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[Preprint]. 2023 Feb 24:2023.02.23.529824. [Version 1] doi: 10.1101/2023.02.23.529824

Fig. 3. Wnt negative regulators Psmd3/7 are targeted and downregulated by Tex10 depletion to compromise PGCLC specification.

Fig. 3.

(A) Venn diagram showing the overlap between Tex10 and H3K4me3 ChIP-seq peaks at the D2PGCLC stage.

(B) Venn diagram showing the intersection between Tex10-bound/H3K4me3-marked genes and genes downregulated upon Tex10 depletion.

(C) Gene ontology biological processes enriched in the 322 genes shown in B.

(D) Genome browser tracks of Tex10 and H3K4me3 ChIP-seq signals near Trim27, Tyro3, Psmd3, and Psmd7 gene loci in D2EBs. Green shaded regions indicate the Tex10 peaks.

(E) ChIP-qPCR validation of Tex10 binding and H3K4me3 mark at the promoter regions of Psmd3 and Psmd7. Tex10 ChIP experiment was performed with an anti-HA tag antibody, and IgG served as a negative control.”

(F) Flow cytometry analysis of PGCLC specification efficiency using cell surface markers SSEA1 and CD61. Percentages of double-positive (SSEA1+ and CD61+) cells are indicated at day 6 of PGCLC induction for control shRNA (shCtrl) and Psmd7 shRNA (shKD). Quantification of double-positive percentages in live cells and numbers per 20,000 analyzed cells are shown with bar plots. N = 2 biological replicates per condition (two control shRNAs vs. two Psmd7 shRNAs), and an unpaired t-test was used to detect significance.

(G) Flow cytometry analysis of PGC specification efficiency using the cell surface markers SSEA1 and CD61. Percentages of double-positive (SSEA1+ and CD61+) cells are indicated at day 6 of PGCLC induction for DMSO, dTAG13, and dTAG13 plus Psmd7 ectopic expression (dTAG13+Psmd7) treatment. Quantification of double-positive percentages in live cells and numbers per 20,000 analyzed cells are shown with bar plots. Two cell clones C1 and C2 were used as biological replicates, and a paired t-test was used to detect significance.