Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Livers from groups of WT and CC1-KO C57BL/6 donor mice, stored in UW solution (4°C/18 hours), were transplanted to WT C57BL/6 recipient mice. OLT and serum samples were analyzed 6 hours after reperfusion. The sham group underwent the same procedures except for OLT. (A) Representative H&E staining (original magnification ×100). (B) Suzuki’s histological grading of liver IRI. (C) sAST and sALT levels (IU/L). (D) Representative TUNEL staining and immunohistochemical staining of OLT-infiltrating CD11b+ and Ly6G+ cells (original magnification ×200). (E) Quantification of TUNEL-positive cells/HPF. (F) Serum HMGB1 (ng/mL) and MCP1 (pg/mL) levels measured by ELISA. (G) Quantification of hepatic CD11b+ and Ly6G+ cells/HPF. (H) Real-time reverse transcription PCR–assisted (qRT-PCR–assisted) detection of mRNA coding for MCP1, CXCL1, CXCL2, and CXCL10 in OLT. Data were normalized to HPRT gene expression. Data are mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B, C, and E–G) or Student’s t test (H), n = 5–6/group.

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Staining, TUNEL Assay, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Groups of WT and CC1-KO liver grafts stored in UW solution (4°C/18 hours) were perfused with physiological saline (2 mL) via a cuff placed at the portal vein to collect liver flush from inferior vena cava. (B) Liver flush samples (20 μL) from cold-stressed WT or CC1-deficient livers were screened by Western blots for HMGB1/Histone H3 levels (n = 4/group, *P < 0.05, Student’s t test). (C) WT or CC1-KO liver grafts were collected after cold storage (4°C/18 hours). Representative (n = 3/group) immunohistochemical staining of CC1/4HNE (a ROS metabolite), CC1/HMGB1, and TUNEL is shown. Arrowheads indicate extranuclear HMGB1 localization. (D) BMDM cultures (WT) were stimulated (6 hours) with liver flush obtained from WT or CC1 KO cold-stored grafts. qRT-PCR–assisted detection of mRNA coding for MCP1, CXCL2, CXCL10 with β2M normalization (n = 4–6, *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Saline, Western Blot, Immunohistochemical staining, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours). Liver samples were collected right after cold storage (before OLT) or 3 hours after reperfusion (post-OLT). (B and C) Western blot–assisted detection and relative intensity ratio of cleaved caspase-3, RIP3, CC1, ASK1, p-p38 in naive liver, cold-stored liver or postreperfusion OLT (WT). Vinculin (VCL) expression served as an internal control and was used for normalization (n = 3/group). (D) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in naive WT or CC1-KO liver. VCL expression served as an internal control and was used for normalization (n = 4/group). (E) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in cold-stored WT or CC1-KO livers. VCL expression served as an internal control and used for normalization (n = 3/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B and C) or Student’s t test (D and E).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Primary mouse hepatocytes (WT) with or without cold stimulation (4°C/4 hours) were incubated for the indicated time periods. Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 2/group). (B–D) Cold-stimulated WT or CC1-KO hepatocytes were pretreated with or without siRNA against ASK1. (B) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 3/group). (C) Representative (n = 3/group) immunohistochemical staining of 4HNE (red, upper panels), HMGB1 (red, middle panels), and dead cell detection (red, lower panels). (D) Quantification of dead cells/HPF (n = 4–5/group). *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test.

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Incubation, Western Blot, Expressing, Control, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A–C) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours) with or without ASK1 inhibitor (10 μg/15 mL). (A) Western blot–assisted detection and relative intensity ratio of CC1 and p-p38. VCL expression served as an internal control and used for normalization (n = 3–4/group). (B) Representative (n = 3/group) immunohistochemical staining of CC1/4HNE and CC1/HMGB1. (C) Liver flush (20 μL) from cold-stressed WT or CC1-KO livers with or without ASK1 inhibitor were analyzed by Western blots for HMGB1 levels (n = 3–4/group). (D–H) Cold-stored (4°C/18 hours) WT or CC1-KO livers were transplanted into recipient mice, and OLT and serum samples were analyzed at 6 hours after reperfusion. Some CC1-KO grafts were preincubated with ASK1 inhibitor (10 μg/15 mL) during cold storage (4°C/18 hours). Separate OLT recipient groups were monitored for 20-day survival. (D) Representative H&E (original magnification ×100) and TUNEL staining. (E) sAST and sALT levels (IU/L; n = 7–8/group). (F) Suzuki’s histological grading of liver IRI (n = 7–8/group). (G) Quantification of TUNEL-positive cells/HPF (n = 7–8/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test. (H) Recipient mice were monitored for 20 days and cumulative survival was analyzed (Kaplan-Meier method). Dotted line: WT → WT; solid line: CC1-KO → WT; bold line: CC1-KO+ASK1 inhibitor → WT (n = 6–9/group; *P < 0.05 vs. CC1-KO → WT, log-rank test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, Expressing, Control, Immunohistochemical staining, Staining, TUNEL Assay

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1, ASK1, and p-p38 levels (see Supplemental Figure 1A). (A) Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups based on the relative CEACAM1/β-actin levels (cutoff = 0.85, median). (B) Western blot–assisted expression of ASK1 and p-p38. Data shown in dot plots and bars indicate mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Four representative Western blots are shown (case 1/2: low CEACAM1, case 3/4: high CEACAM1). (D) Representative (n = 3) CEACAM1/4HNE staining (original magnification ×200). (E) Representative (n = 3) CEACAM1/HMGB1 staining (original magnification ×400).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, Expressing, MANN-WHITNEY, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups, based on the relative CEACAM1/β-actin levels (see Figure 6A). (A) Serum AST levels at POD1–7. (B) Serum ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding clinical cases. Representative (n = 3) TUNEL staining (original magnification ×400). (D) Incidence of EAD (Fisher’s exact test). (E) The cumulative probability of overall graft survival. (F) The cumulative probability of rejection-free graft survival. Solid line indicates low CEACAM1; dotted line indicates high CEACAM1 human OLT patient group (Kaplan-Meier method, log-rank test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Expressing, MANN-WHITNEY, TUNEL Assay, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples were classified into low (n = 30) and high (n = 30) CEACAM1 (CC1) expression groups (see Figure 6A for details). Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding cases, followed by qRT-PCR–assisted detection of mRNA coding for TLR4, CD80, CD86, CXCL10, CD68, Cathepsin G, CD28, CD4, and IL17. Data normalized to GAPDH gene expression are shown in dot plots and bars indicative of mean ± SEM. #P < 0.05 (Mann-Whitney U test).

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1 levels. (A) ROC analysis of CEACAM1/β-actin for predicting EAD. Based on a ROC curve and Youden index on the basis of best accuracy in relation to EAD incidence, the CEACAM1/β-actin cutoff value of 0.71 was determined. AUROC, area under the receiver operating characteristic curve. (B) Based on the optimal cutoff value (0.71), 60 human OLTs were classified into CEACAM1/β-actin less than 0.71 (n = 20) and CEACAM1/β-actin greater than 0.71 cases (n = 40), and the incidence of EAD was evaluated. #P < 0.05 (Fisher’s exact test). (C) Serum AST and ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (D) Stepwise multivariate logistic regression analysis was performed to identify independent risk factors of EAD.

Article Snippet: Mouse and human liver samples were stained with sheep anti-CC1 Ab (AF6480, R&D Systems), rabbit anti-4HNE Ab (ab46545, Abcam), and rabbit anti-HMGB1 Ab (ab79823/EPR3507, Abcam).

Techniques: Western Blot, MANN-WHITNEY

Journal: Cancer Science

Article Title: Transfer RNA ‐derived fragment 5′ tRF‐Gly promotes the development of hepatocellular carcinoma by direct targeting of carcinoembryonic antigen‐related cell adhesion molecule 1

doi: 10.1111/cas.15505

Figure Lengend Snippet: Transfer RNA‐derived fragment 5′tRF‐Gly directly regulates carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) expression in hepatocellular carcinoma cells. (A) Schematic illustration displaying the overlapped target genes of 5′tRF‐Gly by miRanda and RNAhybrid. Five genes were selected by Gene Ontology (GO) enrichment analysis of cell growth, cell junction, and cell–cell adhesion. (B) mRNA expression of five target genes was tested by quantitative PCR in Hep3B2.1‐7 cells after transfection with 5′tRF‐Gly mimics. (C) Protein expression of CEACAM1 was tested in Hep3B2.1‐7 cells after transfection with negative control (NC), 5′tRF‐Gly mimics, locked nucleic acid (LNA)‐NC, or LNA‐5′tRF‐Gly by western blotting. (D) Sequence of binding target of 5′tRF‐Gly in WT or mutant CEACAM1 3′‐UTR. (E) After transfection with NC or 5′tRF‐Gly mimics in 293T/17 cells, the relative luciferase activity of three kinds of mutant or WT CEACAM1 3′‐UTR was detected. (F) RIPA confirmed the binding status between 5′tRF‐Gly and CEACAM1 in untreated and treated Hep3B2.1‐7 cells. * p < 0.05; ** p < 0.01; *** p < 0.0001. n.s., not significant

Article Snippet: The Abs used in this study were rabbit anti‐CEACAM1 (ab108397; Abcam), rabbit anti‐N‐cadherin (22018‐1‐AP; Proteintech), mouse anti‐MMP2 (66366‐1‐IG; Proteintech), mouse anti‐E‐cadherin (60335‐1‐IG; Proteintech), mouse anti‐Cyclin D1 (60186‐1‐IG; Proteintech).

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot, Sequencing, Binding Assay, Mutagenesis, Luciferase, Activity Assay

Journal: Cancer Science

Article Title: Transfer RNA ‐derived fragment 5′ tRF‐Gly promotes the development of hepatocellular carcinoma by direct targeting of carcinoembryonic antigen‐related cell adhesion molecule 1

doi: 10.1111/cas.15505

Figure Lengend Snippet: Silencing carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) promotes the proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells. (A) Higher expression of CEACAM1 is related to longer progression‐free survival of HCC in the Kaplan–Meier Plotter database. (B) Expression level of CEACAM1 protein and mRNA in Hep3b2.1‐7 and Huh‐7 cells after transfection with siRNA of CEACAM1. (C) Proliferation rate of Hep3B2.1‐7 and Huh‐7 cells after CEACAM1 knockdown. (D) Representative images and quantification of Transwell migration and invasion assays after Hep3B2.1‐7 or Huh‐7 cells were transfected with negative control (siNC) or siCEACAM1. Magnification, ×100. * p < 0.05; ** p < 0.01; *** p < 0.0001

Article Snippet: The Abs used in this study were rabbit anti‐CEACAM1 (ab108397; Abcam), rabbit anti‐N‐cadherin (22018‐1‐AP; Proteintech), mouse anti‐MMP2 (66366‐1‐IG; Proteintech), mouse anti‐E‐cadherin (60335‐1‐IG; Proteintech), mouse anti‐Cyclin D1 (60186‐1‐IG; Proteintech).

Techniques: Migration, Expressing, Transfection, Negative Control

Journal: Cancer Science

Article Title: Transfer RNA ‐derived fragment 5′ tRF‐Gly promotes the development of hepatocellular carcinoma by direct targeting of carcinoembryonic antigen‐related cell adhesion molecule 1

doi: 10.1111/cas.15505

Figure Lengend Snippet: Role of transfer RNA‐derived fragment 5′tRF‐Gly on tumor progression is mediated by the downregulation of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1). (A) Alteration of CEACAM1 after cotransfection with locked nucleic acid (LNA)‐5′tRF‐Gly or negative control (LNA‐NC) with either CEACAM1 or NC siRNA. (B) Proliferation of Hep3B2.1‐7 cells after cotransfection with LNA‐5′tRF‐Gly or LNA‐NC with either CEACAM1 or NC siRNA. (C, D) Representative images (C) and quantification (D) of Transwell migration and invasion assays after Hep3B2.1‐7 cells were cotransfected with LNA‐5′tRF‐Gly or LNA‐NC with either CEACAM1 or NC siRNA. (E) Protein expression of the possible downstream genes of CEACAM1 were tested in Hep3B2.1‐7 cells after cotransfection with LNA‐5′tRF‐Gly or LNA‐NC with either CEACAM1 or NC siRNA by western blot analysis. (F) Alterations of possible downstream genes of CEACAM1 in Hep3B2.1‐7 after transfection with NC, 5′tRF‐Gly mimics, LNA‐NC, or LNA‐5′tRF‐Gly. (G) Model diagram of 5′tRF‐Gly/CEACAM1 axis functioning in HCC. * p < 0.05; ** p < 0.01; *** p < 0.0001

Article Snippet: The Abs used in this study were rabbit anti‐CEACAM1 (ab108397; Abcam), rabbit anti‐N‐cadherin (22018‐1‐AP; Proteintech), mouse anti‐MMP2 (66366‐1‐IG; Proteintech), mouse anti‐E‐cadherin (60335‐1‐IG; Proteintech), mouse anti‐Cyclin D1 (60186‐1‐IG; Proteintech).

Techniques: Derivative Assay, Cotransfection, Negative Control, Migration, Expressing, Western Blot, Transfection