Journal: Acta Biochimica et Biophysica Sinica

Article Title: Cooperation between NSPc1 and DNA methylation represses HOXA11 expression and promotes apoptosis of trophoblast cells during preeclampsia

doi: 10.3724/abbs.2023012

Figure Lengend Snippet: Table 1 Sequences of shRNAs used in this study

Article Snippet: Adenoviruses encoding NSPc1 (Ad-NSPc1), HOXA11 (Ad-HOXA11) or a negative control (Ad-GFP) were purchased from Genechem (Shanghai, China), and the short heparin RNAs of NSPc1 (sh-NSPc1-1, 2 and 3), HOXA11 (sh-HOXA11-1, 2 and 3), DNMT3a (sh-DNMT3a-1, 2 and 3) and short heparin RNA of scrambled (sh-NC) were purchased from GenePharma (Shanghai, China).

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Cooperation between NSPc1 and DNA methylation represses HOXA11 expression and promotes apoptosis of trophoblast cells during preeclampsia

doi: 10.3724/abbs.2023012

Figure Lengend Snippet: Table 2 Sequences of primers used for qRT-PCR analysis

Article Snippet: Adenoviruses encoding NSPc1 (Ad-NSPc1), HOXA11 (Ad-HOXA11) or a negative control (Ad-GFP) were purchased from Genechem (Shanghai, China), and the short heparin RNAs of NSPc1 (sh-NSPc1-1, 2 and 3), HOXA11 (sh-HOXA11-1, 2 and 3), DNMT3a (sh-DNMT3a-1, 2 and 3) and short heparin RNA of scrambled (sh-NC) were purchased from GenePharma (Shanghai, China).

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Cooperation between NSPc1 and DNA methylation represses HOXA11 expression and promotes apoptosis of trophoblast cells during preeclampsia

doi: 10.3724/abbs.2023012

Figure Lengend Snippet: NSPc1 promotes apoptosis of trophoblast cells during preeclampsia (A) Immunofluorescent staining of NSPc1 (red) and CK-7 (green) in the placentas of PC and PE pregnancies. Nuclei were stained with DAPI (blue). The white arrows refer to NSPc1-positive trophoblast cells. Scale bar, 20 μm. (B,C) qRT-PCR and western blot analysis were performed to detect the mRNA and protein expression of NSPc1 in the placentas of PE pregnancies ( n=6) and trophoblast cells under hypoxia ( n=3). (D) Western blot analysis was used to detect the protein expressions of Bax, Bcl-2, c-caspase-3 and c-caspase-9 in trophoblast cells infected with adenoviruses encoding NSPc1 (Ad-NSPc1) and short heparin RNA of NSPc1 (sh-NSPc1) under hypoxia ( n=3). (E) Flow cytometry analysis was used to detect the apoptosis rate of trophoblast cells after infection with Ad-NSPc1 and sh-NSPc1 under hypoxia ( n=3). Data are shown as the mean±SD from 3 independent experiments. * P<0.05, ** P<0.01.

Article Snippet: Adenoviruses encoding NSPc1 (Ad-NSPc1), HOXA11 (Ad-HOXA11) or a negative control (Ad-GFP) were purchased from Genechem (Shanghai, China), and the short heparin RNAs of NSPc1 (sh-NSPc1-1, 2 and 3), HOXA11 (sh-HOXA11-1, 2 and 3), DNMT3a (sh-DNMT3a-1, 2 and 3) and short heparin RNA of scrambled (sh-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Staining, Quantitative RT-PCR, Western Blot, Expressing, Infection, Flow Cytometry

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Cooperation between NSPc1 and DNA methylation represses HOXA11 expression and promotes apoptosis of trophoblast cells during preeclampsia

doi: 10.3724/abbs.2023012

Figure Lengend Snippet: NSPc1 specifically represses HOXA11 expression in trophoblast cells during preeclampsia (A) qRT-PCR was used to detect HOX gene cluster mRNA expression in trophoblast cells after infection with sh-NSPc1 under hypoxia, and the expression level of each HOX mRNA was normalized to that of GAPDH ( n=3). (B) The protein expressions of HOXA9, HOXA11, HOXA13, HOXB7, HOXD3 and HOXD8 were detected by western blot analysis in trophoblast cells after infection with sh-NSPc1 under hypoxia ( n=3). (C) The mRNA and protein expression of HOXA11 in trophoblast cells after infection with Ad-NSPc1 under hypoxia was measured by qRT-PCR and western blot analysis, respectively ( n=3). (D,E) The mRNA and protein expression levels of HOXA11 were detected by qRT-PCR and western blot analysis in the placentas of PE pregnancies ( n=6) and trophoblast cells under hypoxia ( n=3). (F) Western blot analysis was used to measure the protein expressions of Bax, Bcl-2, c-caspase-3 and c-caspase-9 in trophoblast cells after infection with adenoviruses encoding HOXA11 (Ad-HOXA11) and short heparin RNA of HOXA11 (sh-HOXA11) under hypoxia ( n=3). (G) Flow cytometry analysis was used to detect the apoptosis rate of trophoblast cells infected with Ad-HOXA11 and sh-HOXA11 under hypoxia ( n=3). Data are shown as the mean±SD from 3 independent experiments. * P<0.05, ** P<0.01.

Article Snippet: Adenoviruses encoding NSPc1 (Ad-NSPc1), HOXA11 (Ad-HOXA11) or a negative control (Ad-GFP) were purchased from Genechem (Shanghai, China), and the short heparin RNAs of NSPc1 (sh-NSPc1-1, 2 and 3), HOXA11 (sh-HOXA11-1, 2 and 3), DNMT3a (sh-DNMT3a-1, 2 and 3) and short heparin RNA of scrambled (sh-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Infection, Western Blot, Flow Cytometry

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Cooperation between NSPc1 and DNA methylation represses HOXA11 expression and promotes apoptosis of trophoblast cells during preeclampsia

doi: 10.3724/abbs.2023012

Figure Lengend Snippet: NSPc1 inhibits HOXA11 expression by promoting the binding of DNMT3a to the HOXA11 promoter (A) Total cell lysates of trophoblast cells under hypoxia were immunoprecipitated with NSPc1 antibodies or control IgG and subjected to western blot analysis using anti-HOXA11 antibodies. (B) A Co-IP assay was performed to determine the endogenous interaction between NSPc1 and DNMT3a in trophoblast cells using antibodies against NSPc1 and DNMT3a. IgG was used as a negative control. (C) ChIP assay was used to detect DNMT3a enrichment at the HOXA11 promoter after trophoblast cells were infected with Ad-NSPc1 or sh-NSPc1 under hypoxia. The ChIP-enriched DNA fragments of the HOXA11 promoter using IgG and anti-DNMT3a antibody were amplified by PCR. Total input (5%) was used as a positive control ( n=3). (D) The mRNA and protein expression levels of HOXA11 in trophoblast cells after transfection with Ad-NSPc1 and sh-DNMT3a under hypoxia were measured by qRT-PCR and western blot analysis ( n=3), respectively. Data are shown as the mean±SD from 3 independent experiments. * P<0.05, ** P<0.01.

Article Snippet: Adenoviruses encoding NSPc1 (Ad-NSPc1), HOXA11 (Ad-HOXA11) or a negative control (Ad-GFP) were purchased from Genechem (Shanghai, China), and the short heparin RNAs of NSPc1 (sh-NSPc1-1, 2 and 3), HOXA11 (sh-HOXA11-1, 2 and 3), DNMT3a (sh-DNMT3a-1, 2 and 3) and short heparin RNA of scrambled (sh-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Binding Assay, Immunoprecipitation, Control, Western Blot, Co-Immunoprecipitation Assay, Negative Control, Infection, Amplification, Positive Control, Transfection, Quantitative RT-PCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Cooperation between NSPc1 and DNA methylation represses HOXA11 expression and promotes apoptosis of trophoblast cells during preeclampsia

doi: 10.3724/abbs.2023012

Figure Lengend Snippet: NSPc1 and DNMT3a synergistically regulate apoptosis of trophoblast cells (A) The protein expressions of Bax, Bcl-2, c-caspase-3 and c-caspase-9 in trophoblast cells transfected with Ad-NSPc1 and sh-DNMT3a under hypoxia were measured by western blot analysis ( n=3). (B) Flow cytometry analysis was used to detect the apoptosis rate of trophoblast cells transfected with Ad-NSPc1 and sh-DNMT3a under hypoxia ( n=3). Data are shown as the mean±SD from 3 independent experiments. * P<0.05, ** P<0.01.

Article Snippet: Adenoviruses encoding NSPc1 (Ad-NSPc1), HOXA11 (Ad-HOXA11) or a negative control (Ad-GFP) were purchased from Genechem (Shanghai, China), and the short heparin RNAs of NSPc1 (sh-NSPc1-1, 2 and 3), HOXA11 (sh-HOXA11-1, 2 and 3), DNMT3a (sh-DNMT3a-1, 2 and 3) and short heparin RNA of scrambled (sh-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Transfection, Western Blot, Flow Cytometry