Journal: Journal of Advanced Research
Article Title: Core fucosylation regulates the ovarian response via FSH receptor during follicular development
doi: 10.1016/j.jare.2024.01.025
Figure Lengend Snippet: FUT8 knockdown attenuates the FSH/FSHR signaling pathway. A, CCK8 cell proliferation assay. Cells were treated with 100 ng/mL rhFSH, and cell proliferation was measured by absorbance at a wavelength of 450 nm. Significant differences are shown as the means ± SD, n = 4. B, Immunofluorescence assay. KGN, KGN-KD and KGN-KD-Re cells were treated with FSH (400 ng/mL) and stained with anti-FSHR (red) (1:500) and DAPI (blue). C, Western blot. Cell lysates of KGN, KGN-KD and KGN-KD-Re cells were resolved by 7.5 % SDS-PAGE, transferred to a PVDF membrane and probed with anti-FSHR (1:2000). GAPDH was used as the reference gene for the cytoplasm. D, Western blot. Cell lysates of Fut8 +/+ and Fut8 −/− ovaries were resolved by 7.5 % SDS-PAGE, transferred to a PVDF membrane and probed with anti-FSHR (1:2000). GAPDH was used as a loading control. E, Quantitative real-time PCR. RNA was isolated from the granulosa cells of MOR and POR patients. The mRNA expression levels of FSHR were compared. The relative expression of mRNAs was normalized to GAPDH. Data are presented as the means ± SEM, n = 3. F, FSH-induced cAMP production was measured using a GloSensor cAMP Assay kit. Cells were seeded in a 6-well plate for 24 h, and then 100 μM of IBMX was added, to inhibit phosphodiesterases, for 20 min. The cAMP levels were measured in KGN, KGN-KD, and KGN-KD-Re cells following stimulation with 100 IU/L of rhFSH or 100 μM of forskolin for 5 min. Data are presented as the means ± SEM, n = 3. G, PKA-CREB signaling pathway and Akt-FoxO3a signaling pathway. Serum-starved KGN, KGN-KD, and KGN-KD-Re cells were treated with FSH for 30 min, and cell lysates were resolved by 10 % SDS-PAGE, followed by transfer to a PVDF membrane. Membranes were probed with anti-phospho-PKA (1:2000) and anti-PKA (1:2000); anti-phospho-CREB (1:2000) and anti-CREB (1:2000); and anti-KITL (1:2000); anti-phospho-Akt (1:2000) and anti-Akt (1:2000); anti-phospho-FoxO3a (1:2000) and anti-FoxO3a (1:2000), and GAPDH was used as a loading control. Quantitative data are represented as the means ± SD, n = 3. H, Western blot. Lysates of Fut8 +/+ and Fut8 −/− ovaries were resolved by 10 % SDS-PAGE, and probed with anti-KITL (1:2000), with GAPDH used as the loading control. Quantitative data are represented as the means ± SD, n = 3. I, Western blot. Lysates of Fut8 +/+ and Fut8 −/− ovaries were resolved by 10 % SDS-PAGE and probed with anti-pFoxO3a (1:2000) and anti-FoxO3a (1:2000). Quantitative data are represented as the means ± SD, n = 3. J, TUNEL assay in the Fut8 +/+ and Fut8 −/− ovarian follicles. Representative micrographs are shown. Quantitative data are represented as the means ± SD, n = 3. K, Western blot analysis. Cell lysates of Fut8 +/+ and Fut8 −/− ovaries were resolved by 7.5 % SDS-PAGE, transferred to a PVDF membrane, and probed with anti-BCL2 and anti-BAX (1:2000). Quantitative data are represented as the means ± SD, n = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Article Snippet: FSH-induced cAMP production was measured using a GloSensor cAMP Assay kit (Promega).
Techniques: Knockdown, Proliferation Assay, Immunofluorescence, Staining, Western Blot, SDS Page, Membrane, Control, Real-time Polymerase Chain Reaction, Isolation, Expressing, cAMP Assay, TUNEL Assay
