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. 2019 Oct 30;8:e49910. doi: 10.7554/eLife.49910

Figure 3. SCGN p.R77H is a hypomorphic allele.

(a) Basal and DHA fatty acid induced GLP-1 release from parental and Scgn deleted (KO) clones. GLP-1 values are normalized to total protein content. (b) Basal and DHA stimulated GLP-1 secretion from rescue clones expressing human SCGNWT or SCGNR77H. (c) Immunofluorescence images showing subcellular localization of SNAP25 and SCGN in parental STC-1 cells and the indicated SCGN KO and rescue cell lines. Scale bar 15 µm. (d) For the experiment depicted in (c), SNAP25 staining intensity ratio between the membranous compartment and the total cellular signal was plotted in the indicated cell lines. Dots indicate individual cells, horizontal bars correspond to the mean within each group. (e) Midbrain size of zebrafish after scgn targeting with morpholinos or rescue with human SCGNWT or SCGNR77H. Dots indicate individual embryos, horizontal bars correspond to the mean within each group. The zebrafish experiments were performed in triplicate. *p<0.05, **p<0.01, ****p<0.0001 unpaired student t test in (a), (b) and (d). ****p<0.0001 multiple comparison ANOVA in (e). Error bars in (a) and (b) represent the S.E.M.

Figure 3—source data 1. Source data for Figure 3A.
elife-49910-fig3-data1.xlsx (8.6KB, xlsx)
DOI: 10.7554/eLife.49910.016
Figure 3—source data 2. Source data for Figure 3B.
elife-49910-fig3-data2.xlsx (11.2KB, xlsx)
DOI: 10.7554/eLife.49910.017
Figure 3—source data 3. Source data for Figure 3D.
elife-49910-fig3-data3.xlsx (10.7KB, xlsx)
DOI: 10.7554/eLife.49910.018
Figure 3—source data 4. Source data for Figure 3E.
elife-49910-fig3-data4.xlsx (9KB, xlsx)
DOI: 10.7554/eLife.49910.019

Figure 3.

Figure 3—figure supplement 1. Scgn deficient clones of STC-1 cells were generated by CRISPR/Cas9 technology.

Figure 3—figure supplement 1.

Protein knockout (KO) was confirmed by immunoblotting. Expression of SCGNWT or SCGNR77H in rescue clones is also shown.
Figure 3—figure supplement 2. SNAP25 co-precipitation from SCGN rescue cell lines.

Figure 3—figure supplement 2.

Re-expressed SCGN was HA tagged.
Figure 3—figure supplement 3. Bright-field images of zebrafish after morpholino injections (MO).

Figure 3—figure supplement 3.

In-situ hybridization of HuC (elavl3) was performed in embryos at 48 hr post fertilization to highlight the developing brain. Control: control MO injection; MO: scgn MO injection; MO+WT: SCGN MO and human SCGN WT mRNA co-injection; MO+R77H: SCGN MO and human SCGN R77H mRNA co-injection. All injections are performed at one-cell stage of the development.