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: The Journal of Biological Chemistry

Article Title: Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents

doi: 10.1016/j.jbc.2024.105632

Figure Lengend Snippet: Western blot analysis with pulldown assay for the binding of (+)-( S )-bakuchiol to PHBs and VDACs in the cell extracts. A – C , the presence of PHB1, PHB2, VDAC1, and VDAC2 in the beads or supernatant samples of the pulldown assay using 17 -conjugated with streptavidin beads from lysates of MDCK ( A ), A549 ( B ), or HeLa ( C ) cells infected with/without A/PR/8/34 virus was analyzed by Western blotting using the specific antibodies in Table S1 . 15 - and 16 -conjugated with streptavidin beads were used as negative controls. Data are representative of three independent experiments, and the results were reproducible. D – F , expression plasmids of PHB1 or PHB2 harboring a FLAG tag at its C terminus, driven by a CMV (pCMV-PHB1-FLAG or pCMV-PHB2-FLAG) were transfected into 293T cells. pCMV-FLAG excluding the PHB gene (pCMV-empty) was used as a negative control. Each supernatant in the transfectant was used for a pulldown assay. The presence of overexpressed PHB1-FLAG and PHB2-FLAG proteins similar to PHB1 and PHB2 in the pulldown samples of 15 -, 16 -, or 17 -conjugated beads or supernatants was analyzed by Western blotting using the specific antibodies in Table S1 ( D ). Signal intensities were measured using ImageJ software, and the protein levels of PHB1- (n = 4 each) ( E ) and PHB2-FLAG (n = 4 each) ( F ) in the 15 -, 16 -, or 17 -conjugated beads were calculated relative to those of pCMV-empty-transfected samples. Data represent the mean ± SEM and are representative of four independent experiments, and the results were reproducible. ∗ p < 0.05 or ∗∗ p < 0.01 denote the significance levels for the indicated comparisons determined through one-way ANOVA, followed by the post hoc Tukey’s test. CMV, cytomegalovirus; MDCK, Madin-Darby canine kidney; PHB, prohibitin; VDAC, voltage-dependent anion channel.

Article Snippet: The expression plasmid of PHB1 (NM_002634, restriction site: Sgf I- Mlu I) harboring a FLAG tag at its C terminus, which was driven by a CMV promoter (pCMV-PHB1-FLAG), was purchased from Origene Inc. (#RC201229).

Techniques: Western Blot, Binding Assay, Infection, Virus, Expressing, FLAG-tag, Transfection, Negative Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents

doi: 10.1016/j.jbc.2024.105632

Figure Lengend Snippet: Immunofluorescence analysis for intracellular interactions with (+)-( S )-bakuchiol and PHB1, PHB2, VDAC2, or mitochondria. MDCK, A549, or HeLa cells were seeded on glass bottom wells at a density of 5 × 10 3 cells. The cells were fixed and subsequently permeabilized. The cells were blocked for endogenous biotin and then treated with 25 μM 15 , 16 , or 17 for 24 h. Biotin-linker compounds was detected with Alexa Fluor 488–conjugated streptavidin ( green ). PHB1 ( A ), PHB2 ( B ), VDAC2 ( C ), or ATP5B ( D ), a mitochondrial maker, were detected using their specific antibodies ( red ). Cell nuclei were stained with DAPI ( blue ). Images were captured on a confocal laser-scanning fluorescence microscope. The indicated images were the merged images of three colors ( green , red , and blue ). The white scale bar in each image represents 50 μm. The white squares in images of 17 -treated cells were enlarged, and white arrows indicate regions of colocalization, as indicated by the yellow color . The white scale bar in the enlarged image represents 10 μm. Data are representative of three or four independent experiments, and the results were reproducible. DAPI, diamidino-2-phenylindole; MDCK, Madin-Darby canine kidney; PHB, prohibitin; VDAC, voltage-dependent anion channel.

Article Snippet: The expression plasmid of PHB1 (NM_002634, restriction site: Sgf I- Mlu I) harboring a FLAG tag at its C terminus, which was driven by a CMV promoter (pCMV-PHB1-FLAG), was purchased from Origene Inc. (#RC201229).

Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents

doi: 10.1016/j.jbc.2024.105632

Figure Lengend Snippet: Effects of PHB2 or VDAC2 knockdown on the inhibition of influenza viral growth by (+)-( S )-bakuchiol. A , B, D , and E , MDCK cells were transfected with the dicer-substrate siRNA (DsiRNA) of 10 nM human PHB2 or 30 nM human VDAC2#1 , #2 , or #3 using reverse transfection and incubated for 48 h. Further, 10 or 30 nM negative control (NC) DsiRNA was used as a negative control. The DsiRNA-treated cells were incubated with 12.5 μM 1 and with 0.1 MOI of A/PR/8/34 virus for 24 h, and 0.125% DMSO was used as a negative control. The cells were lysed in a SDS sample buffer, and the presence of PHB1, PHB2, and influenza NP ( A ) or VDAC2 and influenza NP ( D ) was analyzed using Western blotting, respectively. Signal intensities were measured using ImageJ software, and the protein levels of influenza NP in PHB2 DsiRNA- (n = 4 each) ( B ) or VDAC2# 1 to 3 DsiRNA- (n = 3 each) ( E ) transfected cells were analyzed relative to those in the NC and DMSO-treated cells. The indicated protein levels were normalized to those of β-actin. Data represent the mean ± SEM and are representative of four or three independent experiments, and the results were reproducible. ∗ p < 0.05, ∗∗ p < 0.01, or ∗∗∗ p < 0.001 denote the significance levels for the indicated comparisons through one-way ANOVA, followed by the post-hoc Tukey’s test. ( C and F ) MDCK cells (1 × 10 5 cells/well) were transfected with 10 nM human PHB2 or 30 nM human VDAC2#1 , 2 , or 3 using reverse transfection and incubated for 24 h. Furthermore, 10 or 30 nM DsiRNA was used as the negative control (NC). The DsiRNA-treated cells were infected with 0.0001 MOI of A/PR/8/34 virus for 1 h. Subsequently, 12.5 μM 1 was added to the cells in the medium supplemented with 3 μg/ml L-tosylamido-2-phenyl ethyl chloromethyl ketone–treated trypsin. DMSO (0.125%) was used as the negative control. The cells were incubated for 24, 48, or 72 h. At specified time points, culture-conditioned media were collected from each well and viral titers in the conditioned media were determined in NC- or PHB2 DsiRNA- (n = 11–15 each) transfected and virus-infected cells ( C ), or NC- or VDAC2#1 to 3 DsiRNA- (n = 7–9 each) ( F ) transfected and virus-infected cells. The data represent the mean ± SEM and are representative of five or three independent experiments, and the results were reproducible. ∗ p < 0.05, ∗∗ p < 0.01, or ∗∗∗ p < 0.001 was considered statistically significant for the comparisons of both NC transfection and DMSO treatment by one-way ANOVA, followed by the post hoc Tukey’s test. † p < 0.05, †† p < 0.01, or ††† p < 0.001 was considered statistically significant for the comparison of both NC transfection and bakuchiol treatment by Student’s t test. ‡ p < 0.05 was considered statistically significant for the comparisons of both PHB2 transfection and bakuchiol treatment by Student’s t test. § p < 0.05 was considered statistically significant for the comparison of both NC transfection and bakuchiol treatment by one-way ANOVA with the post hoc Tukey’s test. DMSO, dimethyl sulfoxide; MDCK, Madin-Darby canine kidney; MOI, multiplicity of infection; NP, NP, nucleoprotein; PHB, prohibitin; VDAC, voltage-dependent anion channel.

Article Snippet: The expression plasmid of PHB1 (NM_002634, restriction site: Sgf I- Mlu I) harboring a FLAG tag at its C terminus, which was driven by a CMV promoter (pCMV-PHB1-FLAG), was purchased from Origene Inc. (#RC201229).

Techniques: Inhibition, Transfection, Incubation, Negative Control, Virus, Western Blot, Software, Infection, Comparison

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: RHBDD2: a 5-fluorouracil responsive gene overexpressed in the advanced stages of colorectal cancer.

doi: 10.1007/s13277-012-0503-3

Figure Lengend Snippet: Fig. 1 In silico RHBDD2 expression profile based on oligo-microarray datasets of CRC samples. a Strategy fol- lowed to obtain the compiled dataset. b A positive association between RHBDD2 expression and tumor progression is ob- served, with a significant in- crease in stage C. The asterisk indicates a p value of <0.001 between normal and the stages B, C and D. The triangle indi- cates a p value of <0.01 between stage A and stages C and D

Article Snippet: RHBDD2 protein was detected using a commercially anti-RHBDD2 polyclonal antibody (TA306891, Origene, USA) and horseradish peroxidase (HRP)-conjugated antirabbit secondary antibody (Dako, Denmark, 1:2,000) followed by chemiluminescence autoradiography.

Techniques: In Silico, Expressing, Microarray

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: RHBDD2: a 5-fluorouracil responsive gene overexpressed in the advanced stages of colorectal cancer.

doi: 10.1007/s13277-012-0503-3

Figure Lengend Snippet: Fig. 2 Comparison of RHBDD2 protein expression between normal and CRC samples. Although normal mucosa showed RHBDD2 expression (a), an increase in the intensity of reaction was usually found in colorectal carcinomas (b). Microphotographs are ×40

Article Snippet: RHBDD2 protein was detected using a commercially anti-RHBDD2 polyclonal antibody (TA306891, Origene, USA) and horseradish peroxidase (HRP)-conjugated antirabbit secondary antibody (Dako, Denmark, 1:2,000) followed by chemiluminescence autoradiography.

Techniques: Comparison, Expressing

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: RHBDD2: a 5-fluorouracil responsive gene overexpressed in the advanced stages of colorectal cancer.

doi: 10.1007/s13277-012-0503-3

Figure Lengend Snippet: Fig. 3 RHBDD2 mRNA/ protein expression in CRC samples according Dukes’ stages. a RT-qPCR analysis of RHBDD2 mRNA in a set of CRC samples and the cell line Colo205. b IHC analysis of RHBDD2 protein expression in 105 CRC samples. Samples were clustered into three groups according to Dukes’ stages: a, b and c–d. A positive significant association between intensity of reaction and Dukes’ stages was found (p<0.01). The bottom panel shows microphotographs of CRC tissue sections of the IHC results obtained in the dif- ferent stages. An increase in the intensity of reaction (brown staining) is clearly associated with the advanced stages of the disease. All microphotographs are ×40

Article Snippet: RHBDD2 protein was detected using a commercially anti-RHBDD2 polyclonal antibody (TA306891, Origene, USA) and horseradish peroxidase (HRP)-conjugated antirabbit secondary antibody (Dako, Denmark, 1:2,000) followed by chemiluminescence autoradiography.

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: RHBDD2: a 5-fluorouracil responsive gene overexpressed in the advanced stages of colorectal cancer.

doi: 10.1007/s13277-012-0503-3

Figure Lengend Snippet: Fig. 4 RHBDD2 expression analysis in the Colo205 cell line. a A strong reaction is observed in the cytoplasm of Colo205 cells incubated with RHBDD2 antibody (left) compared with the negative control (right). b RHBDD2 protein is localized in the endoplasmic reticulum of the Colo205 cells. c RHBDD2 mRNA expression in Colo205 cells after being exposed to different concentrations of 5FU. Asterisk indicates a p value of <0.001 in the 5FU-treated cells compared with control. d Western blotting of the 5FU assay

Article Snippet: RHBDD2 protein was detected using a commercially anti-RHBDD2 polyclonal antibody (TA306891, Origene, USA) and horseradish peroxidase (HRP)-conjugated antirabbit secondary antibody (Dako, Denmark, 1:2,000) followed by chemiluminescence autoradiography.

Techniques: Expressing, Incubation, Negative Control, Control, Western Blot

Journal: Neurochemical Research

Article Title: Sphingosine 1-Phoshpate Receptors are Located in Synapses and Control Spontaneous Activity of Mouse Neurons in Culture

doi: 10.1007/s11064-022-03664-3

Figure Lengend Snippet: Primary antibodies used for Western blot (WB) and immunofluorescence microscopy in neurons (IFn) or brain slices (IFs)

Article Snippet: Rabbit anti-S1PR2 , 1:500 (WB)/1:250 (IFn)/1:100 (IFs) , Origene (AP01311PU-N).

Techniques: Western Blot, Immunofluorescence, Microscopy