Additional file 1 of Hepatocyte-specific Sox9 knockout ameliorates acute liver injury by suppressing SHP signaling and improving mitochondrial function
Creators: name: Qin, Dan
name: Wang, Rui
name: Ji, Jinwei
name: Wang, Duo
name: Lu, Yuanyuan
name: Cao, Shiyao
name: Chen, Yaqing
name: Wang, Liqiang
name: Chen, Xiangmei
name: Zhang, Lisheng
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1. Identification of tamoxifen-induced hepatocyte-specific Sox9 deficient mice. (A) Genotyping by sequencing protocol. (B) Plasma AST and ALT levels were measured. N = 5 for Sox9 f/f and N = 9 for H-Sox9 KO group. Figure S2. Sox9 is overexpressed in IRI-induced mouse livers. Wild-type mice were subjected to 1 h of hepatic ischemia or sham surgery, liver samples were collected at 6 h post reperfusion. (A) Sox9 mRNA level was measured (N = 4 per group). (B) Immunofluorescence co-staining for Sox9 and HNF4α. PV, periportal vein. Scale bar, 100 µm. Figure S3. Hepatocyte-specific Sox9 knockout ameliorates PHx-induced acute liver injury. Sox9 f/f and Alb-CreERT2/+; Sox9 f/f mice pre-treated with tamoxifen were subjected to PHx, 48 h later, plasma and liver samples were collected for analysis. (A) Plasma ALT level was measured in Sox9 f/f and H-Sox9 KO mice. N = 4. (B) TNFα and IL-6 mRNA expression were determined by qRT-PCR in Sox9 f/f and H-Sox9 KO mice. N = 4. (C) Representative liver sections stained with H&amp;E. Scale bar, 100 µm. (D) Representative BrdU images in livers of Sox9 f/f and H-Sox9 KO mice induced by PHx. Scale bar, 100 µm. (E) Quantification of the percentage of BrdU+ cells in liver tissues. Figure S4. Hepatocyte-specific Sox9 knockout ameliorates CCl4-induced acute liver injury. Sox9 f/f and Alb-CreERT2/+; Sox9 f/f mice pre-treated with tamoxifen were subjected to CCl4, 36 h later, plasma and liver samples were collected for analysis. (A) Plasma ALT level was measured in Sox9 f/f and H-Sox9 KO mice. N = 4–5. (B) TNFα, IL-6 and IL-1β mRNA expression were determined by qRT-PCR in Sox9 f/f mice and H-Sox9 KO mice. N = 4–5. (C) Representative liver sections stained with H&amp;E. Scale bar, 100 µm. (D) Representative BrdU images in livers of Sox9 f/f and H-Sox9 KO mice induced by CCl4. Scale bar, 100 µm. (E) Quantification of the percentage of BrdU+ cells in liver tissues. Figure S5. Loss of Sox9 in hepatocytes decreases SHP expression, Sox9 overexpression increases SHP expression. (A) Representative confocal images of SHP (Red) and DAPI (blue) staining in livers. Scale bar, 50 μm. (B) Hepatic expression levels of Sox9 and SHP in Sox9 f/f and H-Sox9 KO mice were determined by qRT-PCR analysis following PHx injury. 36B4 was used as a housekeeping gene. N = 4 per group. (C) Hepatic expression levels of Sox9 and SHP in Sox9 f/f and H-Sox9 KO mice were determined by qRT-PCR analysis following CCl4 injury. 36B4 was used as a housekeeping gene. N = 4–5 per group. (D) Expression levels of SOX9 and SHP in AML12 cells were determined by qRT-PCR analysis following SOX9 overexpression (SOX9 OE) or not. GAPDH was used as an internal control. (E) Expression levels of Sox9 and SHP in Hepa1-6 cells were determined by qRT-PCR analysis following Sox9 overexpression or not. 36B4 was used as an internal control. (F) Expression levels of SOX9 and SHP in Huh-7 cells were determined by qRT-PCR analysis following SOX9 overexpression or not. GAPDH was used as an internal control. Figure S6. Overexpression of SOX9 induces cell death in Huh-7 cells treated with indicated concentration of H2O2. (A) Quantitative detection of Red Mito Tracker in Huh-7 cells (Corresponding to Fig. 6C). (B) Quantification of Calcein AM/PI staining in the control group or SOX9 OE group treated with indicated concentration of H2O2. Calcein AM/PI double staining of Huh-7 cells. Compared with the control group, SOX9 OE group resulted in increased cell death. Scale bar, 100 µm. Figure S7. Knocking-down of SHP reduces cell damage by protecting mitochondrial function. Huh-7 cells were transfected with either SOX9 overexpression plasmid (SOX9 OE), vector plasmid (Vector) or both; the latter condition involved co-transfection with siRNA targeting SHP followed by treatment with 400 μM H2O2 for 6 h before the harvest process. (A) Expression level of SOX9 in Huh-7 cells was determined by qRT-PCR analysis. Student’s two-tailed t t-test (unpaired) was used to determine statistical significance differences between groups. Statistical significance was presented at the level of *P &lt; 0.05. (B) Expression level of SHP in Huh-7 cells were determined by qRT-PCR analysis. ANOVA was used to determine statistical significance differences between groups. *p &lt; 0.05 vs. Vehicle + Control siRNA; #p &lt; 0.001 vs. Vehicle + SHP siRNA; + p &lt; 0.001 vs. Sox9 OE + Control siRNA. (C) Ca2+ indicator X-Rhod-1 in control and SOX9 OE Huh-7 cells treated with H2O2. Scale bar, 50 µm. (D) Mito Tracker Red staining of control and SOX9 OE Huh-7 cells treated with H2O2. Scale bar, 100 µm. Figure S8. Knocking-down of SHP mitigates cell death induced by SOX9 overexpression in the presence of H2O2. Huh-7 cells were transfected with either SOX9 overexpression plasmid (SOX9 OE), vector plasmid (Vector) or both; the latter condition involved co-transfection with siRNA targeting SHP followed by treatment with 400 μM H2O2 for 6 h before the harvest process. Representative images displaying TUNEL staining in Huh-7 cells from the indicated experimental groups are presented with a scale bar of 200 µm. Table S1. The primer sequences for genotyping. Table S2. The primer sequences for qRT-PCR and ChIP.