Journal: Journal of Advanced Research

Article Title: A novel effect of sulforaphane on promoting mouse granulosa cells proliferation via the NRF2–TKT pathway

doi: 10.1016/j.jare.2024.09.020

Figure Lengend Snippet: SFN regulates TKT through NRF2-targeted modulation. (A) After 12 hourstreatment, GCs were subjected to assess the expression of Nrf2 , Tkt and Nqo1 in the Control, SFN and SFN+si Nrf2 groups by qPCR. (B) Conducting CUT&Tag sequencing, peak analysis was performed using IGV software, with the Control group in blue and the SFN group in red. (C, D) After Nrf2 knockdown or overexpression, western blot analysis was conducted to evaluate the protein levels of NRF2, NQO1, and TKT. ACTB was used for normalization, and the quantitative grayscale analysis is presented on the right. (E) R5P level detection was employed to evaluate the activity of PPP in all groups. (F, G) Targeted metabolomics analysis was conducted in GCs to examine changes in amino acid and nucleotide levels after 24 h of treatment with either SFN alone or SFN combined with si Nrf2 . Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***p value < 0.001 by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used: ACTB (AC026, Abclonal), PCNA (13110, Cell Signaling Technology), CDK2 (2546, Cell Signaling Technology), cyclin E1 (A14225, Abclonal), G6PD (12263, Cell Signaling Technology), TKT (11039–1-AP, Proteintech), NRF2 (12721, Cell Signaling Technology), Histone 3 (4499, Cell Signaling Technology), TUBA1A (2125, Cell Signaling Technology), and NQO1 (A19586, Abclonal).

Techniques: Expressing, Control, Sequencing, Software, Knockdown, Over Expression, Western Blot, Activity Assay, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: A novel effect of sulforaphane on promoting mouse granulosa cells proliferation via the NRF2–TKT pathway

doi: 10.1016/j.jare.2024.09.020

Figure Lengend Snippet: SFN stimulates GCs proliferation via the NRF2-TKT pathway. (A, B) After a 24-hour treatment with either SFN or a combination of SFN and siNrf2, changes in the cell cycle of GCs were detected using flow cytometry. The data were quantified and analyzed in (B) . (C, D) After treatment of GCs with SFN or DMSO for 12 h, CDK2, cyclin E1 and PCNA protein levels in GCs were detected by western blot, and the quantitative grayscale results are presented on the right. (D) Cell proliferation viability was assessed using the CCK-8 assay after knockdown or overexpression of Nrf2 in GCs, treated with SFN or DMSO for 24 h. (E, F) EdU labeling for 6 h in GCs, followed by fluorescent staining with red indicating EdU and blue representing DAPI, assessed GCs proliferation. (F) displays the proportion of EdU-positive cells. Scale bar: 50 μm. (G) Cell proliferation was detected using the CCK-8 assay in GCs treated with SFN or ML385 for 24 h. (C, D) Proliferation-related protein levels were detected by western blot after a 12-hour treatment of GCs with different treatments, and the quantitative grayscale analysis is presented on the right. (I) Western blot analysis assessed protein levels after a 12-hour treatment of GCs in the empty vector control, OE- Nrf2 , and OE- Nrf2 + si Tkt groups, quantitative grayscale analysis was performed to assess the relative protein levels. (J) The CCK-8 assay detected the cell proliferation activity of Control, OE- Nrf2, and OE- Nrf2 + si Tkt groups after 24 h of GCs treatment. Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***p value < 0.001 by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used: ACTB (AC026, Abclonal), PCNA (13110, Cell Signaling Technology), CDK2 (2546, Cell Signaling Technology), cyclin E1 (A14225, Abclonal), G6PD (12263, Cell Signaling Technology), TKT (11039–1-AP, Proteintech), NRF2 (12721, Cell Signaling Technology), Histone 3 (4499, Cell Signaling Technology), TUBA1A (2125, Cell Signaling Technology), and NQO1 (A19586, Abclonal).

Techniques: Flow Cytometry, Western Blot, CCK-8 Assay, Knockdown, Over Expression, Labeling, Staining, Plasmid Preparation, Control, Activity Assay, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: A novel effect of sulforaphane on promoting mouse granulosa cells proliferation via the NRF2–TKT pathway

doi: 10.1016/j.jare.2024.09.020

Figure Lengend Snippet: In vivo illustrating of the NRF2-TKT pathway in the regulation of GCs proliferation by SFN. (A) Relative protein levels of GCs in each groups were detected by Western blot, and quantitative grayscale values are provided below the blot. (B, C) Following EdU labeling of ovarian follicles, red fluorescence staining was used to evaluate GCs proliferation. (C) represents the analysis of the proportion of EdU-positive cells. Scale bar: 50 μm. (D, E) Immunohistochemical staining results for PCNA in mouse ovarian tissue, with corresponding statistical analysis of the proportion of PCNA-positive cells on the right (E) . Scale bar: 100 μm. (F, G) Immunohistochemical staining outcomes for PCNA in mouse ovarian tissue, accompanied by statistical analysis depicting the proportion of PCNA-positive cells (G) . Scale bar: 100 μm. (H) Relative protein levels of GCs in each groups were detected by western blot, and the quantitative grayscale results are presented on the right. Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***p value < 0.001 by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used: ACTB (AC026, Abclonal), PCNA (13110, Cell Signaling Technology), CDK2 (2546, Cell Signaling Technology), cyclin E1 (A14225, Abclonal), G6PD (12263, Cell Signaling Technology), TKT (11039–1-AP, Proteintech), NRF2 (12721, Cell Signaling Technology), Histone 3 (4499, Cell Signaling Technology), TUBA1A (2125, Cell Signaling Technology), and NQO1 (A19586, Abclonal).

Techniques: In Vivo, Western Blot, Labeling, Fluorescence, Staining, Immunohistochemical staining, Two Tailed Test