Fig 2: GLIS2 deficiency induces activation and MTD of HSCs by inactivating the PPAR-?–mediated lipid storage pathway. (A, B) qRT-PCR and Western blot detection of stellate cell status-related genes (P75NTR, GFAP) and TGF-ß1 receptor (TGFßR) mRNA and protein expression levels (n = 3, SD; *P < .05; **P < .01; ***P < .001). (C) The CCK-8 assay was used to monitor cell proliferation induced by GLIS2 knockout or TGF-ß1 induction (n = 3, SD; *P < .05; **P < .01; ***P < .001). (D) Transwell migration assay was used to detect cell migration induced by GLIS2 knockout or TGF-ß1 induction (n = 3, SD; *P < .05; **P < .01; ***P < .001). (E, F) qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of adipose-related genes (PLIN2 and ADIPOR1) (n = 3, SD; *P < .05; **P < .01; ***P < .001). (G) Oil red O staining was used to detect changes in lipid droplets in GLIS2-SG cells caused by GLIS2 knockout or TGF-ß1 induction. (H, I) qRT-PCR and Western blot were used to determine the mRNA and protein expression levels of PPAR-? targeted genes (FABP4, CD36, and SCD1) in GLIS2-SG cells induced by doxycycline (n = 3, SD; *P < .05; **P < .01; ***P < .001). (J) A dual luciferase assay (n = 3) was used to detect the interaction between GLIS2 and the PPAR-? promoter (n = 3). (K) A chromatin immunoprecipitation (IP) assay revealed an interaction between PPAR-? and the promoters of target genes (FABP4, CD36, and SCD1).

Fig 3: Aerobic exercise activated BDNF signaling in the ovary of DHEA-induced PCOS rats. Rats received DHEA for the induction of polycystic ovarian syndrome, together with or without exercise treatment. (A) mRNA expression of BDNF, TrkB, and p75NTR factors in ovarian tissue was analyzed by real-time PCR. (B,C) The expression of BDNF, TrkB, and p75NTR in ovarian tissue was assessed by Western blot assay. (D,E) The expression of PI3K, AKT, and p-AKT in ovarian tissue was assessed by Western blot assay. (F–I) The expression of p75NTR, NF-?B, p-NF-?B, JNK, and p-JNK in ovarian tissue was assessed by Western blot assay. n = 10 in each group. Data are shown as mean ± SEM. ** p < 0.05, vs. control group; ## p < 0.01, vs. PCOS group.

Fig 4: NT3P75-2 overexpression induces TrkC pathway activation while reducing the activation of P75NTR signal in PC12 cells. a–c Western blot analysis of the protein levels of TrkC, AKT, p-AKT, and ß-actin. Quantification of TrkC and p-AKT protein expression levels (**P < 0.01 by one-way ANOVA followed by Bonferroni’s multiple comparison test; n.s., no significance, n = 3). d–f Western blot analysis of the protein levels of P75NTR, JNK, p-JNK, and ß-actin. Quantification of P75NTR and p-JNK protein expression levels (##P < 0.01, *P < 0.05, ***P < 0.001 by one-way ANOVA followed by Bonferroni’s multiple comparison test, n = 3). g–i Western blot analysis of the protein levels of Bcl-2, Bax, and ß-actin. Quantification of Bcl-2 and Bax protein expression levels (##P < 0.01, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Bonferroni’s multiple comparison test; n.s., no significance, n = 3)

Fig 5: PPAR-? signal inactivation caused by GLIS2 deficiency can be partially reversed by reducing HDAC3. (A) Mapping of knockout vector containing both sgRNA and Cas9 proteins that target mouse HDAC3. (B) Genomic sequencing revealed that sgRNAs targeting HDAC3 can result in indels. (C, D) qRT-PCR and Western blot were used to confirm the knockout effect of HDAC3 (n = 3, SD; *P < .05; **P < .01; ***P < .001). (E) When HDAC3 was knocked out, the binding of HDAC3 and PPAR-? was detected using a co-IP assay in GLIS2-SG cells induced by doxycycline. (F) When HDAC3 was knocked out, the PPAR-? acetylation level was detected in GLIS2-SG cells induced by doxycycline. (G, H) Cell proliferation and migration abilities in GLIS2-SG cells induced by doxycycline were assessed using the CCK-8 assay and the Transwell migration assay when HDAC3 was knocked out. (I, J) When HDAC3 was knocked out, the mRNA and protein expression levels of HSC status–related genes (P75NTR, GFAP) and adipose-related genes (PLIN2 and ADIPOR1) were determined using qRT-PCR and Western blot (n = 3, SD; *P < .05; **P < .01; ***P < .001). (K) Oil red O staining was used to detect changes in lipid droplets in HDAC3 knockout.