antibody wt1  (Novus Biologicals)

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Human primer pairs used to assess the steady-state mRNA level of target genes.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Lentivirus-mediated gene transfection into hAmMSCs. (A) The hAmMSCS 48 h after GFP transfection. Almost all hAmMSCs expressed GFP. (B) SF1 expression in hAmMSCs was confirmed via western blot analysis. (C) WT1+KTS and WT1−KTS mRNA expression in hAmMSCs was confirmed via quantitative PCR using isoform-specific primers. (D) Expression of WT1 in hAmMSCs was confirmed via western blot analysis. (E) WT1+KTS and WT1−KTS were mainly expressed in the cytoplasm and partially in the nucleus. (F) Neither the SF-1- nor WT1-transfected hAmMSCs showed any morphological changes compared with those transfected with GFP. Data are presented as the mean ± SD from three independent experiments. **, p <0.01, ***, p <0.001. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; GFP, green fluorescent protein; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Membrane, Derivative Assay, Wilms Tumor Assay, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Lentivirus-mediated gene transfection into human fibroblasts. (A) SF-1, WT1+KTS, and WT1−KTS mRNA expression in human fibroblasts was confirmed via quantitative PCR. (B) SF-1 and WT1 expression in human fibroblasts was confirmed via western blot analysis. (C) Real-time RT-PCR analysis revealed that neither SF-1 nor WT1 transient expression affected the gene expression of steroidogenic factors in human fibroblasts. (D) SF-1 did not enhance pregnenolone and progesterone production by human fibroblasts. Data are presented as the mean ± SD from three independent experiments. *, p<0.05, **, p<0.01.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Effects of SF-1 and WT1 on progesterone-producing cell lineage. GFP, SF-1, WT1+KTS, and WT1−KTS were transiently expressed in hAmMSCs via lentivirus-mediated gene transfection and analyzed for progesterone-producing capacity. (A) The expression of steroidogenic genes involved in progesterone synthesis in hAmMSCs was measured via quantitative PCR. SF-1 considerably increased the expression of StAR , CYP11A1 , and HSD3B2 in hAmMSCs. In contrast, WT1+KTS and WT1−KTS did not alter the mRNA expression of progesterone-producing factors. (B) SF-1 significantly enhanced pregnenolone and progesterone production by the hAmMSCs. (C) No difference was observed in the mRNA levels of progesterone-producing factors when SF-1 and WT1−KTS were co-expressed compared with those when SF-1 was expressed alone. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; GFP, green fluorescent protein; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; PCR, polymerase chain reaction; SD, standard deviation; KTS, lysine, threonine, and serine.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Membrane, Derivative Assay, Wilms Tumor Assay, Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Effects of SF-1 and WT1 on adrenal steroidogenic cell lineage. (A) SF-1 significantly increased the mRNA expression of NRDB1 ( DAX-1 ) but not of OSR2 , ACTHR , and AR , in hAmMSCs. (B) SF-1, WT1+KTS, and WT1−KTS did not change the mRNA levels of CYP21A2 , CYP11B1 , and CYP11B2 . (C) Co-expression of SF-1 and WT1−KTS did not alter the mRNA levels of adrenal steroidogenic enzymes in hAmMSCs. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; SD, standard deviation; KTS, lysine, threonine, and serine.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay, Standard Deviation

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Effects of SF-1 and WT1 on androgen-producing cell lineage. (A) SF-1 did not change the mRNA levels of NR2F2 , LIFR , PDGFRα , and LHCGR . (B) SF-1 significantly increased the mRNA expression of CYP17A1 in hAmMSCs. In contrast, WT1+KTS and WT1−KTS did not alter the mRNA expression of androgen-producing enzymes. (C) SF-1 enhanced DHEA and androstenedione production, but not testosterone production, by hAmMSCs. (D) No difference was observed in the mRNA levels of androgen-producing factors when SF-1 and WT1−KTS were co-expressed compared with those when SF-1 was expressed alone. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05; N.D., not detectable. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; DHEA, dehydroepiandrosterone; SD, standard deviation; KTS, lysine, threonine, and serine.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay, Standard Deviation

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Effects of SF-1 and WT1 on estrogen-producing cell lineage. (A) WT1−KTS significantly increased the mRNA expression of ALDH1A2 , AMHR2, LHX9, GPX3 , and NR5A2 in hAmMSCs. (B) WT1−KTS, but not WT1+KTS or SF-1, significantly increased the mRNA expression of CYP19A1 in hAmMSCs. SF-1 increased the mRNA level of HSD17B1 . (C) Immunocytochemical analysis confirmed that WT1−KTS increased aromatase expression in hAmMSCs. (D) WT1−KTS and SF-1 did not increase estrogen production by hAmMSCs even in the presence of testosterone. (E) Co-expression of SF-1 and WT1−KTS also did not alter the mRNA levels of estrogen-producing enzymes in hAmMSCs. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; N.S., not significant. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; KTS, lysine, threonine, and serine.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Activation of CYP19A1 PII promoter by WT1–KTS. (A) Tissue-specific promoters of human CYP19A1. The mRNA expression of I.1 (placenta), I.f (brain), and PII (ovary) transcripts was observed in hAmMSCs. Transient expression of WT1−KTS significantly increased the mRNA level of the ovary-specific PII promoter in hAmMSCs. (B) Ovary-specific PII promoter of human CYP19A1. Luciferase assay revealed that WT1−KTS activated the CYP19A1 PII promoter. Data are presented as the mean ± SD from three independent experiments. *, p <0.05. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; KTS, lysine, threonine, and serine.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Activation Assay, Expressing, Luciferase, Membrane, Derivative Assay, Wilms Tumor Assay

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Schema of the hAmMSC differentiation and human steroid hormone biosynthetic pathways. (A) Schematic illustration of the differentiation of hAmMSCs into steroidogenic cells. (B) Schematic diagram of the human steroid hormone biosynthetic pathway. Steroidogenic enzymes shown in blue and red were upregulated by SF-1 and WT1–KTS induction, respectively. Steroid hormones in the boxes marked in blue were produced by SF-1 induction.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Produced