Additional file 1 of Mutated CYP17A1 promotes atherosclerosis and early-onset coronary artery disease
Creators: name: Wu, Ting-Ting
name: Zheng, Ying-Ying
name: Ma, Xiang
name: Xiu, Wen-Juan
name: Yang, Hai-Tao
name: Hou, Xian-Geng
name: Yang, Yi
name: Chen, You
name: Ma, Yi-Tong
name: Xie, Xiang
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Mental Health
subject: Virology
subject: Computational Biology

description: Additional file 1: Figure S1. Schematic of whole-exome sequencing data processing and variant identification. Figure S2. The c.-14G&gt;A variant in CYP17A1. (A) Schematic of identification CYP17A1 as a candidate gene for CAD susceptibility. (B) Pedigrees of the family 2 with c.-14G&gt;A mutation. CYP17A1 genotype (half filled for c.-14G&gt;A carriers, open for wild-type individuals) are shown below each square or circle. Figure S3. CYP17A1 over-expression involved in glycometabolism. (A) Relative mRNA levels of genes related to glycolytic pathway (screened by RNA-seq) in stably expressing C987X mutation or in HEK293T cells with over-expressed CYP17A1 WT or C987X.Vector as control. (B) CYP17A1 promotes the glucose uptake in HEK293T cells. HEK293T cells transfected with plasmids were labeled with intracellular level of 2-NBDG and subjected to measurement of flow cytometry. HEK293T cells cultured in a 6-well culture plate were transiently transfected with 3μg/well of plasmids encoding CYP17A1(WT) or CYP17A1(C987X). After 48h transfection, supernatants were harvested and subjected to the detection of glucose(C) or lactate(D) using assay kits. (E) After 48h transfection, cell lysates were prepared and subjected to western blot analysis. β-actin expression was used as a loading control. The effect of CYP17A1 and C987X mutation on key enzymes of cholesterol synthase in HEK293T and Huh7 cells were detected. (F) The influence of glucose and glutamine on CYP17A1-mediated cell proliferation. HEK293T cells with transient CYP17A1 WT or C987X expression were cultured in three different media: the complete medium with both glucose (Glu+) and glutamine (Gln+); the culture medium with glucose (Glu+) but without glutamine (Gln-); the culture medium without glucose (Glu-) but with glutamine (Gln+). After 24h incubation, cell proliferation in each culture condition was evaluated by CCK8 assay. (G)ATP level was measured by an assay kit. Data are presented as mean ± SD from at least three independent experiments. (H) The expression of CYP17A1 protein in different media, detection by WB. n=3 biological repetition. *, p &lt; 0.0.5; **, p &lt; 0.01; ***, p &lt; 0.005, ****, p &lt; 0.001.n.s., no significance. Figure S4. Identification of CYP17A1-binding proteins. (A)Schematic of the work flow of immunoprecipitation (COIP) followed by tandem mass spectrometry (MS/MS). (B) The top twenty candidate proteins that specifically identified in anti-CYP17A1 IP from HEK293T cell transfected with WT plasmids sample shown in (A). Figure S5. Identification of CYP17A1 functional paths. (A) Schematic of the work flow of combine analysis of CO-IP and RNA-seq. (B) Parts of candidate gene that specifically related to functional paths shown in (A). (C-F) Functional path clusters (the color represents the difference, the deeper the more significant) and interaction network (the color of different nodes represents different clusters, the connection line represents the genetic similarity between terms, and the size of nodes represents the number of genes enriched) in differences unique to wild-type (C and D) or to C987X truncation mutant (E and F). (G)Silver staining of IP. Specific protein (about 35 kD) between CYP17A1 and CYP17A1ΔECD were pointed with an arrow. Figure S6. Diagram of CYP17A1-/-mice. Upper part: (A) Representative photos of of appearance(A) in chow fed CYP17A1-/- heterozygote, homozygous and WT mice at 12 mo of age and relative diagram of the anatomy (B), 9 month of HFD appearance(C) of WT, CYP17A1-/- and CYP17A1-/-ApoE-/- mice. Lower part: Abdominal, epicardial and liver necropsy photos of representative 12-mo-old CYP17A1-/- heterozygote, homozygous and WT mice, showing increased adiposity in CYP17A1-/- vs. WT mice. Livers of CYP17A1-/- mice tend to display increased fat accumulation displaying yellow coloration compared with WT (F vs. C). Figure S7. Characterization of CYP17A1 whole-body knockout mice. (A)WT, CYP17A1-/- mice (n=8) were fed on chow diet or HCD (0.25% cholesterol). Weights were measured at 3,6,9,12 month. Data is expressed as mean ± SD. Statistical analyses, two-way ANOVA. (B)Tissues taken from 12-mo-old WT and CYP17A1-/- mice were immediately weighted by micro balance. Organ coefficient was calculated. Biochemical detector to detect serum AST(C) and ALT(D). (E)Blood pressures were measured indirectly by tail arteries in awake mice. Data are expressed as median with SD. Nonparametric test. #P&lt;0.05, ##P&lt;0.01, ###P&lt;0.001, ####P&lt;0.0001, compared with WT mice fed with HCD diet. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001, ****P&lt;0.0001, compared with WT mice fed with chow diet. ns, not statistically significant compared with WT mice fed with chow diet. NS, not statistically significant compared with WT mice fed with HCD. Figure S8. Adipose and liver histology in CYP17A1-/- mice. Upper part: Hematoxylin and eosin (H&amp;E) staining of gonadal adipose sections from WT (A), CYP17A1-/-(B) mice at 12 mo of age. Lower part: H&amp;E staining of liver sections. H&amp;E staining of liver sections from chow fed female CYP17A1-/- mice at 3, 6,9 and 12 mo-old (A-D). WT female mice at 3, 6, 9and 12 mo(E-H), respectively. All histology images are X40 magnification; scale bar in bottom right =50μm. Figure S9. Metabolic characteristics of CYP17A1 whole-body knockout mice. WT and CYP17A1-/- mice at 6 or 12mo-old (n=6, each month/each genotype) with chow diet, WT and CYP17A1-/- mice a t6 or 9 mo-old (n=6, each month/each genotype with HCD. Plasma DHEA, DHEAS, plasma Estrogen, plasma aldosterone, plasma cortisol, plasma insulin, plasma resistin and plasma 17-hydroxyprogesterone were measured. Data are expressed as mean ± SD. Statistical analyses, two-way ANOVA. #P&lt;0.05, ##P&lt;0.01, ###P&lt;0.001, ####P&lt;0.0001, compared with WT mice fed with HCD diet. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001, ****P&lt;0.0001, compared with WT mice fed with chow diet.