Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A Peptides of DDX3X identified by FAM134B IP-MS. B HEK-293T cells were transfected with Flag-FAM134B and HA-DDX3X. Twenty-four hours later, cells were lysed with IP-lysis, then immunoprecipitated with control IgG, anti-Flag or anti-HA antibodies. Western blot detected the input and immunoprecipitates. C Huh7 and MHCC97-H cells were lysed with IP-lysis, followed by immunoprecipitation with either control IgG, anti-FAM134B, or anti-DDX3X antibodies. Western blot analysis was then performed to detect both the input and the immunoprecipitates. D Purified Flag-DDX3X protein was incubated with purified GST, GST-FAM134B and immobilized on beads. Affinity-isolated samples (top) and 5% of input DDX3X were analyzed by western blot (bottom). E Huh7 and MHCC97-H cells were fixed and incubated with anti-FAM134B and anti-DDX3X antibodies. Colocalization between FAM134B and DDX3X were examined by confocal microscopy. Scale bars, 10 µm. F Schematic diagram of FAM134B interacted with DDX3X. G Volcano plot of DDX3X binding proteins.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: Protein-Protein interactions, Transfection, Lysis, Immunoprecipitation, Control, Western Blot, Purification, Incubation, Isolation, Confocal Microscopy, Binding Assay

Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A Western blot detected protein level of DDX3X in FAM134B overexpression or knockdown cell lines. B RT-qPCR detected DDX3X mRNA level in FAM134B overexpression (Hep3B) or knockdown (MHCC97-H) cell lines. C , D Hep3B cells stably expressing vector or FAM134B overexpression were treated with CHX (20 μM) for 0, 2, 4, 8 h. The degradation kinetics of DDX3X was monitored over time by Western blot. Data are mean ± SEM from three independent experiments, *** P < 0.001. E , F MHCC97-H cells stably expressing shNC or shFAM134B-1 were treated with CHX (20 μM) for 0, 2, 4, 8 h. The degradation kinetics of DDX3X was monitored over time by Western blot. Data are mean ± SEM from three independent experiments, *** P < 0.001. G MHCC97-H cells stably expressing shNC or shFAM134B-1 cells were treated with or without MG132 (10 μM, 4 h). Western blot detected DDX3X protein level. H MHCC97-H cells stably expressing shNC or shFAM134B-1 cells were treated with or without CQ (20 μM, 4 h). Western blot detected DDX3X protein level. I Hep3B cells stably expressing control or FAM134B, and MHCC97-H cells stably expressing control or shFAM134B-1, were collected using IP-lysis buffer. The cell lysates were then immunoprecipitated with either control IgG or anti-DDX3X antibodies. Both immunoprecipitates and input samples were analyzed by Western blot. J HEK-293T cells were transfected with Flag-DDX3X and HA-UB (WT, K48-only, K63-only), with or without Myc-FAM134B. After 24 h, cell lysates were immunoprecipitated with either control IgG or anti-Flag antibodies. Immunoprecipitates and input were analyzed by Western blot.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Control, Lysis, Immunoprecipitation, Transfection

Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A Western blot detected whether DDX3X overexpression and knockdown cell lines were successfully constructed, and detected the phosphorylated AKT (Ser473) level. B − F CCK-8 assay, n = 5 ( B ), soft agar assay, n = 3 ( C , D ) colony formation assay, n = 3 ( E , F ) detected the indicated cell line proliferation rate. Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. G Flow cytometry tested the effect of DDX3X reduction on the cell cycle of MHCC97-H cells. Data are mean ± SEM from three independent experiments, ** P < 0.01. H Western blot detected the expression of cell cycle related proteins in the indicated cells. I − K Transwell assay detected the effect of DDX3X on HCC cells migration and invasion. Data are mean ± SEM from at least three independent experiments, *** P < 0.001.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Over Expression, Knockdown, Construct, CCK-8 Assay, Soft Agar Assay, Colony Assay, Flow Cytometry, Expressing, Transwell Assay, Migration

Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A Western blot detected the protein level of FAM134B, DDX3X, p-AKT (Ser473), AKT in rescue cell lines. B − F CCK-8 assay, n = 5 ( B ), colony formation assay, n = 3 (C and D) investigated whether the promotional effect of FAM134B on HCC proliferation was dependent on DDX3X. Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. E − H Transwell assay investigated whether the promotional effect of FAM134B on HCC migration and invasion was dependent on DDX3X. Data are mean ± SEM from three independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001. I − M The gross image ( I ), tumor volume ( J ), tumor weight ( K ), Ki-67 index ( L ), IHC image (M) of subcutaneous tumor model from shNC, shFAM134B-2 and shFAM134B-2 + DDX3X group, n = 5. Scale bar 100 μm, * P < 0.05, ** P < 0.01, *** P < 0.001. N - O H&E image of lungs ( N ), metastatic modules of lungs ( O ) of teil vein injection lung metastasis model from shNC, shFAM134B-2 and shFAM134B-2 + DDX3X group. n = 5. Scale bar 2000 μm. Data are mean ± SEM, * P < 0.05, ** P < 0.01.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: Western Blot, CCK-8 Assay, Colony Assay, Transwell Assay, Migration, Injection

Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A Western blot detected FAM134B protein level in Huh7 and MHCC97-H cells stably expressing shNC or shDDX3X. B MHCC97-H cells were transfected with vector, Flag-WT DDX3X, Flag-mutant DDX3X at the same concentration. Forty-eight hours later, cells were lysed with RIPA, followed by western blot analysis. C Huh7 cells stably expressing either shNC or shDDX3X were treated with CHX (20 μM) for 0, 2, 4, 8 h. The degradation kinetics of FAM134B was monitored over time by western blot. Data are mean ± SEM from three independent experiments, ns: not significant. D MHCC97-H cells stably expressing either shNC or shDDX3X were treated with CHX (20 μM) for 0, 2, 4, 8 h. The degradation kinetics of FAM134B was monitored over time by western blot. Data are mean ± SEM from three independent experiments, ns: not significant. E MHCC97-H and Huh7 cells were transfected with siNC or siDDX3X. Twenty-four hours later, total RNA was isolated using TRIzol reagent. RT-qPCR was then employed to assess the mRNA levels of DDX3X and FAM134B. Data are mean ± SEM from three independent experiments, *** P < 0.001. F , G Luciferase reporter assay detected the effect of DDX3X on FAM134B promoter activity. Data are mean ± SEM from three independent experiments, ** P < 0.01. H The representative IHC images of DDX3X protein expression in HCC tissues. Scale bar, 40 μm.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Concentration Assay, Isolation, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A Western blot detected the protein expression of DDX3X in 20 paired HCC tissues and adjacent non-tumor tissues, arrows indicated non-specific bands. The numbers indicated the relative amount of DDX3X signal compared to the first lane on the left, normalized to β-actin. B The log2 transformed fold change of DDX3X expression in tumor tissues and non-tumor tissues from ( A ). C The representative images of DDX3X expression in tissue microarray. Scale bar 4000 μm (top), 100 μm (bottom). D DDX3X scoring of paired HCC tissues and adjacent non-tumor tissues. Scale bar represents SEM, *** P < 0.001, n = 122. E Kaplan-Meier curves of the overall survival (OS) rates between groups with differential DDX3X expression. P < 0.0001, n = 112. F Pearson’s correlation analysis of FAM134B and DDX3X protein levels. P = 0.0251. G MHCC97-H and Huh7 cells were transfected with HA-FAM134B. Forty-eight hours later, cells were fixed and incubated with anti-HA and anti-DDX3X antibodies. The expression of HA-FAM134B and DDX3X were observed with fluorescence microscope.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Expressing, Transformation Assay, Microarray, Transfection, Incubation, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Targeting FAM134B-DDX3X axis inhibiting AKT signaling in hepatocellular carcinoma

doi: 10.1038/s41419-025-08080-3

Figure Lengend Snippet: A MHCC97-H, HepG2, Huh7, Hep3B, LM3, PLC/PRF/5 cells were treated with RK-33 at varying concentrations for 24 hours. The cell survival rate was then determined using the CCK-8 assay. Scale bar represents SEM, n = 5. B PLC/PRF/5, MHCC97-H, Huh7 cells were treated the RK-33 at concentration of 0, 5, 10, 15 μM for 24 h. Western blot detected DDX3X protein level. The numbers indicated the relative amount of DDX3X signal compared to the first lane on the left, normalized to GAPDH. C Statistical chart of apoptotic cells from the indicated cell lines. Scale bar represents SEM, n = 3, *** P < 0.001. D − F The gross image ( D ), tumor volume ( E ), tumor weight ( F ) of subcutaneous tumor model from the indicated group, n = 4, scale bar represents SEM, * P < 0.5, ** P < 0.01. G The Liver/body weight ratio from HTVi model, n = 5, scale bar represents SEM, * P < 0.5, ** P < 0.01, *** P < 0.001. H The representative gross images of HTVi model. I Graphical summary of the study. The interaction between FAM134B and DDX3X inhibits DDX3X K48-linked polyubiquitination and promotes its K63-linked polyubiquitination, thereby stabilizing the expression of the DDX3X protein. Subsequently, DDX3X activates the AKT signaling pathway by facilitating the translation of Rac1. Additionally, DDX3X enhances the transcription of FAM134B, suggesting the existence of a positive feedback loop between these two proteins.

Article Snippet: Anti-DDX3X antibody (1:1000, 11115-1-AP), Anti-FAM134B antibody (1:1000, 21537-1-AP), Anti-GAPDH antibody (1:5000, 10494-1-AP), Anti-Rac1 antibody (1:500, 24072-1-AP) were purchased from Proteintech.

Techniques: CCK-8 Assay, Concentration Assay, Western Blot, Expressing