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: PLoS ONE

Article Title: Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

doi: 10.1371/journal.pone.0007475

Figure Lengend Snippet: List of antibodies used for staining.

Article Snippet: Fibroblasts-Reticular , ER-TR7 , Rat IgG2a , Cedarlane, Burlington, NC.

Techniques: Staining, Plasmid Preparation

Journal: PLoS ONE

Article Title: Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

doi: 10.1371/journal.pone.0007475

Figure Lengend Snippet: PBMC were cultured in SFM at 5×10 5 cells/ml in 8-well glass slides for 7 days. Cells were then air-dried, fixed, and stained with antibodies. A) CD13, B) CD14, C) CD45RA, D) CD45RB, E) CD45RO, F) Irrelevant mouse IgG2a control. Cells were then counterstained with hematoxylin to identify nuclei. Positive staining was identified by red staining, with nuclei counterstained blue. Solid arrow points to a fibrocyte, open arrow points to a macrophage, and asterisk indicates a cluster of lymphocytes. Photomicrographs are representative results from at least four different donors. Bar is 50 µm.

Article Snippet: Fibroblasts-Reticular , ER-TR7 , Rat IgG2a , Cedarlane, Burlington, NC.

Techniques: Cell Culture, Staining, Control

Journal: PLoS ONE

Article Title: Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

doi: 10.1371/journal.pone.0007475

Figure Lengend Snippet: PBMC were cultured as described in . Cells were then air-dried, fixed, and stained with antibodies against A) CD18, B) CD68, C) CD163, D) CD206, E) CD209, and F) mouse IgG1 control. Cells were then counterstained with hematoxylin to identify nuclei. Bar is 50 µm.

Article Snippet: Fibroblasts-Reticular , ER-TR7 , Rat IgG2a , Cedarlane, Burlington, NC.

Techniques: Cell Culture, Staining, Control

Journal: PLoS ONE

Article Title: Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

doi: 10.1371/journal.pone.0007475

Figure Lengend Snippet: PBMC were cultured in SFM at 5×10 5 cells/ml in 8-well glass slides for 7 days in the presence of 10% FCS and 10 ng/ml M-CSF. Cells were then air-dried, fixed and stained with 5 µg/ml mouse IgG1 monoclonal antibodies against CD13, CD14, CD32, CD163, CD90, mouse IgG1, proyly-4-hydroxylase, 25F9, S100A8/A9, PM-2K, TE-7, and collagen-I. Cells were then counterstained with hematoxylin to identify nuclei. Inserts indicate staining at higher magnification. Bar is 50 µm.

Article Snippet: Fibroblasts-Reticular , ER-TR7 , Rat IgG2a , Cedarlane, Burlington, NC.

Techniques: Cell Culture, Staining, Bioprocessing

Journal: PLoS ONE

Article Title: Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

doi: 10.1371/journal.pone.0007475

Figure Lengend Snippet: PBMC were cultured in SFM at 5×10 5 cells/ml in 8-well glass slides for 1 hour ( ex vivo ) or 7 days. Cells were then air-dried, fixed, and stained with antibodies against A and B) CD64, C and D) CD32, E and F) CD16, G and H) CD32a, I and J) CD32b, or K and L) goat IgG control antibodies. Cells were then counterstained with hematoxylin to identify nuclei. Asterisks are to the right of macrophages. Bar is 50 µm.

Article Snippet: Fibroblasts-Reticular , ER-TR7 , Rat IgG2a , Cedarlane, Burlington, NC.

Techniques: Cell Culture, Ex Vivo, Staining, Control

Journal: PLoS ONE

Article Title: Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

doi: 10.1371/journal.pone.0007475

Figure Lengend Snippet: PBMC were cultured as described in . Normal human dermal fibroblasts were cultured for 2 days in 8-well glass slides. Cells were then air-dried, fixed, and stained with antibodies. A and B) collagen-I, C and D) collagen IV, E and F) proyly-4-hydroxylase, G and H) rabbit IgG control antibody. Cells were then counterstained with hematoxylin to identify nuclei. Asterisks are to the right of macrophages. Bar is 50 µm.

Article Snippet: Fibroblasts-Reticular , ER-TR7 , Rat IgG2a , Cedarlane, Burlington, NC.

Techniques: Cell Culture, Staining, Control

Journal:

Article Title: Axoplasm Isolation from Peripheral Nerve

doi: 10.1002/dneu.20755

Figure Lengend Snippet: (A) Electron micrograph of negative staining of axoplasm from the isotonic squeeze method shows a nonhomogeous preparation containing vesicular structures of different sizes. (B) Negative staining of axoplasm from the hypotonic extraction procedure shows a more homogenous preparation containing numerous small size vesicle-like structures. Scale bar in both images 200 nm. (CF) Western blot comparison of soluble and pellet fractions of axoplasms extracted by different methods and cleared by regular centrifugation or ultracentrifuge (UC). Albumin and transferrin were used to monitor serum contamination, CNPase and GFAP for Schwann cells and other glia, importins and dynein intermediate chain (IC) for cell body components and retrograde signaling complexes, and general Erk1 and Erk2 MAP kinases (gERK) as a loading control. (G) Levels of different proteins in the soluble fraction from hypotonic extraction axoplasm as a percentage of their level in isotonic squeeze axoplasm. All data was normalized to the average of the control group (isotonic squeeze axoplasm), taken as 100% in each blot. Statistics were by one-sample t-test with hypothesized population mean 100, p-value < 0.05. Note markedly reduced levels of serum and glia contaminants, versus enrichment of dynein associated proteins. 80 μg protein per lane.

Article Snippet: Mouse anti-Dynein intermediate chain clone 74.1 was from Chemicon (MAB1618), rabbit anti-NFH was from Chemicon (AB1989); mouse anti-NFH clone N52 was from Sigma; mouse anti-Importin β clone 31H4 was from Sigma (I2534); mouse anti-CNPase was from Chemicon (MAB326); mouse anti-GFAP clone G-A-5 was from Sigma (G6171); rabbit anti-albumin and rabbit anti-transferrin were from Cedarlane (CLAG5140 and GLAG5240 respectively); mouse anti-tubulin β3 was from Sigma (T2200); and rabbit anti-gERK was from Sigma (M5670).

Techniques: Negative Staining, Western Blot, Centrifugation