Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 promtoes aggregates deaggregation and clearance via autophagy. (a) Cells were treated with MG132 and immunostained with antibodies to ubiquitin (red) and LC3B-II (green). Arrows indicate ubiquitin-positive aggregates that colocalize with LC3B-positive autophagosomes. Scale bars: 10 μm. (b) Confocal images of TRIM44[OE-CON] and TRIM44[OE] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA (10 mM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (c) Confocal images of TRIM44[KD-CON] and TRIM44[KD] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM). Arrows indicate cells with remaining aggregates. Scale bars: 10 µm. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (d, e) Confocal images of WT or ATG5 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (d). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (e). (f, g) Confocal images of WT or BECN1 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells were treated with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (f). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of BECN1 and TRIM44 were assayed by western blot (g). (h, i) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. The status of aggregates after MG132 treatment was quantified in the histogram. Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (h). (j) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) together with MG132 (5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm.

Article Snippet: Anti-TRIM44 polyclonal antibody (Proteintech Group, 11,511-1-AP); anti-Ub antibody (Biolegend, 646,301); anti-mCherry antibody (ThermoFisher, PA5-34,974); anti-VIM antibody (ThermoFisher, MA5-11,883); anti-NFE2L2/NRF2 antibody (ThermoFisher, PA5-27,882); anti-20S proteasome antibody (MilliporeSigma, ST1049); anti-TUBG/γ-Tubulin antibody (MilliporeSigma, T5326); anti-ATG5 antibody (Novus Biologicals, NBP2-24,389); Anti-GFP antibody (Santa Cruz Biotechnology, sc-9996); anti-ACTB/β-Actin antibody (Santa Cruz Biotechnology, sc-47,778), anti-HA antibody (Santa Cruz Biotechnology, sc-805); anti-BECN1 antibody (Cell Signaling Technology, 4122); anti-HDAC6 antibody (Cell Signaling Technology, 7558); anti-LC3B antibody (Cell Signaling Technology, 3868); anti-SQSTM1/p62 antibody (Cell Signaling Technology, 88,588); anti-cleaved PARP antibody (Cell Signaling Technology, 5625); Alexa Fluor 594 donkey anti rabbit IgG(H + L) (Invitrogen, A21207); Alexa Fluor 546 goat anti mouse IgG(H + L) (Invitrogen, A11003).

Techniques: Ubiquitin Proteomics, Transfection, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a Representative immunohistochemistry showing the expression levels of ANXA1, SQSTM1, and p-AKT (S473) in the NPCs with different metastatic potentials. Scale bar = 50 μm. b Correlation analysis of ANXA1 and SQSTM1expressin in 127 NPCs based on immunohistochemistry scores (Spearman’s correlation test). c ANXA1 positively regulating SQSTM1 expression. Western blot analysis showing the expression levels of SQSTM1 in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. d ANXA1 regulating SQSTM1 expression through autophagy. Western blot analysis showing the expression levels of SQSTM1 in the ANXA1 knockdown 5–8F cells and scramble shRNA control cells treated with BAF or CQ (left) or transfected with BECN1 or ATG5 siRNA (right). Scr, scramble. Vector, an empty vector; KD, knockdown; OE, overexpression; BAF, bafilomycin A1; CQ, chloroquine

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Immunohistochemistry, Expressing, Western Blot, Control, Knockdown, shRNA, Transfection, Plasmid Preparation, Over Expression

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a Western blot analysis showing the expression levels of BECN1, SQSTM1 and LC3-II in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. b Electron microscopic examination showing autophagic vacuoles ( red arrows ) in the cytoplasm of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Scale bar = 5 μm. c ( top ) Immunofluorescent staining showing the number of LC3 puncta in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Cells was stained by indirect immunefluorescence using anti-LC3 antibody and observed by confocal microscopy (bottom). The number of LC3 puncta per cell was quantified. Scale bar = 10 μm. d ANXA1 knockdown enhances autophagic flux (left). Western blot analysis showing the LC3-II levels in the ANXA1 KD 5–8F cells and scramble shRNA control cells treated with BAF (middle and right). The number of EGFP-LC3 puncta in the ANXA KD1 5–8F cells and scramble shRNA control cells treated with BAF. Cells were transfected with 1 μg of EGFP-LC3 plasmid for 24 h, and BAF treatment for an additional 24 h, thereafter the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. e Autophagy induction by ANXA1 knockdwon follows an autophagic pathway dependent of BECN1 and ATG5. (left) Western blot analysis showing the LC3-II levels in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with BECN1 or ATG5 siRNA. (middle and right) The number of EGFP-LC3 puncta in the ANXA KD 5–8F cells and scramble shRNA control cells transfected with BECN1 or ATG5 siRNA. Cells were cotransfected with 1 μg of EGFP-LC3 plasmid and siRNA against BECN1 or ATG5 for 24 h, and the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. Mean ± SD and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns, no significance

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Control, Staining, Confocal Microscopy, Knockdown, shRNA, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a Cell migration (top) and invasion (bottom) in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with BECN1 siRNA and negative control siRNA respectively. b Cell migration (top) and invasion (bottom) in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with ATG5 siRNA and negative control siRNA respectively. c Cell migration (top) and invasion (bottom) in the ANXA1 KD 5–8F cells and scramble shRNA control cells treated with 3-MA (3-methyladenine). Mean ± SD ( n = 3 replicates) and statistical significance are denoted; *** P < 0.001; ns, no significance. d The in vivo metastasis assays of ANXA1 KD 5–8F cells stably transfected with lentiviral vector-expressing BECN1 or ATG5 shRNA and control cells using experimental lung metastasis model in nude mice ( n = 10 mice each). The representative photography of lungs (top) and H&E staining of lung sections (middle) from each group showing metastatic tumors (white arrows), and the numbers of surface lung metastases per mouse are shown (bottom). Mean ± SEM ( n = 10) and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns, no significance. Scale bar = 100 μm

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Migration, shRNA, Control, Transfection, Negative Control, In Vivo, Stable Transfection, Plasmid Preparation, Expressing, Staining

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a QRT-PCR showing the mRNA expression levels of ANXA1, E-cadherin, N-cadherin, Vimentin, and Snail in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. b Western blot analysis showing the protein expression levels of E-cadherin, N-cadherin, Vimentin and Snail in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. c Western blot analysis showing the protein expression levels of E-cadherin, N-cadherin, Vimentin, and Snail in the EphA2 KD 5–8F cells and scramble shRNA control cells transfected with BECN1 siRNA or ATG5 siRNA (left), or treated with 3-MA (right). d Western blot analysis showing the Snail protein expression level in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with SQSTM1 expression plasmid, and ANXA1 OE 6–10B cells and vector control cells transfected with SQSTM1 siRNA. e QRT-PCR showing the Snail mRNA expression level in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with SQSTM1 expression plasmid, and ANXA1 OE 6–10B cells and vector control cells transfected with SQSTM1 siRNA. Mean ± SD ( n = 3 replicates) and statistical significance are denoted; * P < 0.05; ** P < 0.01; *** P < 0.001; no, no significance

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, shRNA, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: In this model, ANXA1 activates PI3K/AKT signaling, leading to BECN1 and ATG5-dependent autophagy inhibition; autophagy inhibition makes SQSTM1 accumulation, which inhibits the degradation of Snai1; the increased Snai1 induces EMT-like alterations, and then promotes NPC cell migration and invasion and metastasis

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Inhibition, Migration

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