Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: RNA-seq on placentas from mice exposed to NaAsO 2 . (A–G) All pregnant mice except controls were exposed to NaAsO 2 ( 15 mg / L ) by drinking water throughout pregnancy. All pregnant mice were sacrificed on GD 18. (A) Volcano plot of differentially expressed genes in mouse placentas. A total of 743 genes (red triangle) were up-regulated, and 1,889 genes (green dot) were down-regulated by at least 1.5-fold with a significance level ( p < 0.05 ). (B) KEGG pathway analysis. (C) Top 100 genes were screened based on the p -value ranking of all differential genes and then we screened 40 candidate genes among them, with the average FPKM > 10 of all samples as the filter condition. RNA-seq of mouse placentae from three dams were analyzed per group. (D) Validation of trophoblasts development-related differentially expressed genes by real-time RT-PCR ( n = 6 mice/group). (E) Fold differences in differentially expressed genes between RNA-seq and real-time RT-PCR. (F) Placental CYR61 was detected by immunoblotting ( n = 3 mice/group, repeated three times). (G) Representative pictures of CYR61-positive cells in labyrinth zone and junctional zone. Scale bars, 20 μ m ( n = 6 mice/group). (H–J) HTR-8/SVneo cells were exposed to NaAsO 2 ( 2 μ M ). (H) Cyr61 mRNA was measured by real-time RT-PCR ( n = 6 samples/group). (I) CYR61 protein was detected by immunoblotting ( n = 3 samples/group, repeated three times). (J) CYR61-positive cells were stained with by immunofluorescence ( n = 3 samples/group). All data are expressed as mean ± SEM . The numeric data are shown in Table S8. RNA-seq data are reported in an Excel sheet and uploaded to the National Center for Biotechnology Information (GSE222092). Unpaired two-tailed Student’s t -test was used for D–F, H, I. * p < 0.05 , ** p < 0.01 . Note: As, arsenic; As-H, 15 mg / L NaAsO 2 ; Ceacam 3/13, carcinoembryonic antigen-related cell adhesion molecule 3/13; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FPKM, fragments per kilobase per million; Not DEG, not differential gene expression; RNA-seq, RNA sequencing; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; Sparcl1, SPARC-like protein 1; TGF- β 2 , transforming growth factor β 2 ; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: The role of CYR61 in induced inhibition of migration and invasion in As-exposed human placental trophoblasts. HTR-8/SVneo cells were exposed to NaAsO 2 ( 2 μ M ). (A–D) HTR-8/SVneo cells were transfected with CYR61 overexpression plasmid and then exposed to NaAsO 2 ( 2 μ M ). (A) The efficiency of CYR61 overexpression was determined by real-time RT-PCR ( n = 6 samples/group). (B) Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ( n = 2 samples/group, repeated three times), and β -actin was used as a loading control. (C) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (D) Cell invasion was measured by transwell invasion assay ( n = 3 samples/group, repeated 2 times). (E–H) HTR-8/SVneo cells were transfected with CYR61 knockdown plasmid and then exposed to NaAsO 2 ( 2 μ M ). (E) The efficiency of CYR61 knockdown was determined by real-time RT-PCR ( n = 6 samples/group). (F) Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ( n = 2 samples/group, repeated three times); β -actin was used as a loading control. (G) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (H) Cell invasion was measured by transwell invasion assay ( n = 3 samples/group, repeated 2 times). Scale bars, 200 μ m . All data were expressed as mean ± SEM . The numeric data are shown in Table S8. Unpaired two-tailed Student’s t -test was used for A and E. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for B–D and F–H. * p < 0.05 , ** p < 0.01 . Note: ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; CYR61 OE, CYR61 overexpression; shCYR61, CYR61 shRNA; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Inhibition, Migration, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Transwell Migration Assay, Transwell Invasion Assay, Two Tailed Test, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of m 6 A modification on Cyr61 mRNA stability and CYR61 protein in As-exposed human placental trophoblasts. HTR-8/SVneo cells were exposed to NaAsO 2 ( 2 μ M ). (A) HTR8/SVneo cells were treated with actinomycin D ( 5 μ g / mL ), followed by detection of Cyr61 mRNA at indicated times ( n = 3 samples/group, repeated two times). (B) The m 6 A content was measured by EpiQuik m 6 A methylation quantification kit ( n = 5 samples/group, repeated two times). (C) Schematic representation of the position of m 6 A motifs with CYR61 transcript. (D) The m 6 A -modified Cyr61 levels were measured by MeRIP-qPCR ( n = 3 samples/group). (E) Schematic representation of the interaction between Cyr61 mRNA and IGF2BP1/2/3 proteins. (F) The interaction between Cyr61 mRNA and IGF2BP2 proteins was detected by RIP-qPCR ( n = 3 samples/group). (G) The expression of m 6 A methyltransferases and demethylases was measured by real-time RT-PCR ( n = 6 samples/group). (H) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 samples/group). (I) A schematic model of the reversible m 6 A methylation. SAM is not only a methyl donor for m 6 A methylation but also a cofactor for catalytic domain of Mettl3. (J) Intracellular SAM content was measured by LC-MS/MS ( n = 6 samples/group). All data were expressed as mean ± SEM . The numeric data are shown in Table S8. Unpaired two-tailed Student’s t -test was used for A, B, D, F, G, H, and J. * p < 0.05 , ** p < 0.01 . Note: ActD, actinomycin D; Alkbh5, a-ketoglutarate-dependent dioxygenase alkB homolog 5; ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FTO, fat-mass obesity-associated protein; IGF2BP1/2/3, insulin-like growth factor 2 mRNA-binding protein one-half/3; LC-MS/MS, liquid chromatography–tandem mass spectrometry; m 6 A , N 6 -methyladenosine ; MeRIP-qPCR, methylated RNA immunoprecipitation–qPCR; Mettl3/14, methyltransferase-like 3/14; Rbm15/15b, RNA-binding motif protein 15/15b; RIP-qPCR, RNA-binding protein immunoprecipitation–qPCR; RT-PCR, reverse transcription polymerase chain reaction; SAH, s-adenosyl-l-homocysteine; SAM, s-adenosylmethionine; SEM, standard error of the mean; μ M , μ mol / L ; WTAP, wilms tumor 1-associating protein.

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Modification, Methylation, Expressing, Quantitative RT-PCR, Activity Assay, Inhibition, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Binding Assay, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation, RNA Binding Assay, Reverse Transcription Polymerase Chain Reaction, Wilms Tumor Assay

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of As3MT knockdown on Cyr61 m 6 A modification in As-exposed human placental trophoblasts. HTR8/SVneo cells were exposed to 2 μ M NaAsO 2 in presence or absence of As3MT-shRNA (A) The efficiency of As3MT knockdown was determined by real-time RT-PCR ( n = 6 samples/group). (B) Intracellular SAM content was measured by LC-MS/MS ( n = 6 samples/group). (C) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 samples/group). (D and E) HTR8/SVneo cells were treated with actinomycin D ( 5 μ g / mL ), followed by detection of Cyr61 mRNA at indicated times ( n = 3 samples/group, repeated two times). (F) CYR61, Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ( n = 3 samples/group). (G) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (H) Cell invasion was measured by transwell invasion assay ( n = 3 samples/group, repeated two times). Scale bars, 200 μ m . All data were expressed as mean ± SEM . The numeric data are shown in Table S8. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–H. Asterisks indicate comparison with the control group. Hashtags indicate comparison with the As group. * p and # p < 0.05 , ** p and ## p < 0.01 . Note: ActD, actinomycin D; ANOVA, analysis of variance; As, arsenic; As3MT, arsenite methyltransferase; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MMP2, matrix metalloproteinases 2; RT-PCR, reverse transcription polymerase chain reaction; SAM, s-adenosylmethionine; shAs3MT 1#, As3MT shRNA 1#; shAs3MT 2#, As3MT shRNA 2#; SEM, standard error of the mean; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Modification, shRNA, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Inhibition, Western Blot, Migration, Transwell Migration Assay, Transwell Invasion Assay, Comparison, Liquid Chromatography, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of SAM supplementation on induced inhibition of Cyr61 m 6 A modification in As-exposed human placental trophoblasts. HTR8/SVneo cells were incubated with NaAsO 2 ( 2 μ M ) in presence or absence of SAM ( 10 μ M ). (A) Intracellular SAM content was measured by LC-MS/MS ( n = 6 samples/group). (B) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 samples/group). (C) HTR8/SVneo cells were treated with actinomycin D ( 5 μ g / mL ), followed by detection of Cyr61 mRNA at indicated times ( n = 3 samples/group, repeated two times). (D and E) CYR61, Vimentin, N-cadherin, and MMP2 proteins were determined by immunoblotting ( n = 3 samples/group, repeated three times). (F) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (G) Cell invasion was measured by transwell invasion assay ( n = 6 samples/group, repeated two times). Scale bars, 200 μ m . All data were expressed as mean ± SEM . The numeric data are shown in Table S8. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–G. Asterisks indicate comparison with the control group. Hashtags indicate comparison with the As group. * p and # p < 0.05 , ** p and ## p < 0.01 . Note: ANOVA, analysis of variance; As, arsenic; ActD, actinomycin D; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; MMP2, matrix metalloproteinases 2; RT-PCR, reverse transcription polymerase chain reaction; SAM, s-adenosylmethionine; SEM, standard error of the mean; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Inhibition, Modification, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Western Blot, Migration, Transwell Migration Assay, Transwell Invasion Assay, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of FA supplementation on placental development and fetal growth restriction in As-exposed mice. Pregnant mice were divided into four groups: CTRL, FA, As, and As + FA groups. In the As and As + FA groups, pregnant mice drank ultrapure water containing NaAsO 2 ( 15 mg / L ) throughout pregnancy. In FA and As + FA groups, pregnant mice were administered FA ( 150 μ g / kg ) by gavage daily throughout pregnancy. All pregnant mice were sacrificed on GD18. (A) Fetal weight ( n = 8 dams/group, 12–15 fetuses per dam). (B) Crown–rump length ( n = 8 dams/group, 12–15 fetuses per dam). (C) Placental weight ( n = 8 dams/group, 12–15 fetuses per dam). (D) Placental diameter ( n = 8 dams/group, 12–15 fetuses per dam). (E) Placental cross-section was stained with H&E. Scale bars, 500 μ m (left); 200 μ m (right) ( n = 6 mice/group). (F) The percentage of labyrinth zone area in the entire placenta area ( n = 6 mice/group). (G) The ratio of cross-sectional thickness of labyrinth zone to junctional zone ( n = 6 mice/group). (H) Placental SAM content was determined by LC-MS/MS ( n = 6 mice/group). (I) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 mice/group). (J) Placental CYR61 and MMP2 were measured by immunoblotting ( n = 3 mice/group, repeated two times). All data were expressed as mean ± SEM . The numeric data are shown in Table S8. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–D and F–J. * p < 0.05 , ** p < 0.01 . Note: ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FA, folic acid; H&E, hematoxylin and eosin; JZ, junctional zone; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LZ, labyrinth zone; MMP2, matrix metalloproteinases 2; SAM, s-adenosylmethionine; SEM, standard error of the mean.

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Inhibition, Western Blot, Liquid Chromatography, Mass Spectrometry

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Based on a case–control study: association among maternal urinary As concentration, plasma CYR61 content, and fetal growth restriction. (A) Maternal urinary As concentration in AGA and SGA infants was determined by hydride generation-atomic fluorescence spectrometry ( n = 45 human sample/group). (B) Maternal plasma CYR61 content in AGA and SGA infants was measured by ELISA ( n = 45 human sample/group). (C) The correlation between maternal urinary As concentration and plasma CYR61 content in AGA infants ( n = 45 human sample). (D) The correlation between maternal urinary As concentration and plasma CYR61 content in SGA infants ( n = 45 human sample). (E) Placental CYR61-positive cells in SGA infants and AGA infants were stained by immunohistochemistry ( n = 18 human sample/group). Representative histology images were showed. Blue arrows indicate CYR61-positive cells. Scale bars, 20 μ m . (F) Placental vimentin, MMP2, and MMP9 were detected by immunoblotting ( n = 18 human sample/group). The data in A and B are expressed as mean ± SD . The data in A–D are represented by dots indicating individual values. The data in E and F are expressed as mean ± SEM . The numeric data are shown in Table S8. Two-tailed Student’s t -test was used for A, B, E, and F. The Spearman correlation was used for C and D. * p < 0.05 , ** p < 0.01 . Note: AGA, appropriate for gestational age; As, arsenic; CYR61, cysteine-rich angiogenic inducer 61; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; SEM, standard error of the mean; SGA, small for gestational age.

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Western Blot, Two Tailed Test, Standard Deviation