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. 2021 Sep 29;129(9):097013. doi: 10.1289/EHP8196

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EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted.

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Figure 3A is scale labeled day, ranging from 0 to 10 in unit increments, illustrating Bisphenol A exposure. There is a cartoon illustration titled embryonic stem cell placed between 1 and 2 days, another cartoon illustration titled epiblast-like cell placed approximately between 3.5 to 4 days, and third cartoon illustration titled P G C-like cell placed between 7 and 8 days. Days 0 to 3 are titled uppercase n 2 b27 positive C H I R 99021 P D O 325901 leukemia inhibitory factor, Days 3 to 5 are titled uppercase n 2 b 27 positive Activin A lowercase beta fibroblast growth factor KnockOut Serum Replacement, and Days 5 to 10 are titled G K 15 positive B M P 4 or 8b stem cell factor leukemia inhibitory factor epidermal growth factor. Day 5 represents R N A-Seq (epiblast-like cell), and Day 10 represents R N A-Seq (P G C-like cell). Figure 3B is two heatmaps titled Untreated and 100 nanomolar Bisphenol A plotting, from top to bottom, Nanog, D n m t1, Sox2, Pou5f1, U h r f1, T fap 2c, D p p a 3, D n m t 3A, P r d m 14, D a z l, M y c, D N d1, D d x 4, Td r d 5, and Nanos 3 for counts per million (log to the base 10) ranging from 1.0 to 2.5 in increments of 0.5. Figure 3C is a graph plotting principal component 2, ranging from negative 200 to 200 in increments of 100 (y-axis) across principal component 1, ranging from negative 200 to 200 in increments of 100 (x-axis) for untreated and 100 nanomolar Bisphenol A. Figure 3D is a volcano plot titled 100 nanomolar Bisphenol A versus untreated plotting negative log to the base 10 of uppercase p adjusted, ranging from 0 to 5 in unit increments (y-axis) across log to the base 2 of fold change, ranging from negative 5 to 5 (x-axis). Figure 3E is a set of four Genome browser views, each comprising two views, namely, untreated and 100 nanomolar Bisphenol A, ranging from 0 to 18, 0 to 0.5, 0 to 36, and 0 to 3 (y-axis) for expression data for representative genes, up- (T t r and A f p) and downregulated (Lars2 and 1700013H16Rik) in Day 5 double positive/B lymphocyte-induced maturation protein 1 Blimp1-mVenus and stella-E C F P transgenic reporter cells, respectively. — Transcriptome analysis of PGCLCs derived from EpiLCs exposed to BPA. (A) Graphic showing dosing and differentiation strategy followed by cell collection for RNA-seq library generation. Uncolored cells represent Blimp1-; Stella- /DN/nongerm cells; yellow cells represent $Blimp 1 +; Stella- / SP / presumed$ transitioning germ cells; green cells represent $Blimp 1 +; Stella + / DP / BVSC / d 5$ PGCLCs. [Illustration in part created with ©BioRender – (biorender.com), per the Biorender terms and conditions.] (B) ${Log}_{10} -expression$ level-ranked heatmap of germ cell markers indicated in RNA-seq data generated from d5 aggregate FACS-purified DP/PGCLCs, derived from EpiLCs exposed to control (untreated) or $100 nM$ BPA. Candidate germ cell marker list taken from Hayashi et al. (Hayashi et al. 2011). (C) PCA of transcriptome of d5 DP/BVSC cells, derived from untreated or $100 nM$ BPA-exposed EpiLCs. (D) Volcano plot of RNA-seq data displaying gene expression pattern of d5 DP/PGCLCs, derived from untreated or 100 nM BPA-exposed EpiLCs. Significant differentially expressed genes ( $\log 2 -fold change \geq 2$ , Benjamini-Hochberg–adjusted $p \leq 0.05$ ) are highlighted in red. (E) Genome browser view of expression data for representative genes, up- (Ttr and Afp) and down-regulated (Lars2 and 1700013H16Rik) in d5 DP/BVSC cells, derived from $100 nM$ BPA-exposed EpiLCs. Y-axis values are normalized cpm/ $10^{3}$ . For each locus, top panel represents “Untreated” and bottom panel represents “ $100 nM$ BPA exposed.” For full list of differentially up- and down-regulated genes, see Excel Tables S14 and S15, respectively. Note: $β FGF$ , basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; BMP8b, bone morphogenetic protein 8b; BPA, bisphenol A; BVSC, B lymphocyte-induced maturation protein 1 Blimp1-mVenus and stella-ECFP reporter transgenes; cpm, counts per million; DN, double negative; DP, double positive; EGF, epidermal growth factor; embryonic stem cell; EpiLC, epiblast-like cell; FACS, fluorescence-activated cell sorting; KSR, knockout serum replacement; LIF, leukemia inhibitory factor; PGC, primordial germ cell; PGCLC, PGC-like cell; SCF, stem cell factor.