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Expression analysis of <t>HIF1A</t> in granulosa cells. ( a ) indicates the mRNA expression of HIF1A under different FSH and IGF1 concentrations. ( b ) Indicates the detection of HIF1A protein under normoxia. The lane numbers 1–4 indicate the western runs of individual protein lysates. Columns 1 and 2 represent duplicates of HIF1A probing in FSH (20 ng/ml) and IGF1(50 ng/ml) treated GC under normoxia while columns 3 and 4 represent the Beta actin (BACT) probing in the corresponding samples. The arrow marks in ( b ) indicate the HIF1A (columns 1 and 2) and BACT (columns 3 and 4) protein bands. The image is obtained with the exposure time of 8 seconds in Simple Western instrument. ( c ) illustrates the immunolocalization of HIF1A in different growing ovarian follicles of abattoir ovaries. The brown color immunochemical signal indicates the HIF1A staining inside the follicle. 400x optical magnification was used for primary and secondary and large antral follicle’s wall. 100x optical magnification was used for tertiary follicles in the pictures. Inside dotted box indicates the specific follicle for primary, secondary and tertiary follicle sections whereas dotted box points the GC layer in large antral follicle’s wall. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3) for Fig. 1a. Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.
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Expression analysis of <t>HIF1A</t> in granulosa cells. ( a ) indicates the mRNA expression of HIF1A under different FSH and IGF1 concentrations. ( b ) Indicates the detection of HIF1A protein under normoxia. The lane numbers 1–4 indicate the western runs of individual protein lysates. Columns 1 and 2 represent duplicates of HIF1A probing in FSH (20 ng/ml) and IGF1(50 ng/ml) treated GC under normoxia while columns 3 and 4 represent the Beta actin (BACT) probing in the corresponding samples. The arrow marks in ( b ) indicate the HIF1A (columns 1 and 2) and BACT (columns 3 and 4) protein bands. The image is obtained with the exposure time of 8 seconds in Simple Western instrument. ( c ) illustrates the immunolocalization of HIF1A in different growing ovarian follicles of abattoir ovaries. The brown color immunochemical signal indicates the HIF1A staining inside the follicle. 400x optical magnification was used for primary and secondary and large antral follicle’s wall. 100x optical magnification was used for tertiary follicles in the pictures. Inside dotted box indicates the specific follicle for primary, secondary and tertiary follicle sections whereas dotted box points the GC layer in large antral follicle’s wall. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3) for Fig. 1a. Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.
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Expression analysis of <t>HIF1A</t> in granulosa cells. ( a ) indicates the mRNA expression of HIF1A under different FSH and IGF1 concentrations. ( b ) Indicates the detection of HIF1A protein under normoxia. The lane numbers 1–4 indicate the western runs of individual protein lysates. Columns 1 and 2 represent duplicates of HIF1A probing in FSH (20 ng/ml) and IGF1(50 ng/ml) treated GC under normoxia while columns 3 and 4 represent the Beta actin (BACT) probing in the corresponding samples. The arrow marks in ( b ) indicate the HIF1A (columns 1 and 2) and BACT (columns 3 and 4) protein bands. The image is obtained with the exposure time of 8 seconds in Simple Western instrument. ( c ) illustrates the immunolocalization of HIF1A in different growing ovarian follicles of abattoir ovaries. The brown color immunochemical signal indicates the HIF1A staining inside the follicle. 400x optical magnification was used for primary and secondary and large antral follicle’s wall. 100x optical magnification was used for tertiary follicles in the pictures. Inside dotted box indicates the specific follicle for primary, secondary and tertiary follicle sections whereas dotted box points the GC layer in large antral follicle’s wall. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3) for Fig. 1a. Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.
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Expression analysis of <t>HIF1A</t> in granulosa cells. ( a ) indicates the mRNA expression of HIF1A under different FSH and IGF1 concentrations. ( b ) Indicates the detection of HIF1A protein under normoxia. The lane numbers 1–4 indicate the western runs of individual protein lysates. Columns 1 and 2 represent duplicates of HIF1A probing in FSH (20 ng/ml) and IGF1(50 ng/ml) treated GC under normoxia while columns 3 and 4 represent the Beta actin (BACT) probing in the corresponding samples. The arrow marks in ( b ) indicate the HIF1A (columns 1 and 2) and BACT (columns 3 and 4) protein bands. The image is obtained with the exposure time of 8 seconds in Simple Western instrument. ( c ) illustrates the immunolocalization of HIF1A in different growing ovarian follicles of abattoir ovaries. The brown color immunochemical signal indicates the HIF1A staining inside the follicle. 400x optical magnification was used for primary and secondary and large antral follicle’s wall. 100x optical magnification was used for tertiary follicles in the pictures. Inside dotted box indicates the specific follicle for primary, secondary and tertiary follicle sections whereas dotted box points the GC layer in large antral follicle’s wall. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3) for Fig. 1a. Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.
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Expression analysis of HIF1A in granulosa cells. ( a ) indicates the mRNA expression of HIF1A under different FSH and IGF1 concentrations. ( b ) Indicates the detection of HIF1A protein under normoxia. The lane numbers 1–4 indicate the western runs of individual protein lysates. Columns 1 and 2 represent duplicates of HIF1A probing in FSH (20 ng/ml) and IGF1(50 ng/ml) treated GC under normoxia while columns 3 and 4 represent the Beta actin (BACT) probing in the corresponding samples. The arrow marks in ( b ) indicate the HIF1A (columns 1 and 2) and BACT (columns 3 and 4) protein bands. The image is obtained with the exposure time of 8 seconds in Simple Western instrument. ( c ) illustrates the immunolocalization of HIF1A in different growing ovarian follicles of abattoir ovaries. The brown color immunochemical signal indicates the HIF1A staining inside the follicle. 400x optical magnification was used for primary and secondary and large antral follicle’s wall. 100x optical magnification was used for tertiary follicles in the pictures. Inside dotted box indicates the specific follicle for primary, secondary and tertiary follicle sections whereas dotted box points the GC layer in large antral follicle’s wall. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3) for Fig. 1a. Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.

Journal: Scientific Reports

Article Title: HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

doi: 10.1038/s41598-020-60935-1

Figure Lengend Snippet: Expression analysis of HIF1A in granulosa cells. ( a ) indicates the mRNA expression of HIF1A under different FSH and IGF1 concentrations. ( b ) Indicates the detection of HIF1A protein under normoxia. The lane numbers 1–4 indicate the western runs of individual protein lysates. Columns 1 and 2 represent duplicates of HIF1A probing in FSH (20 ng/ml) and IGF1(50 ng/ml) treated GC under normoxia while columns 3 and 4 represent the Beta actin (BACT) probing in the corresponding samples. The arrow marks in ( b ) indicate the HIF1A (columns 1 and 2) and BACT (columns 3 and 4) protein bands. The image is obtained with the exposure time of 8 seconds in Simple Western instrument. ( c ) illustrates the immunolocalization of HIF1A in different growing ovarian follicles of abattoir ovaries. The brown color immunochemical signal indicates the HIF1A staining inside the follicle. 400x optical magnification was used for primary and secondary and large antral follicle’s wall. 100x optical magnification was used for tertiary follicles in the pictures. Inside dotted box indicates the specific follicle for primary, secondary and tertiary follicle sections whereas dotted box points the GC layer in large antral follicle’s wall. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3) for Fig. 1a. Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.

Article Snippet: Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

Techniques: Expressing, Western Blot, Simple Western, Staining, Cell Culture

Effects of echinomycin on candidate gene expression. Suppression of HIF1 activity using echinomycin altered the mRNA expression of HIF1A ( a ), VEGFA ( b ) and VNN2 ( c ) genes under normoxic and hypoxic conditions. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.

Journal: Scientific Reports

Article Title: HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

doi: 10.1038/s41598-020-60935-1

Figure Lengend Snippet: Effects of echinomycin on candidate gene expression. Suppression of HIF1 activity using echinomycin altered the mRNA expression of HIF1A ( a ), VEGFA ( b ) and VNN2 ( c ) genes under normoxic and hypoxic conditions. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.

Article Snippet: Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

Techniques: Gene Expression, Activity Assay, Expressing, Cell Culture

Effects of HIF1A knockdown on candidate gene expression. Figure depicts the mRNA expression values of HIF1A ( a ), VEGFA ( b ) and VNN2 ( c ) genes upon HIF1A knockdown under normoxic and hypoxic conditions. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Journal: Scientific Reports

Article Title: HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

doi: 10.1038/s41598-020-60935-1

Figure Lengend Snippet: Effects of HIF1A knockdown on candidate gene expression. Figure depicts the mRNA expression values of HIF1A ( a ), VEGFA ( b ) and VNN2 ( c ) genes upon HIF1A knockdown under normoxic and hypoxic conditions. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Article Snippet: Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

Techniques: Knockdown, Gene Expression, Expressing, Cell Culture

Effects of HIF1A knockdown on the expression of steroidogenic genes and steroidogenesis. Suppression of HIF1 expression by knockdown altered STAR ( a ), HSD3B ( b ) and CYP19A1 ( c ) transcript abundance. ( d , e ) show the estradiol and progesterone concentrations in the spent culture media. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Journal: Scientific Reports

Article Title: HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

doi: 10.1038/s41598-020-60935-1

Figure Lengend Snippet: Effects of HIF1A knockdown on the expression of steroidogenic genes and steroidogenesis. Suppression of HIF1 expression by knockdown altered STAR ( a ), HSD3B ( b ) and CYP19A1 ( c ) transcript abundance. ( d , e ) show the estradiol and progesterone concentrations in the spent culture media. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Article Snippet: Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

Techniques: Knockdown, Expressing, Cell Culture

Effects of HIF1A knockdown on cell proliferation. ( a , b ) depicts CCND2 and PCNA mRNA expressions upon HIF1A knockdown in granulosa cells under normoxic and hypoxic conditions. ( c , d ) depict the percent of replicating cells (S-phase) under normoxic and hypoxic conditions, respectively, upon treating with control and HIF1A gapmers. Data were represented in the mean ± SEM values of three independent culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Journal: Scientific Reports

Article Title: HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

doi: 10.1038/s41598-020-60935-1

Figure Lengend Snippet: Effects of HIF1A knockdown on cell proliferation. ( a , b ) depicts CCND2 and PCNA mRNA expressions upon HIF1A knockdown in granulosa cells under normoxic and hypoxic conditions. ( c , d ) depict the percent of replicating cells (S-phase) under normoxic and hypoxic conditions, respectively, upon treating with control and HIF1A gapmers. Data were represented in the mean ± SEM values of three independent culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Article Snippet: Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

Techniques: Knockdown, Control

HIF1 is essential for granulosa cell function. Expression of HIF1A is induced by FSH, IGF1 and low oxygen levels in bovine granulosa cells. HIF1 may regulate steroidogenesis, cell proliferation, angiogenesis and inflammation processes in developing ovarian follicles.

Journal: Scientific Reports

Article Title: HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

doi: 10.1038/s41598-020-60935-1

Figure Lengend Snippet: HIF1 is essential for granulosa cell function. Expression of HIF1A is induced by FSH, IGF1 and low oxygen levels in bovine granulosa cells. HIF1 may regulate steroidogenesis, cell proliferation, angiogenesis and inflammation processes in developing ovarian follicles.

Article Snippet: Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

Techniques: Cell Function Assay, Expressing