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. 2023 Jan 31;131(1):017009. doi: 10.1289/EHP10477

Figure 2.

Figure 2A is a Venn diagram titled Benzo(a)pyrene diol epoxide treated versus untreated displays three circles. The circle on the top is labeled 250, the circle on the left, is labeled 1043, the circle on the right, is labeled 927. The intersection area is labeled 9. The intersection area represents Collagen alpha-1(12), Type 2 iodothyronine deiodinase, Myosin Heavy Chain 10, Phospholipase D1, Pleckstrin and Sec7 Domain Containing 3, Protein Tyrosine Phosphatase Non-Receptor Type 13, single-stranded D N A binding protein 2, and transforming growth factor beta-2. Figure 2B is a String analysis depicting Phospholipase D1, Ras-related C3 botulinum toxin substrate 1 and Cell division control protein 42 homolog. Figure 2C is a set of two western blots, displaying Vector (100), HZ 09 (31), Negative control (100), si1-HZ 09 (163), si2-HZ 09 (187) for Swan 71 and Vector (100), HZ 09 (74), Negative control (100), si1-HZ 09 (144), si2-HZ 09 (179) for H T R-8 or SVneo (columns) and Phospholipase D1, Lowercase beta-tubulin, specificity protein 1, and lowercase beta-tubulin (rows). Figure 2D is a western bolt, displaying Vector (100), HZ 09 (169), Negative control (100), si1-HZ 09 (53), si2-HZ 09 (51) columns and specificity protein 1, Phospholipase D1, and Glyceraldehyde 3-phosphate dehydrogenase, each under Swan 71 and H T R-8 or SVneo (rows). Figures 2E, 2F, 2O, 2P, 2Q, 2R are bar graphs titled chromatin immunoprecipitation in Swan 71, chromatin immunoprecipitation in H T R-8 or SVneo, receptor-interacting protein in Swan 71, receptor-interacting protein in swan 71, receptor-interacting protein in H T R-8 or SVneo, receptor-interacting protein in H T R-8 or SVneo, plotting Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 6 in increments of 2; Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 6 in increments of 2; ribonucleic acid relative expression, ranging from 0 to 6 in increments of 2; ribonucleic acid relative expression, ranging from 0 to 4 in unit increment; ribonucleic acid relative expression, ranging from 0 to 6 in increments of 2; and ribonucleic acid relative expression, ranging from 0 to 4 in unit increments (y-axis) across anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; and anti-Immunoglobulin G and anti-Human antigen R (x-axis) for HZ 09 and Phospholipase D1, respectively. Figures 2H, 2I, 2S, 2T, 2U, 2V are clustered bar graph titled chromatin immunoprecipitation in Swan 71, chromatin immunoprecipitation in Swan 71, receptor-interacting protein in swan 71, receptor-interacting protein in swan 71, receptor-interacting protein in H T R-8 or SVneo, and receptor-interacting protein in H T R-8 or SVneo, plotting Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 4 in unit increments; Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 8 in increments of 2; messenger ribonucleic acid relative expression, ranging from 0 to 4 in increments of 2; messenger ribonucleic acid relative expression, ranging from 0 to 4 in increments of 2; messenger ribonucleic acid relative expression, ranging from 0 to 3 in unit increments; and messenger ribonucleic acid relative expression, ranging from 0 to 4 in increments of 2 (y-axis) across anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; and anti-Immunoglobulin G and anti-Human antigen R (x-axis) for Vector and HZ 09; Negative control and si-HZ09; Vector and HZ 09; HZ 09; Negative control and si1-HZ09; Vector and HZ 09; and Negative control and si1-HZ09, respectively. Figure 2G is a western blot, displaying Vector (100), HZ 09 (34), Negative control (100), si1-HZ 09 (143), si2-HZ 09 (157) columns and specificity protein 1 and lowercase beta-tubulin, each under swan 71 and H T R-8 or SVneo (rows). Figures 2J and 2M, each have a set of two line graphs titled Swan 71, plotting Phospholipase D1 messenger ribonucleic acid remaining, ranging from 0.0 to 1.0 in increments of 0.5 and 0.0 to 1.0 in increments of 0.5 (y-axis) across Time (hour), ranging from 1 to 5 in unit increments (x-axis) for Vector, HZ 09, si1,2-HZ 09, and Negative control. Figures 2K and 2N, each have a set of two line graphs titled H T R-8 or SVneo, plotting Phospholipase D1 messenger ribonucleic acid remaining, ranging from 0.0 to 1.0 in increments of 0.5 and 0.0 to 1.0 in increments of 0.5 (y-axis) across Time (hour), ranging from 1 to 5 in unit increments (x-axis) for Vector, HZ 09, si1,2-HZ 09, and Negative control. Figure 2L is a western blot, displaying Vector (100), Human antigen R (147), Negative control (100), si1-HZ 09 (41), si2-HZ 09 (56) columns and Phospholipase D1 and lowercase beta-tubulin, each under swan 71 and H T R-8 or SVneo (rows). Figure 2W is a western blot titled pull-down by biotin-labeled HZ 09 or Phospholipase D1 messenger ribonucleic acid, displaying input, HZ 09 sense, HZ 09 antisense, input, Phospholipase D1 sense, and Phospholipase D1 antisense (columns) and Human antigen R, under Swan 71 and H T E-8 or SVneo. Figure 2X is a western blot titled pull-down by biotin-labeled Phospholipase D1 messenger ribonucleic acid, displaying input, Vector (Phospholipase D1 sense), HZ 09 (Phospholipase D1 antisense), input, Negative control (Phospholipase D1 sense), and si1-HZ 09 (Phospholipase D1 antisense) (columns) and Human antigen R, under Swan 71 and H T E-8 or SVneo. Figure 2Y is a western blot titled pull-down by biotin-labeled HZ 09, displaying input, Vector (HZ 09 sense), HZ 09 (HZ 09 antisense), input, Negative control (HZ 09 sense), and si1-HZ 09 (HZ 09 antisense) (columns) and Human antigen R, under Swan 71 and H T E-8 or SVneo.

Figure 2A is a Venn diagram titled Benzo(a)pyrene diol epoxide treated versus untreated displays three circles. The circle on the top is labeled 250, the circle on the left, is labeled 1043, the circle on the right, is labeled 927. The intersection area is labeled 9. The intersection area represents Collagen alpha-1(12), Type 2 iodothyronine deiodinase, Myosin Heavy Chain 10, Phospholipase D1, Pleckstrin and Sec7 Domain Containing 3, Protein Tyrosine Phosphatase Non-Receptor Type 13, single-stranded D N A binding protein 2, and transforming growth factor beta-2. Figure 2B is a String analysis depicting Phospholipase D1, Ras-related C3 botulinum toxin substrate 1 and Cell division control protein 42 homolog. Figure 2C is a set of two western blots, displaying Vector (100), HZ 09 (31), Negative control (100), si1-HZ 09 (163), si2-HZ 09 (187) for Swan 71 and Vector (100), HZ 09 (74), Negative control (100), si1-HZ 09 (144), si2-HZ 09 (179) for H T R-8 or SVneo (columns) and Phospholipase D1, Lowercase beta-tubulin, specificity protein 1, and lowercase beta-tubulin (rows). Figure 2D is a western bolt, displaying Vector (100), HZ 09 (169), Negative control (100), si1-HZ 09 (53), si2-HZ 09 (51) columns and specificity protein 1, Phospholipase D1, and Glyceraldehyde 3-phosphate dehydrogenase, each under Swan 71 and H T R-8 or SVneo (rows). Figures 2E, 2F, 2O, 2P, 2Q, 2R are bar graphs titled chromatin immunoprecipitation in Swan 71, chromatin immunoprecipitation in H T R-8 or SVneo, receptor-interacting protein in Swan 71, receptor-interacting protein in swan 71, receptor-interacting protein in H T R-8 or SVneo, receptor-interacting protein in H T R-8 or SVneo, plotting Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 6 in increments of 2; Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 6 in increments of 2; ribonucleic acid relative expression, ranging from 0 to 6 in increments of 2; ribonucleic acid relative expression, ranging from 0 to 4 in unit increment; ribonucleic acid relative expression, ranging from 0 to 6 in increments of 2; and ribonucleic acid relative expression, ranging from 0 to 4 in unit increments (y-axis) across anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; and anti-Immunoglobulin G and anti-Human antigen R (x-axis) for HZ 09 and Phospholipase D1, respectively. Figures 2H, 2I, 2S, 2T, 2U, 2V are clustered bar graph titled chromatin immunoprecipitation in Swan 71, chromatin immunoprecipitation in Swan 71, receptor-interacting protein in swan 71, receptor-interacting protein in swan 71, receptor-interacting protein in H T R-8 or SVneo, and receptor-interacting protein in H T R-8 or SVneo, plotting Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 4 in unit increments; Phospholipase D1 promoter relative fold enrichment, ranging from 0 to 8 in increments of 2; messenger ribonucleic acid relative expression, ranging from 0 to 4 in increments of 2; messenger ribonucleic acid relative expression, ranging from 0 to 4 in increments of 2; messenger ribonucleic acid relative expression, ranging from 0 to 3 in unit increments; and messenger ribonucleic acid relative expression, ranging from 0 to 4 in increments of 2 (y-axis) across anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti- specificity protein 1; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; anti-Immunoglobulin G and anti-Human antigen R; and anti-Immunoglobulin G and anti-Human antigen R (x-axis) for Vector and HZ 09; Negative control and si-HZ09; Vector and HZ 09; HZ 09; Negative control and si1-HZ09; Vector and HZ 09; and Negative control and si1-HZ09, respectively. Figure 2G is a western blot, displaying Vector (100), HZ 09 (34), Negative control (100), si1-HZ 09 (143), si2-HZ 09 (157) columns and specificity protein 1 and lowercase beta-tubulin, each under swan 71 and H T R-8 or SVneo (rows). Figures 2J and 2M, each have a set of two line graphs titled Swan 71, plotting Phospholipase D1 messenger ribonucleic acid remaining, ranging from 0.0 to 1.0 in increments of 0.5 and 0.0 to 1.0 in increments of 0.5 (y-axis) across Time (hour), ranging from 1 to 5 in unit increments (x-axis) for Vector, HZ 09, si1,2-HZ 09, and Negative control. Figures 2K and 2N, each have a set of two line graphs titled H T R-8 or SVneo, plotting Phospholipase D1 messenger ribonucleic acid remaining, ranging from 0.0 to 1.0 in increments of 0.5 and 0.0 to 1.0 in increments of 0.5 (y-axis) across Time (hour), ranging from 1 to 5 in unit increments (x-axis) for Vector, HZ 09, si1,2-HZ 09, and Negative control. Figure 2L is a western blot, displaying Vector (100), Human antigen R (147), Negative control (100), si1-HZ 09 (41), si2-HZ 09 (56) columns and Phospholipase D1 and lowercase beta-tubulin, each under swan 71 and H T R-8 or SVneo (rows). Figure 2W is a western blot titled pull-down by biotin-labeled HZ 09 or Phospholipase D1 messenger ribonucleic acid, displaying input, HZ 09 sense, HZ 09 antisense, input, Phospholipase D1 sense, and Phospholipase D1 antisense (columns) and Human antigen R, under Swan 71 and H T E-8 or SVneo. Figure 2X is a western blot titled pull-down by biotin-labeled Phospholipase D1 messenger ribonucleic acid, displaying input, Vector (Phospholipase D1 sense), HZ 09 (Phospholipase D1 antisense), input, Negative control (Phospholipase D1 sense), and si1-HZ 09 (Phospholipase D1 antisense) (columns) and Human antigen R, under Swan 71 and H T E-8 or SVneo. Figure 2Y is a western blot titled pull-down by biotin-labeled HZ 09, displaying input, Vector (HZ 09 sense), HZ 09 (HZ 09 antisense), input, Negative control (HZ 09 sense), and si1-HZ 09 (HZ 09 antisense) (columns) and Human antigen R, under Swan 71 and H T E-8 or SVneo.

Expression levels of members of PLD1/RAC1/CDC42 pathway regulated by lnc-HZ09 in human trophoblast cells. (A) The significantly down-regulated mRNAs in the intersection of mRNA sequencing data of BPDE-treated vs. untreated Swan 71 cells, lnc-HZ09-overexpressed vs control Swan 71 cells, and RM vs HC villous tissues. (B) String analysis of PLD1, RAC1 and CDC42. (C) Representative western blot analysis of the protein levels of PLD1, RAC1 and CDC42 in Swan 71 or HTR-8/SVneo cells with overexpression or knockdown of lnc-HZ09, with GAPDH as internal standard. The relative intensity of each band was quantified and the mean±SD of three replicates was shown in Figure S3A. (D) Representative western blot analysis of the protein levels of SP1 and PLD1 in Swan 71 or HTR-8/SVneo cells with overexpression or knockdown of SP1, with GAPDH as internal standard. The relative intensity of each band was quantified and the mean±SD of three replicates was shown in Figure S3G. (E–F) SP1 ChIP assay analysis (each n=3) of the relative enrichment of SP1 in the promoter region of PLD1 gene in Swan 71 (E) or HTR-8/SVneo (F) cells. (G) Representative western blot analysis of the protein levels of SP1 in Swan 71 or HTR-8/SVneo cells with overexpression or knockdown of lnc-HZ09, with β-tubulin as internal standard. The relative intensity of each band was quantified and the mean±SD of three replicates was shown in Figure S3H. (H–I) SP1 ChIP assay analysis (each n=3) of the relative enrichment of SP1 in the promoter region of PLD1 gene in Swan 71 cells with overexpression (H) or knockdown (I) of lnc-HZ09. (J–K) The mRNA stability of PLD1 (each n=3) in Swan 71 (J) or HTR-8/SVneo (K) cells with overexpression or knockdown of lnc-HZ09. (L) Representative western blot analysis of the protein levels of PLD1 in Swan 71 or HTR-8/SVneo cells with overexpression or knockdown of HuR, with β-tubulin as internal standard. The relative intensity of each band was quantified and the mean±SD of three replicates was shown in Figure S5E. (M–N) The mRNA stability of PLD1 (each n=3) in Swan 71 (M) or HTR-8/SVneo (N) cells with overexpression or knockdown of HuR. (O–R) RIP assay analysis (each n=3) of the relative levels of lnc-HZ09 (O and Q) or PLD1 mRNA (P and R) that was pulled down by HuR protein in Swan 71 (O and P) or HTR-8/SVneo (Q and R) cells. (S–V) RIP assay analysis (each n=3) of the relative levels of PLD1 mRNA that was pulled down by HuR protein in Swan 71 (S and T) or HTR-8/SVneo (U and V) cells with overexpression (S and U) or knockdown (T and V) of lnc-HZ09. (W) Representative western blot analysis of HuR that was pulled down by biotin-labeled lnc-HZ09 or PLD1 mRNA in Swan 71 or HTR-8/SVneo cells in pull-down assays. (X) Representative western blot analysis of HuR that was pulled down by biotin-labeled PLD1 mRNA in Swan 71 or HTR-8/SVneo cells with overexpression or knockdown of lnc-HZ09 in pull-down assays. (Y) Representative western blot analysis of HuR that was pulled down by biotin-labeled lnc-HZ09 in Swan 71 or HTR-8/SVneo cells with overexpression or knockdown of PLD1 in pull-down assays. The summary data of these bar charts and diagrams were shown in Excel Table S1. The expression level in NC or Vector group was set as “1” in all of mRNA stability assays; the levels of DNA or RNA pulled down by IgG were set as “1” in all of ChIP and RIP assays, respectively; and the band intensity in NC or Vector group was set as ‘100’ in all of western blot assays. C,D,G,L,W–Y show the representative data from three independent experiments. Data in (E–F, H–K, M–V) show mean±SD of three independent experiments. Two-tailed Student’s t-test for (E–F,H–I,O–V); *p<0.05, **p<0.01. Note: BPDE, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide; ChIP, chromatin immunoprecipitation; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HC, healthy control; HuR, overexpression of HuR; HZ09, overexpression of lnc-HZ09; NC, negative control of siRNA; ns, nonsignificance; PLD1, phospholipase D hydrolyze 1; RIP, RNA immunoprecipitation; RM, recurrent miscarriage; SD, standard deviation; si-HuR, knockdown of HuR; si-SP1, knockdown of SP1; SP1, overexpression of SP1; Vector, empty vector of pcDNA3.1; si-HZ09, knockdown of lnc-HZ09.