Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: Expression and localization of CA2 in the human placentas and Serum. (A) Heatmap of carbonic anhydrase family expression in placental transcriptomes from normal and preeclamptic pregnancies. (B) Box plot analysis of carbonic anhydrase family expression in placental transcriptomes from normal and preeclamptic pregnancies. (C) MRNA expression level of CA2 in PE and normal placentas detected by RT‐qPCR in the discovery cohort. (D) Representative western blot bands for CA2. (E) Protein expression level of CA2 in PE and normal placentas detected by Western Blot in the discovery cohort. (F) Serum expression level of CA2 in PE and normal women detected by ELISA in the serum validation cohort. (G) Immunohistochemistry method to detect the expression localization of CA2 in placentas from normal and preeclamptic pregnancies. Scale bars: 3 mm (overview), 200 μm (magnified field). All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Expressing, Analysis, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: Correlation analysis between CA2 expression and clinical indicators. (A) Correlation of CA2 levels with SBP. (B) Correlation of CA2 levels with DBP. (C) Correlation of CA2 levels with Fetal Weight. (D) Correlation of CA2 levels with APGAR‐1 min. (E) Correlation of CA2 levels with APGAR‐5 min. (F) Correlation of CA2 levels with Proteinuria. (G) Correlation of CA2 levels with maternal blood creatinine levels before delivery. (H) Correlation of CA2 levels with blood urea levels. (I) Correlation of CA2 levels with ALT. (J) Correlation of CA2 levels with AST. (K) Correlation of CA2 levels with PT levels. (L) Correlation of CA2 levels with APTT. (M) Correlation of CA2 levels with platelet levels. Correlation analyses were performed using Pearson's correlation coefficient. ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Analysis, Expressing

Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: Preeclampsia modeling and placental CA2 expression in L‐NAME mouse model. (A) Experimental workflow of the L‐NAME‐induced preeclampsia model. (B) Changes in systolic blood pressure in L‐NAME and CONTROL groups. (C, E) Body weights of fetal mice in L‐NAME and CONTROL groups. (D) Placental efficiency of fetal mice in L‐NAME and CONTROL groups. (H) SFLT‐1 levels of mice in L‐NAME and CONTROL groups. (F) The ratio of urinary albumin and creatine of mice in L‐NAME and CONTROL groups. (G, I) Representative renal and placental stained with hematoxylin & eosin (H&E) in L‐NAME and CONTROL groups. Scale bars: 250 μm. (J) Ratio of palcental labyrinth and junction zone of mice in L‐NAME and CONTROL groups. (K) MRNA expression level of placental CA2 in L‐NAME and CONTROL groups detected by RT‐qPCR. (L) Representative western blot bands for CA2. (M) Protein expression level of placental CA2 in L‐NAME and CONTROL groups detected by Western Blot. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Expressing, Control, Staining, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: Effects of CA2 on trophoblast cell function. (A) RT‐qPCR analysis to detect the expression of mRNA level after overexpression of CA2 in HTR‐8/SVneo cells. (B) Representative western blot bands for CA2. (C) Western Blot analysis to detect the expression of protein level after overexpression of CA2 in HTR‐8/SVneo cells. (D) Effect of CA2 overexpression on the proliferative capacity of HTR‐8/SVneo cells. (E) Impact of CA2 overexpression on the migration and invasion abilities of HTR‐8/SVneo cells. (F) Statistical analysis of the Transwell migration and invasion assays. (G) Effect of CA2 overexpression on the apoptotic properties of HTR‐8/SVneo cells. (H) Statistical analysis of the apoptosis assay. pEX‐3‐control: Empty vector control group; pEX‐3‐CA2: CA2 overexpression group. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Cell Function Assay, Quantitative RT-PCR, Analysis, Expressing, Over Expression, Western Blot, Migration, Apoptosis Assay, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: VEGFA overexpression rescues the impaired proliferation, migration and invasion of HTR‐8/SVneo cells induced by CA2 overexpression. (A) Heatmap showing hierarchical clustering of differentially expressed genes between the control and CA2 overexpression groups. (B) Volcano plot of differentially expressed genes. (C) Western Blot analysis of CA2 and VEGFA to detect the protein expression level on the of HTR‐8/SVneo cells in each expremental group. (D) Proliferative capacity analysis of HTR‐8/SVneo cells in the control group、CA2 overexpression group and CA2 and VEGFA co‐overexpression group. (E) Migration and invasion abilities of HTR‐228/SVneo cells in the control group, CA2 overexpression group and CA2 and VEGFA co‐overexpression group. (F) Statistical analysis of the Transwell migration and invasion abilities in the control group, CA2 overexpression group and CA2 and VEGFA co‐overexpression group. pEX‐3‐control 1/2/3: Empty vector control group; pEX‐3‐CA2 1/2/3: CA2 overexpression group; pEX‐3‐control: Empty vector control group; pEX‐3‐ CA2: CA2 overexpression group; pEX‐3‐ CA2+ pEX‐3‐ VEGFA: CA2 and VEGFA co‐overexpression group. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Over Expression, Migration, Control, Western Blot, Analysis, Expressing, Plasmid Preparation

Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: HIF‐1α activates the transcription of CA2. (A, B) RT‐qPCR analysis of CA2 and HIF‐1α to detect the expression of mRNA level under hypoxic or normoxia exposure in HTR‐8/Svneo cells. (C) Representative western blot bands. (D, E) Western Blot analysis of CA2 and HIF‐1α to detect the expression of protein level under hypoxic or normoxia exposure in HTR‐8/Svneo cells. (F, G) RT‐qPCR analysis of CA2 and HIF‐1α to detect the expression of mRNA level after overexpression HIF‐1α in HTR‐8/Svneo cells. (H) Representative western blot bands. (I, J) Western Blot analysis of CA2 and HIF‐1α to detect the expression of protein level after overexpression HIF‐1α in HTR‐8/Svneo cells. (K) Luciferase activity of HTR‐8/SVneo cells with or without overexpression HIF‐1α. (L) Luciferase activity of HTR‐8/SVneo cells with or without exposing hypoxic environment. pcDNA3.1‐control: Empty vector control group; pcDNA3.1‐HIF‐1α: HIF‐1α overexpression group; CA2 promoter + Vecter: Plasmid with CA2 promoter + empty vector control group; CA2 promoter + HIF‐1α OE: Plasmid with CA2 promoter + HIF‐1α overexpression group; CA2 promoter + Vecter + hypoxia 12 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 12 h; CA2 promoter + Vecter + hypoxia 24 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 24 h; CA2 promoter + Vecter + hypoxia 36 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 36 h. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Quantitative RT-PCR, Analysis, Expressing, Western Blot, Over Expression, Luciferase, Activity Assay, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

doi: 10.1096/fj.202602024R

Figure Lengend Snippet: Inhibition of CA2 partially improves outcomes in a L‐NAME‐induced PE mouse model. (A) Experimental workflow for LNP‐NC and LNP‐siCA2 treatment in L‐NAME PE mice. (B) MRNA expression level of placental CA2 in LNP‐NC and LNP‐siCA2 groups detected by RT‐qPCR. (C) Representative immunofluorescence staining of CA2 in LNP‐NC and LNP‐siCA2 groups. Scale bars: 500 μm. (D) Quantitative analysis of mean fluorescence intensity of CA2 immunofluorescence signals n LNP‐NC and LNP‐siCA2 groups. (E) Changes in systolic blood pressure in LNP‐NC and LNP‐siCA2 groups. (F) The ratio of urinary albumin and creatine of mice in LNP‐NC group and LNP‐siCA2 group. (G) Representative renal stained with hematoxylin & eosin (H&E) in LNP‐NC and LNP‐siCA2 groups. (H) SFLT‐1 levels of mice in LNP‐NC and LNP‐siCA2 groups. (I, K) Body weights of fetal mice in LNP‐NC and LNP‐siCA2 groups. (J) Placental efficiency of fetal mice in LNP‐NC group and LNP‐siCA2 group. (L) Representative placental stained with hematoxylin & eosin (H&E) in LNP‐NC and LNP‐siCA2 groups. Scale bars: 250 μm. (M) Ratio of palcental labyrinth and junction zone of mice in LNP‐NC and LNP‐siCA2 groups. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Analysis, Fluorescence, Reverse Transcription Polymerase Chain Reaction