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. 2018 Sep 25;7:e38839. doi: 10.7554/eLife.38839

Figure 3. SURF4 mutagenesis causes an accumulation of intracellular PCSK9-eGFP.

(A) Individual sgRNA sequencing counts for SURF4-targeting sgRNA in eGFP high and eGFP low populations for each of 4 biologic replicates. Adjusted p values were calculated using DESeq2. (B) Flow cytometry histograms of PCSK9-eGFP and A1AT-mCherry fluorescence in reporter cells transfected with plasmids delivering Cas9 and SURF4-targeting sgRNA or empty vector. (C) Quantification of intracellular fluorescence for cells treated with empty vector (n = 3) or unique SURF4-targeting sgRNAs (n = 6). (D) Quantification of intracellular fluorescence for clonal cell lines each containing frameshift-causing indels at two different SURF4 target sites (n = 7 wild-type clones, n = 3 clones generated from each SURF4-targeting sgRNA). (E) Flow cytometry histograms for cells expressing PCSK9-eGFP-2A-A1AT-mCherry and deleted for SURF4 with or without stable expression of a wild-type or FLAG-tagged SURF4 cDNA. (F) Time course of intracellular accumulation of tetracycline-inducible PCSK9-eGFP on WT, SURF4-deficient, or SURF4 rescue background (n = 3 biologic replicates for each cell line at each time point). *p<0.05 by Student’s t-test. Error bars represent standard deviations.

Figure 3.

Figure 3—figure supplement 1. ER stress markers.

Figure 3—figure supplement 1.

Clonal cell lines with a stably integrated tetracycline-inducible PCSK9 cDNA on either WT or SURF4 mutant background were treated with tetracycline or vehicle control and analyzed by immunoblotting for various markers of ER stress (A), quantified by densitometry (B). Error bars represent standard deviations.
Figure 3—figure supplement 2. SURF4-deficient genotypes.

Figure 3—figure supplement 2.

Clonal cell lines were genotyped by PCR amplification of sgRNA target site and Sanger sequencing of the amplicon with either TIDE decomposition of individual alleles from the amplicon, or ligation of the amplicon into cloning plasmids and Sanger sequencing of multiple individual clones. Sequence corresponding to sgRNA is underlined and expected double-strand break site is indicated (^).