Fig 1: Src-homology-2-containing phosphotyrosine phosphatase (SHP2) knockdown reduced estrogen receptor alpha (ERa) transcription of the NPPC and NPR2 genes and aromatase activity of granulosa cells. (A) SHP2 siRNA was applied to COV434 cell line, and SHP2 protein expression was analyzed via western blotting. (B) To check the localization of ERa, an immunofluorescence experiment was performed using SHP2-knockdown COV434 cells. The results suggest that ERa nuclear localization was significantly reduced with SHP2 knockdown. (C) NPPC/NPR2 mRNA expressions were analyzed in follicle-stimulating hormone- and E2-stimulated SHP2-knockdown COV434 cell line. (D) To examine the effect of SHP2 knockdown on the functional activity of granulosa cells, the SF1, Wnt4, and p-mTOR proteins were analyzed via western blotting, and a significant reduction in all proteins was observed with SHP2 knockdown. ß-Actin was used as the loading control for western blotting. Bands were quantified using ImageJ software, and the differences are represented by histograms. The experiments were repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 2: In vitro, the CRISPR/dCas9 system leads to rapid epigenetic remodelling at target loci. A, C, E, Schematic representation of the target genomic loci. The sgRNA target sites upstream of the transcription start site (red bars) and the ChIP-qPCR regions are indicated (black bars). B, D, F, Activation of the endogenous targets Nr5a1, Gata4 and Dmrt1 in human foreskin fibroblasts resulted in significant enrichment of H3K4me3 and H3K27ac at multiple loci at day 4 after infection (**P < 0.01, ***P < 0.001 n = 3 biological replicates). All fold enrichments are relative to IgG (negative control) and normalized to GAPDH
Fig 3: The single guide RNA (sgRNA) mediated CRISPR/dCas9 synergistic activation mediator (SAM) system for target gene activation. A, Schematic diagram of target gene activation, indicating that the SAM complex, consisting of three components, binds to the endogenous target gene promoter and transcriptional activation occurs. B, Three sgRNAs targeted each of the endogenous human Nr5a1, Gata4 and Dmrt1 gene promoters. TSS, transcription start site. C, Schematic representation of lenti-dCas9-VP64 plasmids expressing dCas9-effector fusion proteins. D, Schematic representation of lenti-MS2-P65-HSF1 (MPH) plasmids expressing the transcriptional activation complex. E, The lenti-3ßHSD-EGFP plasmid reporter system in which 3ßHSD gene promoter fragments were cloned upstream of EGFP. F, Schematic representation of the Lenti sgRNA(MS2)-Puro vector. For the cloning of these designed sgRNAs, the insertion site of the guidance sequence lies downstream of the U6 promoter
Fig 4: Reduced Menin in the hypothalamus accelerates systemic aging. (A–C) Menin protein expression and mRNA levels in the hypothalamus of young (3M) and old (20M) mice; n = 4 mice. (D) Menin protein expression levels in 7 regions of young (3M) and old (20M) mice brain; n = 4 mice. Actin serves as a loading control. (E–G) Inflammatory factors protein expression and mRNA levels in the hypothalamus of young (3M) and old (20M) mice; n = 3 mice. (H) Generation of conditional Men1-knockout mice by crossing Men1f/f mice with SF1-Cre mice (ScKO). (I–K) The protein and mRNA levels of Menin in the hypothalamus of 6 months ScKO and control mice; n = 3 mice. (L) Representative hypothalamus brain sections from 6 months control and ScKO mice stained with Menin (green) and SF1 (red) antibody. Section was counterstained with DAPI (blue). Scale bar, 100 µm, 20 µm; n = 5 mice. (M–O) Inflammatory factors protein expression and mRNA levels in the hypothalamus of ScKO (6M) and control (6M) mice; n = 3 mice. (P) Schematic diagram of open circuit indirect calorimetry. (Q) Day and night respiratory quotients (RER) of age-matched male ScKO mice (10M) and control mice (10M) were measured. Exact dietary (R), water intake (S), and activity (T) were also measured. (U) Lifespan of these mice (Control mice, n = 14; ScKO mice, n = 16). (V–Z) Male mice were sacrificed at 10 months for measuring muscle (quadriceps) fiber size (V), bone mass (2), dermal thickness (X, Y), and tail tendon breaking time (Z); n = 3~9 mice. Scale bar, 20 µm. (AA–AC) Ten months male ScKO and age-matched control mice behavior in Morris water maze tests. (AD) Ten months male ScKO and control mice behavior in T maze. (AE) Ten months male ScKO and control mice behavior in Y maze. (AF, AG) Electrophysiological recording from ScKO and control mice. Representative whole-cell recordings on SF-1 neurons in the hypothalamus of ScKO and control mice are shown on panel AF. Quantitation of their mEPSC and mIPSC frequency and amplitude are showed in panel AG (n = 20 cells from 3 mice). Mouse number used in measuring energy expenditure by open circuit indirect calorimetry: ScKO mice: n = 16 mice; Control mice: n = 16 mice. Mouse number used in behavior tests: Control: n = 24 mice, ScKO mice: n = 17 mice. Data represent mean ± SEM, n.s.: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, Kaplan–Meier survival estimate for survival curve. Unpaired t test for behavioral statistics. Statistical applications between groups across multiple time points were analyzed by repeated-measures ANOVA. Other statistical applications were analyzed by one-way ANOVA with Tukey’s post hoc analysis. The underlying data of Fig 1 can be found in S1 Information. mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; SF-1, steroidogenic factor-1.
Fig 5: miR-92a-3p regulated TCF21 and SF-1 expression in ESCs. (a) The predicted miR-92a-3p binding sites of the TCF21 3’UTR. (b and c). ESCs were transfected with the indicated mimic or inhibitor for 48 h and harvested for real-time RT-PCR (n = 3; ***, P < 0.001, **, P < 0.01, *, P < 0.05; t-test). (d) ESCs were transfected with the indicated mimic or inhibitor for 48 h. Cells were harvested for western blotting performed with the indicated antibodies (n = 3)
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