Fig 2: Loss of CIC alters gene expression following differentiation. (A) PCA plot comparing control (red) and CIC knockout BeWo cells (blue) treated with DMSO (circle) or 40 µM forskolin (FSK) (triangle). (B) Volcano plot comparing control and CIC knockout BeWo cells treated with DMSO. (C) Volcano plot comparing control and CIC knockout BeWo cells treated with 40 µM forskolin (FSK). (D) Pathway analysis comparing control and CIC knockout BeWo cells treated with 40 µM forskolin (FSK). (E) Heat map of relative gene expression changes of DMSO and 40 µM forskolin treated control and CIC knockout BeWo cells. (F) CGA, CGB2, HSD11B2, ERVW-1, ERVFRD-1, and TEAD4 gene expression by qPCR in control and CIC knockout BeWo cells treated with DMSO or 40 µM Forskolin (FSK). n = 6; Data are representative of mean +/- SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

Fig 3: Loss of CIC impairs biochemical differentiation. (A) Representative western blot image of CIC and total protein expression in empty-vector, non-targeting, and CIC knockout BeWo cells treated cells. Please see Supplemental Fig. 4 for images of uncropped blots. (B) ELISA of HCG production in empty-vector, non-targeting, and CIC knockout BeWo cells treated with DMSO or 40 µM Forskolin (FSK). n = 3 biological replicates. (C) qPCR analysis of CGA, CGB2, ERVW-1, and ERVFRD-1 gene expression in empty vector, non-targeting, and CIC knockout BeWo cells treated with DMSO or 40 µM Forskolin (FSK). n = 3 biological replicates. Data are representative of mean +/- SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

Fig 4: Trophoblast differentiation decreases expression of the mitochondrial citrate carrier. (A) Schematic of glucose metabolism to acetyl-CoA. Pyruvate is transported via the mitochondrial pyruvate carrier into the inner mitochondrial membrane where it is converted to acetyl-CoA, fueling the tricarboxylic acid cycle (TCA). Citrate is transported by the citrate carrier (CIC) to the cytoplasm where ATP citrate lyase (ACLY) converts it to acetyl-CoA, which can be used for histone acetylation. Acetate may contribute to Acetyl-CoA pools through the activity of Acetyl-CoA Synthetase 2 (ACSS2). Figure generated with Biorender. (B) qPCR analysis showing relative expression of SLC25A1, ACLY and ACSS2 mRNA following 48 h treatment of BeWo cells with DMSO or 40 µM Forskolin (FSK). n = 4 biological replicates. (C) Representative western blot images of HCG, CIC, ACLY, ACSS2 proteins and total protein expression in BeWo cells treated with DMSO or 40 µM forskolin. Please see Supplemental Fig. 2 for images of uncropped blots. (D) Quantification of CIC, ACLY, and ACSS2 protein expression in BeWo cells treated with DMSO or 40 µM Forskolin (FSK). n = 5 biologic replicates. Data are representative of mean +/- SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.