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

Figure 2.

Figure 2A 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. Figure 2B is a set of three scatter–bar plots titled double negative (B lymphocyte-induced maturation protein 1 negative stella negative), single positive (B lymphocyte-induced maturation protein 1 positive stella negative), and double positive (B lymphocyte-induced maturation protein 1 positive stella positive) plotting Cell Number (times 10 begin superscript 5 end superscript), ranging from 0 to 1.5 in increments of 0.5, Cell Number (times 10 begin superscript 4 end superscript), ranging from 0 to 4 in unit increment, and Cell Number (times 10 begin superscript 3 end superscript), ranging from 0 to 5 in unit increments (y-axis) across A, B, C, D, E, and F (x-axis) for Water, 1 nanomolar, 10 nanomolar, 100 nanomolar, 1 micromolar, and 10 micromolar, respectively. Figure 2C is a F A C S contour plot having 1 row and three columns, namely, Water, 1 nanomolar, and 100 nanomolar. Row 1 indicates cell populations plotting stella negative E C F P (y-axis) across B lymphocyte-induced maturation protein 1 negative m Venus (x-axis). Figure 2D is a set of representative histograms titled double negative (B lymphocyte-induced maturation protein 1 negative stella negative), single positive (B lymphocyte-induced maturation protein 1 positive stella negative), and double positive (B lymphocyte-induced maturation protein 1 positive stella positive) plotting Bisphenol A, namely, 100 nanomolar, 1 nanomolar, and water (bottom to top).(y-axis) across CellTrace Yellow, respectively. Figures E, F, and G each are a set of four scatter–bar plots titled Undivided, 1 Division, 2 Divisions, and 3 Divisions plotting percentage of double negative (B lymphocyte-induced maturation protein 1 negative stella negative), ranging from 0.0 to 0.4 in increments of 0.1, 0 to 12 in increments of 4, 55 to 70 in increments of 5, and 15 to 35 in increments 5(y-axis); plotting percentage of single positive (B lymphocyte-induced maturation protein 1 positive stella negative), ranging from 0.0 to 2.5 in increments of 0.5, 20 to 32 in increments of 4, 52 to 62 in increments of 2, and 10 to 18 in increments 2 (y-axis); and plotting percentage of double positive (B lymphocyte-induced maturation protein 1 positive stella positive), ranging from 0 to 15 in increments of 5, 36 to 46 in increments of 2, 39 to 45 in unit increments, and 0 to 15 in increments 5 (y-axis) across A, B, and C (x-axis) for Water, 1 nanomolar, and 100 nanomolar, respectively.

Proliferation analysis of different cell populations in day 5 aggregates derived from EpiLCs following 24-h BPA exposure. (A) Graphic illustrating BPA exposure and cell differentiation strategy. Uncolored cells represent Blimp1-; Stella-/DN/nongerm cells; yellow cells represent Blimp1+;Stella-/SP/presumed transitioning germ cells; green cells represent Blimp1+;Stella+/DP/BVSC/d5 PGCLCs. [Illustration in part created with ©BioRender (biorender.com), per the Biorender terms and conditions.] (B) Scatter-bar plots showing absolute numbers of cells in the different gated subpopulations indicated following exposure of EpiLCs to different BPA concentrations indicated. Plots represent mean±standard deviation. n=8. For calculated one-way ANOVA with Šidák-adjusted p-values for comparison to control and cell numbers, see Table 2 and Excel Table S8, respectively. (C) FACS contour plots showing cell populations indicated from d5 aggregates. EpiLCs were treated with the conditions indicated for 24 h prior to aggregate formation. (D) Representative histograms showing CellTrace™ Yellow staining profile of the different cell populations indicated. The Y-axis represents the number of cells, whereas the X-axis represents the fluorescence intensity. Recurring cell divisions create the appearance of secondary peaks, which widens the original peak of undivided cells. (E–G) Scatter-bar plots showing proportion of cells in the different populations indicated that remain undivided or have undergone one, two, or three cell divisions in day 5 aggregates. Number of divisions calculated using FlowJo Proliferation tool (version 10; FlowJo, LLC). Scatter plots show mean±standard deviation. n=3–4. For calculated one-way ANOVA with Šidák adjusted p-values for comparison and cell proportions, see Table 3 and Excel Table S11, respectively. Note: ANOVA, analysis of variance; βFGF, 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; DAPI, 4′,6-diamidino-2-phenylindole; DN, double negative; DP, double positive; EGF, epidermal growth factor; ESC, embryonic stem cell; EpiLC, epiblast-like cell; FACS, fluorescence-activated cell sorting; LIF, leukemia inhibitory factor; KSR, knockout serum replacement; PGC, primordial germ cell; PGCLC, PGC-like cell; SP, single positive.