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. Author manuscript; available in PMC: 2021 Jun 14.
Published in final edited form as: Cell Rep. 2021 May 18;35(7):109145. doi: 10.1016/j.celrep.2021.109145

Figure 2. Generation of GATA6−/− pluripotent stem cell lines.

Figure 2.

(A) CRISPR-Cas9-derived indels in K3 pluripotent stem cells using guide RNAs (gRNAs) targeting (I) exon 2 and (II) exon 4 of the GATA6 gene. Red denotes gRNA sequence; green denotes PAM sequence.

(B) Western blot analysis of GATA6 and β-actin in GATA6+/+, GATA6Ex2Δ31/Δ38 and GATA6Ex4Δ1/Δ2 cell lines at day 2 of differentiation.

(C) Graph showing qRT-PCR analysis of definitive endoderm-enriched mRNAs in definitive endoderm normalized to the housekeeping mRNA RPL13a. Mean values, n = 3, mean ± SD. Student’s t test: *p < 0.05.

(D) Doxycycline-inducible GATA6-3xFLAG cDNA plasmid.

(E) Western blot analysis of GATA6 and β-actin protein expression in GATA6+/+ and GATA6Ex4Δ1/Δ2;ind GATA6 pluripotent cells with and without doxycycline at day 2 of differentiation. Long, short, and exogenous short 3× FLAG epitope-tagged GATA6 isoforms are indicated.

(F) Immunofluorescence analysis of GATA6, FLAG, and DAPI in GATA6+/+ and GATA6Ex4Δ1/Δ2;ind GATA6 cells with and without doxycycline at day 2 of differentiation. Scale bars, 100 μm. GATA6-positive population: flow cytometry analysis, n = 3, mean ± SD.

(G) Differentiation protocol used to analyze GATA6 function.

(H) Heatmap with hierarchical clustering of GATA6-dependent genes during definitive endoderm formation in GATA6+/+ and GATA6Ex4Δ1/Δ2;ind GATA6 pluripotent cells with and without doxycycline. GATA6-dependent genes: false discovery rate (FDR) < 0.05: GATA6+/+ versus GATA6Ex4Δ1/Δ2;ind GATA6 (—dox), and GATA6Ex4Δ1/Δ2;ind GATA6 (—dox) versus GATA6Ex4Δ1/Δ2;ind GATA6 (+dox) at days 2 and 4 using gene-specific analysis (GSA; RNA-seq, n = 2).