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

Figure 1.

Figure 1A is scale labeled day, ranging from 0 to 5 in unit increments, illustrating Bisphenol A exposure. There is a cartoon illustration titled embryonic stem cell placed between 1 and 2 days, and another cartoon illustration titled epiblast-like cell placed approximately between 3.5 to 4 days. Days 0 to 3 are titled uppercase n 2 b27 positive C H I R 99021 P D O 325901 leukemia inhibitory factor, and Days 3 to 5 are titled uppercase n 2 b 27 positive Activin A lowercase beta fibroblast growth factor KnockOut Serum Replacement. Figure 1B is a scatter–bar plot plotting Cell Number (times 10 begin superscript 5 end superscript) ranging from 0 to 15 in increments of 5 (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. Figure 1C is a representative F A C S contour plots having three rows and six columns. The six columns are as follows: Water, 1 nanomolar, 10 nanomolar, 100 nanomolar, 1 micromolar, and 10 micromolar. The first row indicates cell staining profile plotting 5’-bromo-2’-deoxyuridine (y-axis) across 4′,6-diamidino-2-phenylindole (x-axis). The second row indicates distribution of proliferating cells plotting lowercase gamma uppercase h 2 a x (y-axis) across 5’-bromo-2’-deoxyuridine (x-axis). The third row indicates distribution of cells plotting lowercase gamma uppercase h 2 a x (y-axis) across cleaved poly (A D P-ribose) polymerase (x-axis). Figures D to I are scatter–bar plots titled S-phase or M, Apoptotic, lowercase gamma uppercase h 2 a x, Proliferating and D N A damage (5’-bromo-2’-deoxyuridine positive lowercase gamma uppercase h 2 a x positive), Viable and D N A (cleaved poly (A D P-ribose) polymerase negative lowercase gamma uppercase h 2 a x positive), and early apoptotic and D N A damage (cleaved poly (A D P-ribose) polymerase positive lowercase gamma uppercase h 2 a x positive) plotting percentage of live events ranging from 40 to 70 in increments of 5, 0 to 20 in increments of 5, 50 to 90 in increments of 10, 30 to 70 in increments of 10, 40 to 80 in increments of 10, and 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.

Proliferation analysis of EpiLCs to BPA for 24 h. (A) Graphic illustrating BPA exposure and cell differentiation strategy. [Illustration in part created with ©BioRender (biorender.com), per the Biorender terms and conditions.] (B) Scatter-bar plot indicating cell counts of EpiLCs exposed to the different BPA concentrations indicated: error bars represent mean±standard deviation. n=12 for each condition. One-way ANOVA p-value<0.01. One-way ANOVA with Šidák-adjusted p-values for comparison to control: >0.05 (vs. 1 nM); <0.05 (vs. 10 nM); <0.05 (vs. 100 nM); <0.05 (vs. 1μM); and <0.01 (vs. 10μM). For numerical values of data, see Excel Table S2. (C) Representative FACS contour plots showing distribution of live-gated events. First row indicates cell staining profile following incubation with DAPI and proliferation, measured using anti-BrdU antibody following a 30-min pulse with the thymidine analogue BrdU. Second row indicates distribution of proliferating cells labeled with anti-γH2AX antibody, a marker of DNA damage. Third panel indicates distribution of cells labeled with γH2AX antibody and anti-cPARP antibody, a marker of apoptosis. Numbers indicate proportion of live-gated events within the regions indicated. (D–I) Scatter-bar plots indicating proportion of live-gated events in the different populations indicated. Data indicate mean±standard deviation. n=12–15. For complete set of one-way ANOVA and calculated one-way ANOVA with Šidák-adjusted p-values for each treatment condition compared with control, see Table 1. For numerical values of data, see Excel Table S7. Note: ANOVA, analysis of variance; βFGF, basic fibroblast growth factor; BPA, bisphenol A; DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; EpiLC, epiblast-like cell; FACS, fluorescence-activated cell sorting; LIF, leukemia inhibitory factor; KSR, knockout serum replacement. Number of asterisks on plots indicate level of statistical significance: *(p<0.05); **(p<0.01); ***(p<0.001).