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. Author manuscript; available in PMC: 2018 Dec 11.
Published in final edited form as: Cell Rep. 2018 Nov 6;25(6):1469–1484.e5. doi: 10.1016/j.celrep.2018.10.036

Figure 2. Generation of DGUOKΔ14/Δ5 iPSC-Derived Hepatocyte-like Cells.

Figure 2.

(A) Schematic illustration of DGUOK exon 4 showing the CRISPR/Cas9 guide (red) and nucleotide sequences of DGUOK wild-type (DGUOK+/+) and mutant alleles (DGUOKΔ14/Δ5). Black arrow showing relative position of PCR primers used to identify indels.

(B) Image of the polyacrylamide gel showing DGUOK exon 4 amplicons from control iPSCs (+/+) and iPSCs harboring compound heterozygous deletions of 14 and 5 bp within DGUOK exon 4 (Δ14/Δ5).

(C) Immunoblot to detect DGUOK in either control (+/+) or DGUOKΔ14/Δ5 hepatocytes. HSP90 was used as a loading control.

(D) Quantification of DGUOK protein by densitometry of immunoblots. DGUOK protein was normalized to total protein (n = 3 biological replicates, mean ± SEM, ****p ≤ 0.0001).

(E) Bar graph showing relative steady-state level of DGUOK mRNA in DGUOK+/+ and DGUOKΔ14/Δ5 iPSCs (n = 3 biological replicates, mean ± SEM, ****p ≤ 0.0001).

(F) Representative fluorescent images of DAPI (blue), HNF4A (green), and albumin (red) staining on day 20 wild-type and DGUOKΔ14/Δ5 hepatocyte-like cells. Scale bar, 50 μm.

(G) Bar graph showing relative expression levels of hepatic genes in DGUOK+/+ and DGUOKΔ14/Δ5 hepatocytes at day 20 of differentiation (n = 3 biological replicates, mean ± SEM).