Journal: International journal of oncology

Article Title: An asymmetrically dimethylarginated nuclear 90 kDa protein (p90aDMA) induced by interleukin (IL)-2, IL-4 or IL-6 in the tumor microenvironment is selectively degraded by autophagy.

doi: 10.3892/ijo.2016.3450

Figure Lengend Snippet: Figure 5. The p90aDMA and p70aDMA proteins accumulate in the nucleus of cancer cells and degrade in response to cellular stresses. (A) Nuclear and cytoplasmic extracts were prepared after MDA-MB‑231 cells were cultured with IL-2 or IL-6 as indicated. Fractions were analyzed by western blot analysis using antibody to ASYM24. TBP was probed as a loading control. (B and C) MDA-MB‑231 cells or HeLa cells were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. (D) HeLa cells were cultured in HBSS medium for 24 h to induce autophagy or left in the complete culture medium. The cells were harvested and nuclear and cytoplasmic extracts were prepared for western blot analysis using antibodies to LC3 and ASYM24, respectively. Fractionation efficiency was determined by localization of β-actin (cytoplasm) and TBP (nucleus).

Article Snippet: Target Source Host Dilution Catalog SYM11 Merck Millipore, MA, USA Rabbit 1:1,000 07-413 ASYM24 Merck Millipore, MA, USA Rabbit 1:500 07-414 LC3B Cell Signaling Technology, Inc. (CST), MA, USA Rabbit 1:1,000 #3868 ATG5 Cell Signaling Technology, Inc. (CST), MA, USA Rabbit 1:1,000 #12994 TBP Proteintech, Wu Han, China Rabbit 1:500 22006-1-AP β-actin Zen Bioscience, Cheng Du, China Mouse 1:20,000 70068 that were significantly reduced regardless of the interleukin stimulation (Fig. 2).

Techniques: Cell Culture, Western Blot, Control, Fractionation

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: The Journal of Biological Chemistry

Article Title: LY6D-induced macropinocytosis as a survival mechanism of senescent cells

doi: 10.1074/jbc.RA120.013500

Figure Lengend Snippet: LY6D-induced vacuoles are not derived from autophagy. A , U2OS cells transfected with pcDNA3-HA-LY6D were incubated with 50-nM LysoTracker for 2 h and observed under fluorescence microscope. Arrowheads indicate examples of cytoplasmic vacuoles. Bars, 20 μm. B , U2OS cells pretreated with 10-μM 3-MA for 6 h were then transfected with pcDNA3-HA-LY6D and subjected to quantification of vacuole-forming cells. C , U2OS cells transfected with siRNA for ATG5 and with pcDNA3-HA-LY6D were subjected to immunoblot analysis. D , U2OS cells transfected with siRNA for ATG5 and with pBABEpuro GFP-LC3 as an autophagy marker, cultured in either the growth medium (10% FBS) or serum starvation medium (0.5% FBS) for 18 h, and subjected to quantification of LC3-dot–positive cells. Representative microscopic images ( left ) and the percentage of LC3-dot–positive cells ( right ) are shown. Bars , 50 μm. E , U2OS cells transfected as in panel C were subjected to quantification of vacuole-forming cells. Representative microscopic images ( left ) and the percentage of vacuole-forming cells ( right ) are shown. Bars , 50 μm. Data are mean ± SD ( n = 3 independent cultures except for that in panel B , where n = 4). Statistical significance is shown using the Student’s t-test analysis; ∗∗ p < 0.01; n.s., not significant ( p > 0.05). LY6D, lymphocyte antigen 6 complex, locus D; 3-MA, 3-methyladenine; FBS, fetal bovine serum.

Article Snippet: Anti-LY6D antibody (sc-373838), HRP-conjugated anti-rat antibody (sc-2032), anti-Pan P-Tyr PY99 antibody (sc-7020), anti-c-Src antibody (sc-19), anti-c-Yes antibody (sc-8403), anti-c-Fgr antibody (sc-130), anti-Lyn antibody (sc-015), anti-Hck antibody (sc-072), and anti-Blk antibody (sc-329) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-ATG5 (#12994), anti-P-Erk (T202/204) antibody (#9101S), anti-Erk antibody (#9102S), anti-P-Akt (S473) antibody (#9271S), and anti-Akt antibody (#9272S) were from Cell Signaling Technology (Beverly, MA); anti–γ-tubulin antibody (T6557) and anti–FLAG M2 monoclonal antibody (F3165) were from Sigma Aldrich; anti–P-SFK (Y418) antibody (44--660G) and anti–P-SFK (Y529) antibody (44--662G) were from Thermo Fisher Scientific (Waltham, MA); anti-HA 3F10 monoclonal antibody (1867423) and anti-HA 12CA5 monoclonal antibody (11666606) were from Roche (Basel, Switzerland); HRP-conjugated anti-mouse antibody (W4021) was from Promega; anti-Ras antibody (#06-570) was from Merck Millipore; anti-Fyn antibody (PC32) was from Oncogene science (Uniondale, NY); anti-caveolin 1 antibody was from BD Biosciences (San Jose, CA).

Techniques: Derivative Assay, Transfection, Incubation, Fluorescence, Microscopy, Western Blot, Marker, Cell Culture