Journal: Autophagy

Article Title: Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

doi: 10.1080/15548627.2025.2507617

Figure Lengend Snippet: Autophagy is impaired in WABS patient and DDX11 KO RPE-1 cells. (A) Fibroblasts from three different WABS patients and from a healthy donor (HF1) were left in full medium or serum starved for 16 h. LC3-II level was assessed by western blot analysis. TUBA/tubulin was used as a loading control. (B) Graphs show the quantifications of LC3-II:TUBA. (C,D) fibroblasts from three different WABS patients and from a healthy donor (HF1) were treated with BAF A 1 for 16 h. LC3-II level was assessed by western blot analysis. TUBA was used as loading control. Graphs show the quantifications of LC3-II:TUBA. (E) RNA-seq analysis shows no difference in transcripts per kilobase million/TPM of autophagy genes ( ATG16L1 , ATG5 , ATG12 , BECN1 , SQSTM1 ) between control (CTRL) and DDX11 KO ( DDX11 KO) RPE-1 cells. (F) Western blot analysis revealed no difference in the expression level of the indicated autophagy proteins between control (CTRL) and DDX11 KO ( DDX11 KO) RPE-1 cells. TUBA was used as loading control. (G,H) control (CTRL) and DDX11 KO ( DDX11 KO) RPE-1 cells were grown in full-medium (FM) or serum starved for 16 h (NO FBS). LC3-II was analyzed by western blot. TUBA was used as loading control. Graphs show the quantifications of LC3-II:TUBA. (I,J) Control (CTRL) and DDX11 KO ( DDX11 KO) RPE-1 cells were treated with DMSO or BAF A 1 for 6 h. LC3-II level was analyzed by western blot. TUBA was used as loading control. Graphs show the quantifications of LC3-II:TUBA. (K–M) DDX11 KO (KO) RPE-1 cells were transfected with a vector expressing DDX11-Flag wild-type protein (WT) or the DDX11-Flag K50R helicase-dead mutant (K50R) to perform rescue experiments. After 24 h, control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO (KO + WT and KO + K50R) RPE-1 cells were treated with DMSO or BAF A 1 for 6 h. LC3 level (LC3-I and LC3-II) was analyzed by western blot. Graphs show the quantifications of LC3-II:TUBA. (N) Control (CTRL) and DDX11 KO ( DDX11 KO) RPE-1 cells were transfected with a vector expressing EGFP-HTT-74Q for 48 h to assess HTT mutant protein clearance. (O) Graph shows increased percentage of HTT-positive cells in the DDX11 KO RPE-1 line. Number of cells counted n = 50 in triplicates. Scale bar: 10 µm.

Article Snippet: The following antibodies were used: mouse monoclonal anti DDX11 (Santa Cruz Biotechnology, sc271711); mouse monoclonal anti-Flag antibody (Merck, F1804); rabbit polyclonal anti-LC3 (Novus Biologicals, NB100–2220); mouse monoclonal anti-LAMP1 (Cell Signaling Technology, 15665); rabbit polyclonal anti-SQSTM1 (MBL International, PM045); mouse monoclonal anti-TUBA/α-tubulin antibody (Merck, T6199); rabbit polyclonal anti-ATG16L1 antibody (MBL Life Sciences, PM040); rabbit monoclonal anti-ATG5 antibody (Cell Signaling Technology, 129945); rabbit polyclonal anti-BECN1/BECLIN1 antibody (Cell Signaling Technology, 3738S); rabbit polyclonal anti-AKT antibody (Cell Signaling Technology, 9272S); rabbit polyclonal anti-phospho-AKT (Ser473) antibody (Cell Signaling Technology, 9271 L); rabbit polyclonal anti-BRIP1/FANCJ antibody (Novus Biologicals, NBP1–31883); mouse monoclonal anti-histone H3 antibody (Abcam, ab24834); rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956); mouse and rabbit HRP-conjugated secondary antibody (Merck, A9044 and 12–348, respectively); Alexa Fluor 488 anti-rabbit antibody, Alexa Fluor 555 anti-mouse antibody and Alexa Fluor 633 anti-mouse antibody (Thermo Fisher Scientific, A-11008, A-21137, A-21146, respectively).

Techniques: Western Blot, Control, RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Autophagy

Article Title: Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

doi: 10.1080/15548627.2025.2507617

Figure Lengend Snippet: DDX11 regulates ATG16L1 localization. (A,B) Control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO ( DDX11 KO + WT DDX11 ) RPE-1 cells were transfected with ATG16L1-GFP for 24 h. Confocal imaging shows accumulation of ATG16L1 in the perinuclear area that was reverted by re-expressing the DDX11-Flag wild-type protein. Total cells analyzed: n = 50 per experiment, performed in triplicates. Scale bar: 10 µm. (C) Co-immunoprecipitation experiment using an anti-ATG16 antibody in control (CTRL) and DDX11 KO RPE-1 cells. Western blot analysis shows co-immunoprecipitation of ATG5-ATG12 in both control and DDX11 KO ( DDX11 KO) RPE-1 cells indicating that, in the absence of DDX11, the ATG12–ATG5-ATG16L1 complex formation is not affected. Asterisks denote the IgG heavy chains. (D) Control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO ( DDX11 KO + WT DDX11 ) RPE-1 cells were transfected with a vector expressing GFP-ATG16L1 for 48 h. Co-immunoprecipitation experiment was performed using anti-GFP antibody conjugated beads. The indicated proteins were detected by western blot of the pulled-down sample using specific antibodies. (E) Control (CTRL), DDX11 KO ( DDX11 KO) and DDX11-complemented DDX11 KO ( DDX11 KO + WT DDX11 ) RPE-1 cells were transfected with GFP-ATG16L1 and mCherry-LC3 for 24 h. Then, cells were processed as described for panel A. Confocal imaging reveals that ATG16L1 and LC3 do not colocalize in DDX11 KO cells. This phenotype is reversed by re-expressing the DDX11-Flag wild-type protein. Scale bar: 10 µm.

Article Snippet: The following antibodies were used: mouse monoclonal anti DDX11 (Santa Cruz Biotechnology, sc271711); mouse monoclonal anti-Flag antibody (Merck, F1804); rabbit polyclonal anti-LC3 (Novus Biologicals, NB100–2220); mouse monoclonal anti-LAMP1 (Cell Signaling Technology, 15665); rabbit polyclonal anti-SQSTM1 (MBL International, PM045); mouse monoclonal anti-TUBA/α-tubulin antibody (Merck, T6199); rabbit polyclonal anti-ATG16L1 antibody (MBL Life Sciences, PM040); rabbit monoclonal anti-ATG5 antibody (Cell Signaling Technology, 129945); rabbit polyclonal anti-BECN1/BECLIN1 antibody (Cell Signaling Technology, 3738S); rabbit polyclonal anti-AKT antibody (Cell Signaling Technology, 9272S); rabbit polyclonal anti-phospho-AKT (Ser473) antibody (Cell Signaling Technology, 9271 L); rabbit polyclonal anti-BRIP1/FANCJ antibody (Novus Biologicals, NBP1–31883); mouse monoclonal anti-histone H3 antibody (Abcam, ab24834); rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956); mouse and rabbit HRP-conjugated secondary antibody (Merck, A9044 and 12–348, respectively); Alexa Fluor 488 anti-rabbit antibody, Alexa Fluor 555 anti-mouse antibody and Alexa Fluor 633 anti-mouse antibody (Thermo Fisher Scientific, A-11008, A-21137, A-21146, respectively).

Techniques: Control, Transfection, Imaging, Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: Autophagy

Article Title: Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

doi: 10.1080/15548627.2025.2507617

Figure Lengend Snippet: Schematic model describing a putative role of DDX11 in autophagy pathway regulation. DDX11 exerts its function as a DNA helicase in the nuclear compartment. Within the cytoplasm, DDX11 cooperates with SQSTM1 and ATG16L1 to promote phagophore formation as well as the autophagic flux (bold green and blue dotted arrows, respectively; left side ). Absence of DDX11 affects intracellular localization of the ATG16L1 autophagy precursor, which in turn impairs LC3 lipidation and autophagosome fusion with lysosomal compartment, ultimately reducing the autophagic flux (thin green and blue dotted arrows, respectively; right side ).

Article Snippet: The following antibodies were used: mouse monoclonal anti DDX11 (Santa Cruz Biotechnology, sc271711); mouse monoclonal anti-Flag antibody (Merck, F1804); rabbit polyclonal anti-LC3 (Novus Biologicals, NB100–2220); mouse monoclonal anti-LAMP1 (Cell Signaling Technology, 15665); rabbit polyclonal anti-SQSTM1 (MBL International, PM045); mouse monoclonal anti-TUBA/α-tubulin antibody (Merck, T6199); rabbit polyclonal anti-ATG16L1 antibody (MBL Life Sciences, PM040); rabbit monoclonal anti-ATG5 antibody (Cell Signaling Technology, 129945); rabbit polyclonal anti-BECN1/BECLIN1 antibody (Cell Signaling Technology, 3738S); rabbit polyclonal anti-AKT antibody (Cell Signaling Technology, 9272S); rabbit polyclonal anti-phospho-AKT (Ser473) antibody (Cell Signaling Technology, 9271 L); rabbit polyclonal anti-BRIP1/FANCJ antibody (Novus Biologicals, NBP1–31883); mouse monoclonal anti-histone H3 antibody (Abcam, ab24834); rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956); mouse and rabbit HRP-conjugated secondary antibody (Merck, A9044 and 12–348, respectively); Alexa Fluor 488 anti-rabbit antibody, Alexa Fluor 555 anti-mouse antibody and Alexa Fluor 633 anti-mouse antibody (Thermo Fisher Scientific, A-11008, A-21137, A-21146, respectively).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Betulinic Acid Suppresses UBE2T Expression via MAPK / ERK Inhibition to Block FANCI and FANCD2 Monoubiquitination in Glioblastoma

doi: 10.1111/jcmm.71000

Figure Lengend Snippet: Ba suppresses FANCI/FANCD2 monoubiquitination, FANCD2 foci formation, and downstream DNA repair complex assembly in glioma cells. (A–C) Western blot analysis of FANCI and FANCD2 monoubiquitination in U87MG (A), LN229 (B), and U251 (C) cells treated with cisplatin (CDDP) or BA plus CDDP. Cisplatin induces robust monoubiquitination of both FANCI and FANCD2, whereas BA pretreatment markedly diminishes these modifications (red arrows), indicating inhibition of FA pathway activation. β‐Actin served as a loading control. (D–F) Immunofluorescence analysis show the FANCD2 nuclear foci formation in U87MG (D), LN229 (E), and U251 (F) cells. Blue: DAPI‐stained nuclei; green: FANCD2 Foci. Quantification (right) revealed a significant decrease in the percentage of FANCD2‐positive nuclei in BA + CDDP‐treated cells compared with CDDP alone. Data are presented as mean ± SEM ( n = 3 independent experiments); * p < 0.05 vs. NC, # p < 0.05 vs. CDDP. (G–I) Co‐immunoprecipitation (Co‐IP) assays examining FANCD2‐associated repair proteins. In all three glioma cell lines, cisplatin enhances the interaction between FANCD2 and REV1, BRCA1 and ERCC1, reflecting activation of translesion synthesis (TLS) and homologous recombination (HR) branches of DNA repair. BA pretreatment substantially weakens these interactions, suggesting that BA not only blocks FANCD2 monoubiquitination but also disrupts the recruitment of downstream repair effectors.

Article Snippet: Primer sequence targeted human UBE2T ( NM_014176 , OriGene, Cat. No. HP210681 ) and β‐Actin ( NM_001101 , OriGene, Cat. No. HP204660 ) were used.

Techniques: Western Blot, Inhibition, Activation Assay, Control, Immunofluorescence, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Translesion Synthesis, Homologous Recombination

Journal: Journal of Cellular and Molecular Medicine

Article Title: Betulinic Acid Suppresses UBE2T Expression via MAPK / ERK Inhibition to Block FANCI and FANCD2 Monoubiquitination in Glioblastoma

doi: 10.1111/jcmm.71000

Figure Lengend Snippet: Ba selectively downregulates UBE2T expression through transcriptional suppression rather than mRNA stability, translation, or protein degradation. (A) WB assay show the expression of Fanconi anaemia (FA) pathway components in U87MG, LN229, and U251 glioma cells. Only UBE2T shows a marked reduction in protein abundance following BA pretreatment prior to cisplatin (CDDP) exposure (red arrows). (B–D) WB assay show that overexpression (OE) of UBE2T restores ID2 monoubiquitination suppressed by BA (red arrows), whereas USP1 knockdown (KD) or pharmacological inhibition with ML323 fail to rescue UBE2T levels in U87MG (B), LN229 (C), and U251 (D) glioma cells. β‐Actin served as a loading control. (E–G) Actinomycin D (ActD) chase assays show comparable mRNA decay kinetics (K values) for UBE2T in CDDP and BA + CDDP groups across U87MG (E), LN229 (F), and U251 (G) glioma cells, indicating unaffected mRNA stability. (H) Polysome profiling reveals similar proportions of UBE2T transcripts in polysome and monosome fractions, suggesting no translational defect. (I) Cycloheximide (CHX) chase and MG132 proteasome inhibition assays show no significant changes in UBE2T protein stability in BA‐treated cells, excluding post‐translational degradation as the cause of UBE2T reduction.

Article Snippet: Primer sequence targeted human UBE2T ( NM_014176 , OriGene, Cat. No. HP210681 ) and β‐Actin ( NM_001101 , OriGene, Cat. No. HP204660 ) were used.

Techniques: Expressing, Quantitative Proteomics, Over Expression, Knockdown, Inhibition, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Betulinic Acid Suppresses UBE2T Expression via MAPK / ERK Inhibition to Block FANCI and FANCD2 Monoubiquitination in Glioblastoma

doi: 10.1111/jcmm.71000

Figure Lengend Snippet: Effects of BA on YY1/FAS, ERβ, insulin/IGF‐1, MAPK/ERK, and Notch signalling pathways regulating UBE2T expression in glioma cells. (A) WB assay show that BA suppresses the YY1/FAS axis, decreasing cleaved caspase‐8 and YY1 nuclear accumulation (red arrows); FASL co‐treatment failed to restore UBE2T (black arrows). (B) WB assay show that BA inhibits ERβ nuclear localization and ERK1/2 phosphorylation (red arrows); ERβ activation by DPN re‐stimulated p‐ERK1/2 and elevated UBE2T (red arrows). (C) WB assay show that the insulin/IGF‐1 pathway is unaffected by BA (black arrows), but IGF‐1 treatment enhances IGF‐1Rβ phosphorylation, increases p‐ERK1/2, and up‐regulates UBE2T (red arrows). (D) WB assay show that direct ERK activation (ERK agonist) increases UBE2T expression (red arrows), confirming ERK as a convergent effector. (E) BA does not alter Notch signalling, and DLL4‐Fc–induced Notch activation shows no impact on UBE2T. β‐Actin served as loading controls of total protein, and Lamin B1 for nuclear extracts. (F–H) The relative mRNA levels of UBE2T are detected by qPCR in U87MG (F), LN229 (G) and U251 glioma cell lines (H) additionally treated with agonists of the pathway mentioned above. “*” indicates significant difference compared with BA+CDDP group.

Article Snippet: Primer sequence targeted human UBE2T ( NM_014176 , OriGene, Cat. No. HP210681 ) and β‐Actin ( NM_001101 , OriGene, Cat. No. HP204660 ) were used.

Techniques: Expressing, Phospho-proteomics, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Betulinic Acid Suppresses UBE2T Expression via MAPK / ERK Inhibition to Block FANCI and FANCD2 Monoubiquitination in Glioblastoma

doi: 10.1111/jcmm.71000

Figure Lengend Snippet: ERK activation reverses the tumour‐suppressive effect of BA in cisplatin‐treated xenografts. (A) Representative images of xenograft tumours and quantitative analysis of tumour volumes in four groups: Control (NC), cisplatin (CDDP), BA + CDDP, and BA + CDDP + ERK agonist (ERKA). BA + CDDP treatment markedly reduces tumour growth compared with CDDP alone, while ERK activation restores tumour size to near‐control levels. Data are shown as mean ± SEM; * p < 0.05 vs. NC, # p < 0.05 vs. CDDP, † p < 0.05 vs. BA + CDDP. (B) Western blot analysis of xenograft lysates showing decreased UBE2T as well as monoubiquitination of FANCI and FANCD2 in the BA + CDDP group, along with suppressed ERK1/2 phosphorylation; ERK agonist treatment restores p‐ERK1/2 and UBE2T expression. β‐Actin served as the loading control for total protein. (C, D) Immunohistochemical staining of xenograft tumour sections confirming reduced UBE2T (C) and p‐ERK1/2 (D) expression in the BA + CDDP group, with re‐elevation following ERK agonist co‐treatment. Scale bar = 20 μm.

Article Snippet: Primer sequence targeted human UBE2T ( NM_014176 , OriGene, Cat. No. HP210681 ) and β‐Actin ( NM_001101 , OriGene, Cat. No. HP204660 ) were used.

Techniques: Activation Assay, Control, Western Blot, Phospho-proteomics, Expressing, Immunohistochemical staining, Staining