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7-DHC supplementation delays oxidative aging of granulosa cells induced by Vdr deficiency Mice and KGN cells were treated with 7-DHC. (A) KGN cell extracts were conducted western blots <t>showing</t> <t>Nrf2,</t> SOD2, HO-1, p53, and p21 protein levels. GAPDH was considered as the loading control. Densitometric analysis was used to assess protein expression relative to GAPDH. (B) CCK8 experiment determines the effect of 7-DHC on cell proliferation. (C) Il-6, p53, and p21 mRNA levels in ovary tissue by real-time RT-PCR, calculated as relative to β-actin mRNA. (D) The ROS level was measured using flow cytometry and mean fluorescence intensity (MFI) showed positive areas for ROS. (E) mRNA levels of Amh, Amhr2, Hsd17b1, and Cyp19a1 in VDR KO KGN cells were analyzed using real-time RT-PCR, calculated as relative to β-actin mRNA. (F) Representative microscopic images of immunofluorescence showed ovary staining for <t>PCNA</t> and p21. Statistical analysis of PCNA- and p21-positive cell ratio. There were three biological replicates in each experiment. Results are expressed as the means ± SEM of six determinations per group. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with VDR KO KGN cells or Vdr −/− , unpaired Student’s t-test.
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7-DHC supplementation delays oxidative aging of granulosa cells induced by Vdr deficiency Mice and KGN cells were treated with 7-DHC. (A) KGN cell extracts were conducted western blots <t>showing</t> <t>Nrf2,</t> SOD2, HO-1, p53, and p21 protein levels. GAPDH was considered as the loading control. Densitometric analysis was used to assess protein expression relative to GAPDH. (B) CCK8 experiment determines the effect of 7-DHC on cell proliferation. (C) Il-6, p53, and p21 mRNA levels in ovary tissue by real-time RT-PCR, calculated as relative to β-actin mRNA. (D) The ROS level was measured using flow cytometry and mean fluorescence intensity (MFI) showed positive areas for ROS. (E) mRNA levels of Amh, Amhr2, Hsd17b1, and Cyp19a1 in VDR KO KGN cells were analyzed using real-time RT-PCR, calculated as relative to β-actin mRNA. (F) Representative microscopic images of immunofluorescence showed ovary staining for <t>PCNA</t> and p21. Statistical analysis of PCNA- and p21-positive cell ratio. There were three biological replicates in each experiment. Results are expressed as the means ± SEM of six determinations per group. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with VDR KO KGN cells or Vdr −/− , unpaired Student’s t-test.
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7-DHC supplementation delays oxidative aging of granulosa cells induced by Vdr deficiency Mice and KGN cells were treated with 7-DHC. (A) KGN cell extracts were conducted western blots <t>showing</t> <t>Nrf2,</t> SOD2, HO-1, p53, and p21 protein levels. GAPDH was considered as the loading control. Densitometric analysis was used to assess protein expression relative to GAPDH. (B) CCK8 experiment determines the effect of 7-DHC on cell proliferation. (C) Il-6, p53, and p21 mRNA levels in ovary tissue by real-time RT-PCR, calculated as relative to β-actin mRNA. (D) The ROS level was measured using flow cytometry and mean fluorescence intensity (MFI) showed positive areas for ROS. (E) mRNA levels of Amh, Amhr2, Hsd17b1, and Cyp19a1 in VDR KO KGN cells were analyzed using real-time RT-PCR, calculated as relative to β-actin mRNA. (F) Representative microscopic images of immunofluorescence showed ovary staining for <t>PCNA</t> and p21. Statistical analysis of PCNA- and p21-positive cell ratio. There were three biological replicates in each experiment. Results are expressed as the means ± SEM of six determinations per group. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with VDR KO KGN cells or Vdr −/− , unpaired Student’s t-test.
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primary antibody against pcna - by Bioz Stars, 2026-02
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7-DHC supplementation delays oxidative aging of granulosa cells induced by Vdr deficiency Mice and KGN cells were treated with 7-DHC. (A) KGN cell extracts were conducted western blots showing Nrf2, SOD2, HO-1, p53, and p21 protein levels. GAPDH was considered as the loading control. Densitometric analysis was used to assess protein expression relative to GAPDH. (B) CCK8 experiment determines the effect of 7-DHC on cell proliferation. (C) Il-6, p53, and p21 mRNA levels in ovary tissue by real-time RT-PCR, calculated as relative to β-actin mRNA. (D) The ROS level was measured using flow cytometry and mean fluorescence intensity (MFI) showed positive areas for ROS. (E) mRNA levels of Amh, Amhr2, Hsd17b1, and Cyp19a1 in VDR KO KGN cells were analyzed using real-time RT-PCR, calculated as relative to β-actin mRNA. (F) Representative microscopic images of immunofluorescence showed ovary staining for PCNA and p21. Statistical analysis of PCNA- and p21-positive cell ratio. There were three biological replicates in each experiment. Results are expressed as the means ± SEM of six determinations per group. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with VDR KO KGN cells or Vdr −/− , unpaired Student’s t-test.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of vitamin D receptor induces premature ovarian insufficiency through compromising the 7-dehydrocholesterol-dependent anti-aging effects

doi: 10.3389/fcell.2025.1545167

Figure Lengend Snippet: 7-DHC supplementation delays oxidative aging of granulosa cells induced by Vdr deficiency Mice and KGN cells were treated with 7-DHC. (A) KGN cell extracts were conducted western blots showing Nrf2, SOD2, HO-1, p53, and p21 protein levels. GAPDH was considered as the loading control. Densitometric analysis was used to assess protein expression relative to GAPDH. (B) CCK8 experiment determines the effect of 7-DHC on cell proliferation. (C) Il-6, p53, and p21 mRNA levels in ovary tissue by real-time RT-PCR, calculated as relative to β-actin mRNA. (D) The ROS level was measured using flow cytometry and mean fluorescence intensity (MFI) showed positive areas for ROS. (E) mRNA levels of Amh, Amhr2, Hsd17b1, and Cyp19a1 in VDR KO KGN cells were analyzed using real-time RT-PCR, calculated as relative to β-actin mRNA. (F) Representative microscopic images of immunofluorescence showed ovary staining for PCNA and p21. Statistical analysis of PCNA- and p21-positive cell ratio. There were three biological replicates in each experiment. Results are expressed as the means ± SEM of six determinations per group. *P < 0.05, **P < 0.01, ***P < 0.001. Compared with VDR KO KGN cells or Vdr −/− , unpaired Student’s t-test.

Article Snippet: Primary antibodies against PCNA (10205-2-AP, Proteintech Biotechnology Inc., China), Nrf2 (#12721, Cell Signaling Technology, United States), p21 (#2946, Cell Signaling Technology, United States), γ-H2A.X (AF1201, Beyotime Biotechnology, Shanghai, China) were used.

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Immunofluorescence, Staining