Fig 2: Analysis of potential mechanism for KN93 retarding ovarian follicle development. (A) Interaction analysis of differential proteins in four key KEGG enriched pathways. Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps have direct interactions, which was marked through the pale green elliptic region. (B) Model of KN93 retarding follicular development. After CaMKII was inhibited by the inhibitor KN93, seven interacting proteins were upregulated (Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps), then downregulated oocyte marker DDX4 and granulosa cell marker FOXL2 in follicles, ultimately slowing down primordial follicles formation and activation, thereby leaded to the delay of the ovarian follicle development. Nsdhl, NADP-dependent steroid dehydrogenase-like; Lss, lanosterol synthase; Fdft1, farnesyl-diphosphate farnesyltransferase; Cyp51a1, cytochrome P450 family 51 family A member 1; Hmgcs1, hydroxymethylglutaryl-CoA synthase 1; Fasn, fatty acid synthase; Fdps, dimethylallyltranstransferase; CaMKII, calmodulin-dependent protein kinase II; DDX4, DEAD-box helicase 4; FOXL2, forkhead box L2.

Fig 3: Loss of POST1 decreases the expression of SREBP target genes. (A) Heat map showing gene expression profile of HeLa and HeLa/POST1 KO cells. Cells were cultured in the medium containing 10% fetal bovine serum (FBS), or the depletion medium (5% lipoprotein-deficient serum plus 10 µmol/L mevalonate and 1 µmol/L lovastatin), or the depletion medium supplemented with 25-hydroxycholesterol (25-HC) for 16 h. The mRNA expression levels were normalized to those of HeLa cells in FBS condition. Colors indicate the gene expression range with the least expression in blue and highest expression in red. (B) Quantitative real-time PCR analysis of HeLa and HeLa/POST1 KO cells under different culture conditions. Data were normalized to HeLa cells in FBS condition and presented as mean ± SD (n = 3 independent trials). (C) Immunoblot analysis of HeLa and HeLa/POST1 KO cells under different culture conditions. ACC1, acetyl-coA carboxylase 1; CYP51A1, cytochrome P450 family 51 subfamily A member 1; FASN, fatty acid synthase; FDFT1, farnesyl-diphosphate farnasyltransferase 1; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; INSIG, insulin-induced gene; LDLR, low-density lipoprotein receptor; LSS, lanosterol synthase; SCAP, SREBP-cleavage activating protein; SCD1, stearoyl-CoA desaturase 1; SQLE, squalene epoxidase

Fig 4: Proteomic techniques revealed differential proteins influenced by KN93. (A) Principal component analysis of the identified proteins in the KN93 treatment group and control group. Red and blue dots represent the KN93 treatment group and control group, respectively. (B) Volcano plot indicated the differential proteins between KN93 treatment group and control group. Red dots indicated significantly upregulated proteins with adjusted P-values <0.01, blue dots indicated significantly down-regulated proteins with adjusted P-values <0.01 and gray dots indicated non-significantly expressed proteins. (C) Differential proteins were verified by western blotting, which was then (D) quantified. GAPDH was used as a loading control. The data are presented as the mean ± SD (n=3). **P<0.01,***P<0.001. NS, no significant difference; CYP51A1, cytochrome P450 family 51 family A member 1; FADD, fas-associated death domain; NCAM1, neural cell adhesion molecule 1; Cyct, cytochrome c, testis; IGFBP3, insulin-like growth factor binding protein 3; PLS1, plastin-1; PDZK1, PDZ domain-containing 1; ZP2, zona pellicida sperm-binding protein; YBX2, Y-box binding protein 2; BNIP3L, Bcl-2-interacting protein 3-like.

Fig 5: Expression of the genes involved in P450 pathway. Relative mRNA levels of (A) CYP2A5 (n = 5), (B) CYP51 (n = 8), (C) CYP3A25 (n = 6), (D) CYP2C54 (n = 8), (E) CYP3A11 (n = 6), (F) CYP2C70 (n = 5)."p < 0.05 vs Normal, ***p < 0.001 vs Normal, #p < 0.05 vs Model, ##p < 0.01 vs Model, ###p < 0.001 vs Model.