Journal: Journal of Gastrointestinal Oncology

Article Title: DDX18 promotes growth and metastasis of hepatocellular carcinoma via activating EMT and MAPK signaling

doi: 10.21037/jgo-2025-339

Figure Lengend Snippet: DDX18 interacts with REXO4. (A) Box plots illustrate that REXO4 mRNA levels were significantly elevated in LIHC tissues compared to normal tissues. (B) Kaplan-Meier curve analysis assessed the overall survival rates of HCC patients with high versus low REXO4 expression. (C) Co-IP experiments confirmed the interaction between endogenous DDX18 and REXO4 in HCC cells. (D) Immunofluorescence staining detected the expression of DDX18 (green) and REXO4 (red) in HCC cells. Original magnification ×200. Co-IP, co-immunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; HCC, hepatocellular carcinoma; HR, hazard ratio; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; LIHC, liver hepatocellular carcinoma; mRNA, messenger RNA; N, normal; T, tumor; TPM, transcripts per million.

Article Snippet: The primary antibodies used were: DDX18 (ab128197, Abcam, Boston, MA, USA), REXO4 (18890-1-AP, Proteintech, Wuhan, China), E-cadherin (20874-1-AP, Proteintech), vimentin (60330-1-Ig, Proteintech), extracellular regulated protein kinase (ERK)1/2 (11257-1-AP, Proteintech), phosphorylated-ERK1/2 (28733-1-AP, Proteintech), c-Jun N-terminal kinase (JNK) (80024-1-RR, Profit, Wuhan, China), mitogen-activated extracellular signal-regulated kinase (MEK)1/2 (11049-1-AP, Proteintech), phosphorylated-MEK1/2 (ab278723, Abcam).

Techniques: Expressing, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Immunoprecipitation, Western Blot

Journal: Journal of Gastrointestinal Oncology

Article Title: DDX18 promotes growth and metastasis of hepatocellular carcinoma via activating EMT and MAPK signaling

doi: 10.21037/jgo-2025-339

Figure Lengend Snippet: Knockdown of REXO4 abrogates DDX18-enhanced growth, migration, and invasion of HCC cells in vitro . (A,B) Colony formation assays evaluated the impact of REXO4 on colony formation induced by DDX18 in HCC cells. The colonies were stained with 1% crystal violet. (C,D) Wound-healing assays demonstrated the effects of REXO4 on DDX18-induced wound healing in HCC cells. Scale bar: 100 µm. (E,F) Transwell assays investigated the influence of REXO4 on DDX18-induced migration and invasion of HCC cells. Crystal violet staining. Scale bar: 50 µm. **, P<0.01. HCC, hepatocellular carcinoma; OV, overexpression; sh, short hairpin.

Article Snippet: The primary antibodies used were: DDX18 (ab128197, Abcam, Boston, MA, USA), REXO4 (18890-1-AP, Proteintech, Wuhan, China), E-cadherin (20874-1-AP, Proteintech), vimentin (60330-1-Ig, Proteintech), extracellular regulated protein kinase (ERK)1/2 (11257-1-AP, Proteintech), phosphorylated-ERK1/2 (28733-1-AP, Proteintech), c-Jun N-terminal kinase (JNK) (80024-1-RR, Profit, Wuhan, China), mitogen-activated extracellular signal-regulated kinase (MEK)1/2 (11049-1-AP, Proteintech), phosphorylated-MEK1/2 (ab278723, Abcam).

Techniques: Knockdown, Migration, In Vitro, Staining, Over Expression

Journal: Journal of Gastrointestinal Oncology

Article Title: DDX18 promotes growth and metastasis of hepatocellular carcinoma via activating EMT and MAPK signaling

doi: 10.21037/jgo-2025-339

Figure Lengend Snippet: DDX18 regulates the EMT process and MAPK signaling pathway in a REXO4-dependent manner in HCC cells. (A) Western blot analysis determined the effects of REXO4 siRNA on the EMT process induced by DDX18 overexpression in HCC cells. (B,C) Western blot analysis elucidated the role of REXO4 in DDX18-mediated activation of the ERK/MAPK pathway in HCC cells. EMT, epithelial-mesenchymal transition; ERK, extracellular regulated protein kinase; HCC, hepatocellular carcinoma; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated extracellular signal-regulated kinase; siRNA, small interfering RNA; OV, overexpression; p-, phosphorylated-; sh, short hairpin.

Article Snippet: The primary antibodies used were: DDX18 (ab128197, Abcam, Boston, MA, USA), REXO4 (18890-1-AP, Proteintech, Wuhan, China), E-cadherin (20874-1-AP, Proteintech), vimentin (60330-1-Ig, Proteintech), extracellular regulated protein kinase (ERK)1/2 (11257-1-AP, Proteintech), phosphorylated-ERK1/2 (28733-1-AP, Proteintech), c-Jun N-terminal kinase (JNK) (80024-1-RR, Profit, Wuhan, China), mitogen-activated extracellular signal-regulated kinase (MEK)1/2 (11049-1-AP, Proteintech), phosphorylated-MEK1/2 (ab278723, Abcam).

Techniques: Western Blot, Over Expression, Activation Assay, Small Interfering RNA

Journal: Journal of Gastrointestinal Oncology

Article Title: DDX18 promotes growth and metastasis of hepatocellular carcinoma via activating EMT and MAPK signaling

doi: 10.21037/jgo-2025-339

Figure Lengend Snippet: DDX18 modulates HCC growth in vivo . (A) Representative images of five mice from the shNC, shDDX18#2, or shDDX18#2 + OV-REXO4 groups are shown. (B,C) Subcutaneous tumor volumes and weights were measured. **, P<0.01. HCC, hepatocellular carcinoma; NC, negative control; OV, overexpression; sh, short hairpin.

Article Snippet: The primary antibodies used were: DDX18 (ab128197, Abcam, Boston, MA, USA), REXO4 (18890-1-AP, Proteintech, Wuhan, China), E-cadherin (20874-1-AP, Proteintech), vimentin (60330-1-Ig, Proteintech), extracellular regulated protein kinase (ERK)1/2 (11257-1-AP, Proteintech), phosphorylated-ERK1/2 (28733-1-AP, Proteintech), c-Jun N-terminal kinase (JNK) (80024-1-RR, Profit, Wuhan, China), mitogen-activated extracellular signal-regulated kinase (MEK)1/2 (11049-1-AP, Proteintech), phosphorylated-MEK1/2 (ab278723, Abcam).

Techniques: In Vivo, Negative Control, Over Expression

Journal: Journal of advanced research

Article Title: PROTAC derivatization of natural products for target identification and drug discovery: Design of evodiamine-based PROTACs as novel REXO4 degraders.

doi: 10.1016/j.jare.2023.10.014

Figure Lengend Snippet: Fig. 2. REXO4 is a target of 3-fluoro-10-hydroxylevodiamine. (A) The chemical structures of EVO-PROTAC 13c and 13e. The up-regulated (red) and down-regulated (light blue) proteins whose fold change (EVO-PROTAC/negative control) exceed the value of 1.5 (P < 0.05) after the treatment with EVO-PROTAC 13c (B) and 13e (D). (C) The partial common proteins degraded by EVO-PROTACs by taking an intersection. The expression level of REXO4 protein in HCT116 cells after the treatment of EVO-PROTAC 13c (E), negative control 14a (F) and thalidomide (G) for 24 h. (H) The expression level of REXO4 protein after the pretreatment with proteasome inhibitor MG132 (2 lM) or the neddylation inhibitor MLN4924 (2 lM) for 2 h before the incubation with either vehicle (DMSO) or EVO-PROTAC 13c (0.4 lM) for 24 h. (I) Western blot analysis of REXO4 protein levels in the presence of 3-fluoro-10-hydroxylevodiamine treatment for 4 h and subsequently heated at different temperatures (49–68 C). (J) MST assay between REXO4 and 3-fluoro-10-hydroxylevodiamine. (K) Quantitative real-time PCR analysis of the mRNA level of REXO4 after 24 h of treatment with either vehicle (DMSO), 3- fluoro-10-hydroxylevodiamine (0.8 lM) or EVO-PROTAC 13c (0.8 lM) in HCT116 cells. Data were normalized with GAPDH. (L) The expression level of REXO4 protein in HCT116 cells and REXO4 KD HCT116 cells. (M and N) The IC50 values and wound heal inhibition of 3-fluoro-10-hydroxylevodiamine against HCT116 cells and REXO4 KD HCT116 cells. (O and P) The concentration-dependent increase of ROS level after 24 h of treatment with 3-fluoro-10-hydroxylevodiamine at various concentration. (Q) The expression level of c-H2AX protein after the treatment with 3-fluoro-10-hydroxylevodiamine for 24 h.

Article Snippet: Subsequently, primary antibodies against REXO4 (Proteintech, 18890-1-AP, 1:1000), cH2AX (Abcam # ab2893, 1:1000) and anti-GAPDH (Abcam #ab181602, 1:10,000) were incubated with various membranes overnight at 4 C. The fluorescent secondary antibodies were then used to incubate with them for 2 h after three times washing with TBST, followed by the analysis using a LI-COR Odyssey imaging system.

Techniques: Negative Control, Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Inhibition, Concentration Assay

Journal: Journal of advanced research

Article Title: PROTAC derivatization of natural products for target identification and drug discovery: Design of evodiamine-based PROTACs as novel REXO4 degraders.

doi: 10.1016/j.jare.2023.10.014

Figure Lengend Snippet: Fig. 4. The in vivo antitumor activity, REXO4 degradation and toxicity of EVO-PROTAC 13c. (A) The schematic illustration of the in vivo tumor-inhibition experiment and the general treatment procedure. (B) The comparison of tumor weight in each group. (C) The macroscopic views of dissected xenograft tumor tissues in each group. (D) The expression levels of REXO4 in the tumors after IP administration with EVO-PROTAC 13c. (E) The changes in body weight during the treatment. (F) The H&E staining of major organs and the tumor after the treatment with EVO-PROTAC 13c or compound 2. (G) The IHC of Ki67 and Tunel staining of tumor tissues after the treatment with EVO- PROTAC 13c or compound 2. The figure was prepared with BioRender.com.

Article Snippet: Subsequently, primary antibodies against REXO4 (Proteintech, 18890-1-AP, 1:1000), cH2AX (Abcam # ab2893, 1:1000) and anti-GAPDH (Abcam #ab181602, 1:10,000) were incubated with various membranes overnight at 4 C. The fluorescent secondary antibodies were then used to incubate with them for 2 h after three times washing with TBST, followed by the analysis using a LI-COR Odyssey imaging system.

Techniques: In Vivo, Activity Assay, Inhibition, Comparison, Expressing, Staining, TUNEL Assay

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Construction of an interference sequence to downregulate the expression of REXO4

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Sequencing, Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: HCC tissues exhibited REXO4 overexpression. Levels of REXO4 mRNA expression in HCC and normal liver tissue samples were assessed in GEO datasets. (A) GSE112613 (tumor =5; normal =5); (B) GSE138178 (tumor =49; normal =49); (C) fifty paired tissues from the Affiliated Hospital of Nantong University. **, P<0.01. HCC, hepatocellular carcinoma.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: REXO4 was significantly upregulated in HCC cell lines (Huh7, MHCC-97H, HepG2, and HCCLM3) compared with normal LO2 liver cell lines. (A) The expression levels of REXO4 in HCC and normal liver cells were determined by RT-qPCR; (B) Western blotting showed the protein level of REXO4 compared with β-actin. *, P<0.05; **, P<0.01. HCC, hepatocellular carcinoma.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Clinicopathological characteristics in relation to REXO4 expression status in HCC patients

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: REXO4 upregulation associated with a poorer HCC patient prognosis. (A) The expression of REXO4 in HCC tissues was significantly higher than that in normal tissues. Data from GEPIA; (B) Kaplan-Meier plots summarizing results from the analysis of the correlation between mRNA expression level and patient survival with a comparison of the probability of survival. HCC, hepatocellular carcinoma.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Expressing, Comparison

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: The significant related pathways identified by GSEA. (A,B) The most involved significant pathway included fatty acid metabolism, linoleic acid metabolism, and PPAR signaling pathway; (C) expression of REXO4 in NAFLD patients obtained from GSE138052. *, P<0.05. GSEA, gene set enrichment analysis; NAFLD, non-alcoholic fatty liver disease.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Oil red O staining of low and high expression of REXO4 in HCC patient tissues. (A-C) Patients with low expression of REXO4 showed less connection with lipids. (D-F) Patients with high expression of REXO4 showed higher connection with lipids. HCC, hepatocellular carcinoma.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Staining, Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Immune cell infiltration analyses of REXO4 in HCC patients. (A) The immune cells has a significant correlation with REXO4; (B) those associated with high expression were macrophages, NK cells, CD4+ T cells, and CD8+ T cells.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Construction of knocked down LM3 and Huh7 cell lines. (A,B) The knockdown effect of siRNAs targeting REXO4 in HCCLM3 and Huh7 at the mRNA and protein level. **, P<0.01.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Knockdown

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Downregulating the expression of REXO4 inhibited the proliferation and migration of HCC cells. (A,B) Silencing of REXO4 inhibited cell growth of HCC as indicated by CCK8 assays and colony formation assays (crystal violet staining); (C) knockdown of REXO4 impeded cell invasion in Huh7 and LM3 cell lines (transwell assay; crystal violet staining; scale bars, 100µm; magnification, 100×). **, P<0.01. HCC, hepatocellular carcinoma; CCK8, Cell Counting Kit-8.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Expressing, Migration, Staining, Knockdown, Transwell Assay, Cell Counting

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: Wound-healing assays were performed to evaluate the migration ability. (A,B) Suppression of REXO4 expression weakened migration in LM3 and Huh7 cell lines (shoot with a 4× objective lens; scale bars: 500 µm). **, P<0.01. NC, negative control.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: Migration, Expressing, Negative Control

Journal: Journal of Gastrointestinal Oncology

Article Title: REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression

doi: 10.21037/jgo-21-819

Figure Lengend Snippet: REXO4 promotes liver cancer tumor formation in vivo. (A) LM3 cells transfected with NC siRNA or REXO4 siRNA (si-REXO4-1) were injected into nude mice (n=5) respectively, which were euthanized after 30 days. (B,C) Tumor volume and weight were measured. **, P<0.01. NC, negative control.

Article Snippet: After blocking, the PVDF membrane was washed with 1× TBST, and membranes were then incubated overnight at 4 °C in a primary antibody against REXO4 (1:1,000, 18890-1-AP, Proteintech) and GAPDH (1:2,000, sc-20357, Santa Cruz Biotechnology).

Techniques: In Vivo, Transfection, Injection, Negative Control