Additional file 2 of Hepatocyte SGK1 activated by hepatic ischemia-reperfusion promotes the recurrence of liver metastasis via IL-6/STAT3
Creators: name: Li, Xiangdong
name: Wang, Ziyi
name: Jiao, Chenyu
name: Zhang, Yu
name: Xia, Nan
name: Yu, Wenjie
name: Chen, Xuejiao
name: Wikana, Likalamu Pascalia
name: Liu, Yue
name: Sun, Linfeng
name: Chen, Minhao
name: Xiao, Yuhao
name: Shi, Yuhua
name: Han, Sheng
name: Pu, Liyong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 2: Figure S1. Gal-siSGK1 reverses the activation of inflammatory factors in liver after IR. Livers of wild type mice were first subjected to 70% warm ischemia, followed by reperfusion for 6h. (A) qRT-PCR analysis of IL-1β, TNF-α, INOS, CXCL2, CXCL10 and IL-10 in ischemic livers (n=6samples/group). (B) Serum IL-1β level was detected by Elisa (n=4samples/group). (C) qRT-PCR analysis of CXCR2, CXCR4 in ischemic livers (n=6samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S2. Gal-siSGK1 alleviates apoptosis in vitro cell lines after H/R. (A) Using Gal-siSGK1, and then the apoptosis of AML cell line after H/R were detected by flow cytometry (n=3samples/group). (B) The apoptosis of LO2 cell line after H/R were detected by flow cytometry (n=3samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S3. STAT3 inhibitor alleviates CRLM after IR. Before constructing the mouse IR model, STAT3 inhibitor was injected intraperitoneally. (A) The efficiency of STAT3 knockout was detected by WB (n=3samples/group). (B) Detection of mouse liver function by examining serum ALT and AST (n=4samples/group). (C) HE staining to detect liver tissue damage (n=3samples/group). Scale bars, 50μm. (D) Tunel staining to detect hepatocyte apoptosis (n=4samples/group). Scale bars, 50μm. (E) Quantification of Tunel staining (n=4samples/group). Gal-siSGK1 and GSK-650394 were used to knock down mouse SGK1, respectively, and then a mouse CRLM+IR model was constructed. (F) Representative images of tumor (n=4samples/group). (G) HE staining of mouse liver. (n=3samples) Scale bars, 500μm. (H) Liver surface metastases number of stoves (n=4samples). (I) Quantification of tumor area (n=4samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S4. Overexpression of SAA in patients with CRLM depends on SGK1. Liver specimens from CRLM patients and peripheral blood 1 day after surgery were collected. (A) Immunohistochemistry of liver SAA paratumor tissue, healthy liver tissue as control group (n=4samples/group). (B) Immunohistochemical quantification (n=4samples/group). (C) Serum SAA level from patients with CRLM was examined 1 day after surgery by Elisa and the correlation analysis of liver SGK1 relative expression with serum SAA levels (n=16samples). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S5. Liver metastasis regulated by hepatocyte SGK1 is independent of NRF2 crosstalk. Using Gal-siSGK1 or Gal-siCtrl and then a mouse IR model was constructed. (A) Expression of NRF2 in hepatocytes was detected by western blot (n=3samples/group). (B) qRT-PCR analysis of NRF2 in hepatocytes isolated from ischemic livers (n=4samples/group). (C) Immunofluorescence staining of SGK1, NRF2 and HNF-4α in ischemic livers or control livers, scale bars, 50μm (n=4samples/group). (D) Immunohistochemistry staining of NRF2 in ischemic livers or sham livers, scale bars, 20μm. (n=4samples/group). (E) Gal-siSGK1 or Gal-siCtrl were used to knock down SGK1 in hepatocytes，and then a mouse CRLM+IR model was constructed. Immunofluorescence staining of SGK1, NRF2 and HNF-4α, scale bars, 200μm (n=4samples/group). (F) Immunohistochemistry staining of NRF2 in CRLM livers, scale bars, 50μm. (n=4samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001****p &lt; 0.0001. Figure S6. Administration of GSK-650394 attenuates liver IR injury and CRLM progression in mice compared to specific knockout of hepatocyte SGK1. Gal-siSGK1 and GSK-650394 were used to knock down mouse SGK1, respectively, and then a mouse CRLM+IR model was constructed. (A) Physical pictures of the tumor (n=6samples/group). (B) Number of metastases on liver surface (n = 6samples/group). (C and D) Representative images of bioluminescence and statistics were shown (n = 4samples/group). (E) Survival (n = 8samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S7. SGK1 induces the generation of NETs by activating ERK. Mouse neutrophils were isolated in vitro and placed in a six-well plate first. Then GSK-650394 was added. To induce NETs, LPS (100ng/ml), PMA (200nM) and co-culture with tumor cell supernatants were used. (A) Expression of ERK1/2, p-ERK1/2 was detected by western blot (n=3samples/group). (B) Quantification of p-ERK/ERK. (n=3samples/group). (C) MEK inhibitor (U0126, MCE, China) was used to block ERK. NETs were then induced as mentioned before and were detected by scanning electron microscopy. Scale bars, 10μm. (n=4samples/group). (D) Expression of CitH3 in CRLM-IR mice liver treated with U0126 in vivo was detected by western blot (n=3samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S8: Recruitment of Neutrophils in mice undergoing CRLM+IR is blocked by GSK-650394. GSK-650394 was injected intraperitoneally into mice to construct a mouse CRLM+IR model, and the mouse livers were harvested two weeks later. (A) Immunohistochemical detection of ly6G, mice injected intraperitoneally with DMSO as control group, (n=3samples/group). Scale bars, 50μm. (B) Immunohistochemical quantification (n=3samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S9: GSK-650394 inhibits key biochemical signatures of PMN-MDSCs. GSK-650394 was injected intraperitoneally into mice to construct a mouse CRLM+IR model. The livers and the serum were harvested two weeks later. (A) The mRNA level of S100A8、S100A9、ARG1、STAT3 in livers were detected by qPCR (n=4samples/group). (B) The expression of serum PGE2 and were detected by Elisa (n=4samples/group). All data represent the mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001.