencing. B, Venn diagram for the differentially expressed genes within treatment groups. C, The top 25 up- and down-regulated genes in S pneumoniae infected pcsk9tpu-13/tpu-13 zebrafish in comparison with the WT controls are depicted using a heat-map. Statistics were done using DESeq2 and adjusted using the Benjamini-Hochberg (BH) method. D, The relative expression of selected genes up- or down-regulated in the RNA sequencing data (ldlrap1a, hamp and socs3a) was quantified using qPCR. Gene expression levels were normalized to eef1a1l1 expression and target genes were run once as technical duplicates

Silencing PCSK9 influences the expression of genes of the innate immune response in HepG2 cells. The relative expression levels of PCSK9, HAMP, C7, SOCS3, TNF, TNFAIP3, C6 and LDLRAP1 were determined in control (n = 3) and PCSK9 siRNA (n = 3) transfected HepG2 cells using qPCR. Gene expression levels were normalized to GAPDH expression and target genes were run once as technical duplicates. A 2-tailed t test was used for statistics

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Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Liver Int.