Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: BH3 mimetics induce autophagy and apoptosis in the presence of Cdk inhibitors. (A) Human myeloma U266 cells were exposed to 500 nM GX-015-070 (GX) with or without 100 nM flavopiridol (FP) or 4 nM bortezomib (btz) as a positive control, followed by immunoblot analysis for LC3 (16 h) and PARP cleavage (24 h). In parallel, U266 cells were stably transfected with pEGFP-LC3, followed by exposure (16 h) to 500 nM GX with or without 100 nM FP, and then analyzed for GFP-LC3 puncta by confocal microscopy (bar = 10 μm). F+G, FP plus GX; CF, cleaved fragment; UT, untreated; DAPI, 4′,6-diamidino-2-phenylindole. (B) U266 cells were exposed (24 h) to 500 nM GX with or without 100 nM FP, followed by TUNEL staining using fluorescence microscopy (bar = 40 μm). (C) U266 cells were treated (16 h) with 500 nM GX with or without 100 nM FP in the presence or absence of 10 nM bafilomycin A1 (Baf A1), followed by analysis of autophagic flux by immunoblotting for LC3 (top). LC3-II was quantified relative to actin levels (bottom). Results represent fold increases over the vehicle-treated control (Veh) (means ± SD for three experiments). (D) Alternatively, U266 cells were transiently transfected with a pBABE-puro mCherry-EGFP-LC3B plasmid. After 6 h, cells were treated with 500 nM GX with or without 100 nM FP for an additional 16 h, followed by analysis of autophagic flux by confocal microscopy (bar = 5 μm).

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Positive Control, Western Blot, Stable Transfection, Transfection, Confocal Microscopy, TUNEL Assay, Staining, Fluorescence, Microscopy, Control, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Cotreatment with BH3 mimetics and Cdk inhibitors results in inefficient autophagy. (A) U266 cells were exposed (16 h) to 500 nM GX with or without 100 nM FP and then examined by electron microscopy (bar = 1 μm). Asterisks indicate deformed mitochondria. N, nucleus; M, mitochondrion; L, lysosome; G, Golgi apparatus; C, centrosome; A, autophagosome; AL, autolysosome; AV, autophagic vacuoles with clear content (empty). (B) In parallel, a filter trap assay using dot or slot blots probed with an antiubiquitin antibody (α-ubi) was performed to monitor the intracellular accumulation of SDS-insoluble ubiquitin-positive protein aggregates. (C) U266 cells were treated (16 h) with 500 nM GX with or without 100 nM FP or 5 nM SCH727965, after which immunoblot analysis was performed to monitor the accumulation of polyubiquitinated (polyUbi) proteins using an antiubiquitin antibody. (D and E) Alternatively, cells were stained with antiubiquitin antibody (D) to monitor intracellular ubiquitin-positive protein aggregates (arrows) or with LysoTracker (E) to visualize lysosomes (arrows).

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Electron Microscopy, TRAP Assay, Ubiquitin Proteomics, Western Blot, Staining

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Cdk inhibition downregulates SQSTM1/p62 but fails to affect LC3 processing during autophagy. (A) U266 cells were treated with 500 nM GX with or without 100 nM FP for 6, 16, 24, and 48 h, after which immunoblot analysis was performed to monitor LC3 processing and p62 expression. (B) Blots of p62 were quantified relative to tubulin (Tub) values (fold increase over the vehicle-treated control) (results represent means ± SD for three experiments). (C) RPMI8226 and U266 cells were treated (16 h) with GX (500 nM) with or without FP (100 nM) or SCH727965 (5 nM), after which LC3-II and p62 levels were determined by immunoblot analysis. (D) U266 cells were treated (6 h) with 500 nM GX (as indicated on the x axis) with or without 100 nM FP, after which qPCR was used to monitor p62 mRNA levels (fold increase over the vehicle-treated control) (means ± SD for three experiments). (E) U266 cells were stably transfected with constructs encoding shRNA targeting Cdk9 (left) or its partner cyclin T1 (right), which form the P-TEFb complex, and a scrambled sequence (scr) as a negative control. The cells were then exposed (16 h) to GX, followed by immunoblot analysis to monitor the expression of Cdk9 (p42 and p55 isoforms) or cyclin T1, CTD phosphorylation (p-CTD) (serine 2) of RNA polymerase II, LC3 processing, and p62 expression. The vertical lines (LC3) indicate where additional sample lanes were removed from the images; the horizontal line (phosphorylated CTD and cyclin T1) indicates the splice site in the composite image derived from a single blot.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Inhibition, Western Blot, Expressing, Control, Stable Transfection, Transfection, Construct, shRNA, Sequencing, Negative Control, Phospho-proteomics, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: SQSTM1/p62 downregulation results in cargo loading failure and inefficient autophagy. (A) U266 cells were exposed (16 h) to 100 nM FP plus 500 nM GX in the presence or absence of 7.5 μM spautin-1 (SPT) or 500 μM 3-methyladenine (3-MA), after which LC3 processing and p62 expression were monitored by immunoblot analysis. The vertical line (p62) indicates where additional sample lanes were removed from the image. Values indicate quantification of p62 relative to values for tubulin (fold increase over the untreated control). (B) U266 cells were stably transfected with constructs encoding shRNA targeting Ulk1 or a scrambled sequence as a negative control. Cells were then exposed (16 h) to 500 nM GX with or without 100 nM FP, followed by immunoblot analysis using the indicated antibodies. The vertical line (LC3) indicates where additional sample lanes were removed from the image. Values indicate quantification of p62 relative to tubulin values (fold increase over the untreated control). (C) U266 cells were stably transfected with constructs encoding shRNA targeting p62 or a scrambled sequence and then treated (16 h) with the indicated concentrations of GX (nM), followed by immunoblot analysis for p62 expression, LC3 processing, and intracellular accumulation of polyubiquitinated proteins. (D) U266 cells stably transfected with shRNA directed against p62, Ulk1, Cdk9, cyclin T1, or the scrambled sequence as a control were exposed (16 h) to 500 nM GX in the presence or absence of 100 nM FP or 5 nM SCH727965, followed by a filter trap assay using an antiubiquitin antibody to monitor ubiquitin-positive protein aggregates. Values indicate quantification of the amount of total ubiquitin-positive proteins in SDS-insoluble aggregates (values represent fold increases over the untreated controls of shNC cells). (E) U266 cells stably transfected with shRNA directed against Cdk9, p62, or the scrambled sequence as a control were exposed (16 h) to 500 nM GX and examined by electron microscopy (bar = 2 μm).

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Expressing, Western Blot, Control, Stable Transfection, Transfection, Construct, shRNA, Sequencing, Negative Control, TRAP Assay, Ubiquitin Proteomics, Electron Microscopy

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Expression of SQSTM1/p62 diminishes the increased lethality of BH3 mimetics in p62-defective cells. (A and B) U266 cells stably transfected with shRNA directed against Cdk9 or a scrambled sequence (A) or wild-type (p62+/+) and p62 knockout (p62−/−) MEFs (B) were transiently transfected with a pBABE-puro mCherry-EGFP-LC3B plasmid. After 6 h, cells were treated with GX (U266 cells, 500 nM; MEFs, 200 nM) for an additional 16 h, followed by analysis of autophagic flux using confocal microscopy (bar = 5 μm [A] or 10 μm [B]). Values indicate the number of autophagosomes (A) (yellow) and autolysosomes (AL) (red). (C) U266 cells stably transfected with p62 shRNA were transiently transfected with a construct encoding GFP-tagged p62 or GFP. After 6 h, cells were treated (24 h) with 500 nM GX, followed by 7-aminoactinomycin D (7AAD) staining to monitor cell death by confocal microscopy (left). Arrows indicate GFP-positive/7AAD-negative cells. Dead (7AAD-positive) cells in the GFP-positive population were then quantified by using flow cytometry (right). (D) Immunoblotting analysis was performed to validate p62 expression in wild-type and p62 ko MEFs (inset). MEFs (left, wt; right, p62 ko) were then exposed to 200 nM GX with or without 100 nM FP for 24 h, followed by 7AAD staining to determine the percentage of cell death by flow cytometry. (E) p62 ko MEFs were transiently transfected with GFP-tagged p62 or GFP (inset) (bar = 30 μm). After 6 h, cells were treated with 200 nM GX with or without 100 nM FP, after which the percentage of cell death was determined by flow cytometry (left, GFP; right, GFP-p62) as described above.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Sequencing, Knock-Out, Plasmid Preparation, Confocal Microscopy, Construct, Staining, Flow Cytometry, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: Cdk9 inhibition upregulates NBK/Bik in cells exposed to BH3 mimetics. (A and B) U266 and RPMI8226 cells were treated (24 h) with the indicated concentrations of GX with or without FP (A), SCH727965 (5 nM) (B), or bortezomib (btz) (4 nM) as a positive control, after which immunoblot analysis was performed to monitor Bik expression and/or PARP cleavage. *, nonspecific band. (C) U266 cells stably transfected with shRNA directed against Cdk9, cyclin T1, or a scrambled sequence as a control were exposed (24 h) to 500 nM or 750 nM GX, followed by immunoblot analysis to monitor the expression of Bik and cleavage of caspase 3 and PARP. (D) After U266 cells were treated (24 h) with the indicated concentrations of GX (nM) with or without FP (nM), the ER membrane fraction was isolated and subjected to immunoblot analysis to monitor the subcellular localization of Bik. The same membranes were probed by an anticalnexin antibody as a loading control for ER membranes. (E) U266 cells were treated (16 h) with 500 nM GX plus 100 nM FP in the presence or absence of 1 μM CHX (top) or 300 nM MG-132 (bottom), followed by immunoblot analysis to monitor Bik expression.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Inhibition, Positive Control, Western Blot, Expressing, Stable Transfection, Transfection, shRNA, Sequencing, Control, Membrane, Isolation

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: NBK/Bik upregulation occurs during autophagy in association with loading failure. (A) U266 cells were treated with the indicated concentrations of GX with or without FP (100 nM) for 6, 16, 24, and 48 h, after which Bik expression was monitored by immunoblot analysis. (B) Blots of Bik after treatment with 500 nM GX with or without FP were quantified relative to tubulin values (values represent fold increases over the vehicle-treated controls) (means ± SD for three experiments). (C) U266 cells were cotreated (24 h) with 500 nM GX and 100 nM FP in the presence or absence of 7.5 μM spautin-1 (SPT), 50 μM CQ, or 500 μM 3-MA. After treatment, Bik expression and PARP cleavage were determined by immunoblot analysis. (D and E) U266 cells stably transfected with shRNA directed against Ulk1 (D), beclin-1 and Atg5 (E), or the scrambled sequence as a control were exposed (24 h) to 500 nM GX with or without 100 nM FP, followed by immunoblot analysis to monitor Bik expression and PARP cleavage. Vertical lines indicate where additional sample lanes were removed from the images. (F) U266 cells stably transfected with shRNA of p62 or the scrambled sequence as a control were exposed (24 h) to 500 or 750 nM GX, followed by immunoblot analysis for Bik expression and PARP cleavage. (G) U266 cells were exposed (16 h) to 500 nM GX with or without 100 nM FP or 5 nM SCH727965 (top), and U266 cells stably transfected with shRNA directed against p62, Cdk9, cyclin T1, or the scrambled sequence as a control were treated (16 h) with 500 nM GX with or without 100 nM FP or 5 nM SCH727965 (bottom). After drug treatment, a filter trap assay using anti-Bik antibody (α-Bik) was performed to monitor the amount of Bik in SDS-insoluble protein aggregates. (H) U266 cells were exposed (16 h) to 500 nM GX with or without 100 nM FP or 5 nM SCH727965, followed by immunoprecipitation (IP) using anti-Bik antibody and subsequent immunoblot (IB) analysis using antiubiquitin antibody (α-ubi). Immunoprecipitations without anti-Bik antibody (− Ab) (lane 1) or cell lysate (− lysate) (lane 2) were used as controls. IgG(H), IgG heavy chain.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, shRNA, Sequencing, Control, TRAP Assay, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/MCB.01383-13

Figure Lengend Snippet: NBK/Bik plays a functional role in triggering apoptosis in vitro and in vivo. (A and B) U266 cells were stably transfected with a construct encoding shRNA directed against Bik or a scrambled sequence as a control and then treated (24 h) with 500 nM GX with or without 100 nM FP or 4 nM bortezomib (btz) as a positive control. Immunoblot analysis (A) and flow cytometry (B) were conducted to monitor the expression of the indicated proteins or to determine percentage of apoptotic (annexin V-positive) cells (values represent means ± SD for three experiments). (C) Athymic NCr-nu/nu mice subcutaneously inoculated in the flank with 5 × 106 RPMI8226 cells were treated with GX (3 mg/kg of body weight intramuscularly) with or without FP (5 mg/kg i.p.). Tumors were excised at day 28 after tumor cell inoculation and then homogenized and subjected to immunoblot analysis using the indicated antibodies. (D) NOD/SCID/gamma (NSG) mice were subcutaneously inoculated in each flank with 1 × 107 U266 cells stably transfected with shRNA directed against Bik (right flank) or a scrambled sequence as a control (left flank). FP (3 mg/kg i.p.) with or without GX (3 mg/kg i.p.) was then administered daily for a total of 11 days. Images of tumors removed from two representative mice were captured at day 49 after tumor cell inoculation (bar = 20 mm). (E) NSG mice were injected i.v. via the tail vein with 5 × 106 U266 cells carrying luciferase, after which FP (3 mg/kg i.p.) with or without GX (3 mg/kg i.p.) was administered daily for a total of 19 days. The bioluminescent images of representative mice were captured at day 35 after inoculation of cells. Lumbar vertebrae removed from mice were immunohistochemically (IHC) stained with anti-human CD138 antibody (bar = 10 μm) or hematoxylin and eosin (H.E.) (bar = 20 μm). m, megakaryocyte; v, blood vessel.

Article Snippet: Human multiple myeloma U266 and RPMI8226 cells were obtained from the ATCC and maintained as described previously ( 5 ), and both cell lines were authenticated (Basic STR Profiling Service, ATCC 135-X) by the ATCC before this study was completed.

Techniques: Functional Assay, In Vitro, In Vivo, Stable Transfection, Transfection, Construct, shRNA, Sequencing, Control, Positive Control, Western Blot, Flow Cytometry, Expressing, Injection, Luciferase, Staining

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Cells were transiently transfected with a plasmid encoding the red fluorescence protein and LC3 fusion protein (RFP-LC3). After 48 h post-transfection, cells were counterstained with Hoechst 33342 (blue). RFP-LC3 (red) shows a homogeneous staining in the cytoplasm of MDA-MB-231 cells, indicating the absence or low level of autophagosomes. Both BT474 and AU565 cells show punctate or dotted staining patterns of RFP-LC3 which is a typical marker of autophagosome structures. Scale bar, 10 μm. (B) Numbers of RFP-LC3 dots (Puncta) in the transfected cells as in A. Shown are the average numbers of puncta per cell estimated by examining at least ten images per cell line (mean ± SD).

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Staining, Marker

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Electron microscopy (EM) images show the ultrastructural features of untreated cells or cells treated with 3-methyladenine (3-MA) at 10 mM for 24 h (Bar =1 μm). Arrows denote the autophagosome ultrastructures in cytoplasm. Lower panel shows the average number of autophagosome structures per view (371 μm 2 ) obtained by examining at least 50 images per testing sample. *p<0.0001. (B) EM images of parental cells and cells transfected with siRNA specific to the autophagy regulatory genes ATG7, Beclin 1, and LC3, respectively. Images are representatives of at least 50 captures. Lower panel shows the average number of autophagosome structures as determined in (B) for individual samples. *p<0.0001. (C) Nude mice were injected s.c. with BT474 or MDA-MB-231 cells per the protocol described in the Materials and Methods. When tumors reached 0.6 cm 3 in size, tumor tissues were harvested and analyzed by EM imaging. Bar =1 μm. Lower panel shows the quantification of autophagosome numbers in the respective tissues.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Electron Microscopy, Transfection, Injection, Imaging

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A & B) The indicated cell lines were transiently transfected with a scramble siRNA (siCtrl) as a negative control or siRNA against ATG7 for 72 h, followed by incubation with rhTRAIL (100 ng/ml) for an additional 24 h. The resultant cells were analyzed by flow cytometry for apoptosis (A) or immunoblotting for caspase cleavage (B). (C & D) BT474 cells were transiently transfected with a control siRNA (siCtrl) or siRNA specific to Beclin 1, LC3 or in combination. After 48 h post-transfection, cells were analyzed for apoptosis and caspase activation. *p<0.0001.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Transfection, Negative Control, Incubation, Flow Cytometry, Western Blot, Control, Activation Assay

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Flow cytometry analysis of death receptor expressions on cell surface. Cells were transiently transfected with a control siRNA (siCtrl) or siATG7. After 72 h post transfection, cells were incubated with PE-conjugated antibodies to DR4 (IgG1) or DR5 (IgG2b) or corresponding control IgG isotypes. Shown are representative histograms of siCtrl cells stained with control PE-IgG1 or PE-IgG2b (purple), PE-anti-DR4 or PE-anti-DR5 (green), and siATG7 transfected cells stained with PE-anti-DR4 or PE-anti-DR5 (yellow). The right-shift of a histogram peak indicates the increase in surface expression of the receptors. (B) Western blots of DR4 and DR5 total proteins. Knockdown of ATG7 is indicated by the decrease in ATG7 protein and the simultaneous loss of LC3-II. (C) Confocal images show the redistribution of DR4 from cytosol to plasma membrane upon inhibition of autophagy. Stable BT474/RFP-LC3 cells were left untreated or treated with 3-MA (10 mM) for 24 h. Alternatively, cells were transiently transfected with siCtrl or siATG7 for 48 h. Resultant cells were transfected with plasmids for GFP-DR4 or GFP-DR5 for 18 h. (D) Functional DISC formation assay. The indicated cells were transiently transfected with siATG7 for 72 h and incubated with (His) 6 -TRAIL (1 μg/ml) for 1 h. Affinity isolated DISC complexes were analyzed by western blotting. As a positive control, MDA-MB-231 cells (expressing both DR4 and DR5 on cell surface and are sensitive to TRAIL) recruited adaptor protein FADD and pro-caspase-8 into the DISC complexes upon TRAIL treatment. Only little DISC components were detected in parental BT474 and AU565 cells which are deficient in surface DR4/DR5 (Fig. ). Knockdown of ATG7 enhanced FADD and caspase 8, particularly the cleaved forms p43/41 and p21/18, in the DISC complexes, while had no effect on their total protein expressions.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Flow Cytometry, Transfection, Control, Incubation, Staining, Expressing, Western Blot, Knockdown, Clinical Proteomics, Membrane, Inhibition, Functional Assay, Tube Formation Assay, Isolation, Positive Control

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Cells were treated with bafilomycin A1 (BafA1) at 100 nM for 8 or 24 h, and the resultant whole cell extracts were analyzed by immunoblotting for LC3, p62, DR4 and DR5. Actin was used as a loading control. (B) Relative protein levels were estimated by densitometry analysis of the blots in A, and normalized to the corresponding actin intensity. The level of each protein in the untreated cells (time 0) was arbitrarily set as 1. Shown are representatives of two independent experiments. (C) MDA-MB-231/RFP-LC3 cells and BT474/RFP-LC3, both stably express RFP-LC3 protein, were treated with BafA1 (100nM) for the indicated times, countered stained with Hoechst 33342 (blue), and analyzed by confocal microscopy. Scale bar, 10 μm. MDA-MB-231 cells accumulated punctate structures at a much higher rate compared to BT474 cells.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Western Blot, Control, Stable Transfection, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: In MDA-MB-231 cells, TRAIL binds DR4 and/or DR5 expressed on cell surface, thereby recruiting adaptor protein Fas-associated death domain (FADD) and pro-caspase 8 into a death inducing signaling complex (DISC). Within the DISC, caspase 8 undergoes self-cleavage and activation which triggers the caspase cascade, cleavage of structural proteins, and eventually apoptosis. Both BT474 and AU565 cells are characterized by high basal level of autophagosomes that sequester DR4 and DR5, which may contribute to their deficiency on cell surface. Disruption of autophagosome structures (e.g. by 3-MA or siATG7) restores the surface expression of DR4 and DR5 which make the cells susceptible to TRAIL induced apoptosis.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Activation Assay, Disruption, Expressing

Journal: Cell Reports

Article Title: Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

doi: 10.1016/j.celrep.2019.05.095

Figure Lengend Snippet: Small Extracellular Vesicles (sEVs) and Soluble Factors Form Part of the Senescent Secretome and Mediate Paracrine Senescence in Normal HFFF2s (A) Schematic representation of the proof-of-concept experiments performed to show that sEVs form part of the senescent secretome. HFFF2 human primary fibroblasts expressing a vector encoding an inducible form of H-RAS G12V ER:RAS (iRAS) or an empty vector (iC) were treated with 200 nM 4OHT for 2 days and allowed to produce conditioned media (CM) for a further 3–5 days. This CM was taken from iC or iRAS HFFF2s and tested for the ability to induce senescence in HFFF2 as a whole ( Figure S1 A) or ( B–1E) processed by serial ultracentrifugation to evaluate the effect of the different fractions: supernatant (SN), large extracellular vesicles (MVs), or sEVs to induce paracrine senescence in HFFF2s. (B and C) HFFF2 fibroblasts were treated for 72 h with the different fractions of the CM (SN, MV, or sEV) from iC or iRAS cells, and the endogenous expression of different markers of senescence was determined as shown in (B) representative pictures and by (C) quantifying the percentage of cells staining positive for different antibodies by IF. The graphs represent the means ± SDs of 2–6 independent experiments. Scale bars: 100 μm for BrdU and p53 and 30 μm for p-γH2AX and p16 INK4A . (D and E) HFFF2 cells were treated twice for 72 h with the different fractions of the CM, replated, and counted on different days. (D) Scheme of the experiments performed. (E) Growth curves showing the mean of 3 independent experiments. See also Figure S1 .

Article Snippet: p16 INK4A , Abcam , Cat# ab108349, RRID: AB_10858268.

Techniques: Expressing, Plasmid Preparation, Staining

Journal: Cell Reports

Article Title: Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

doi: 10.1016/j.celrep.2019.05.095

Figure Lengend Snippet: Inhibition of the Enzyme Neutral Sphingomyelinase, N-SMase, Prevents Paracrine Senescence (A) Schematic representation of the experimental settings to determine whether inhibition of N-SMase influences paracrine senescence. iRAS cells were treated with 200 nM 4OHT for 2 days, followed by treatment with different concentrations of Torin-2 (25 and 50 nM) or 2 independent N-SMase inhibitors: GW4869 (1 and 10 μM) and SpE (2 and 5 μM) for 3 days. After the incubation with the inhibitors, cells were washed and allowed to produce fresh CM for 72 h. Normal HFFF2s were then incubated with this CM for a further 72 h. (B) CM-treated HFFF2 fibroblasts were then stained to assess for the percentage of cells expressing markers of senescence: incorporation of BrdU and p-γH2AX by IF (means ± SEMs of 3–4 experiments; one-way ANOVA). (C) Representative pictures for p-γH2AX by IF of HFFF2s treated with the CM from iRAS with or without SpE or GW4869. Scale bar, 50 μm. (D) Schematic representation of the experimental settings and timings to test the implication of small EVs using the Transwell system with a membrane pore size of 0.4 μm. (E and F) The lower chamber was stained to quantify the percentage of cells incorporating BrdU and expressing p16 INK4A by IF. Representative pictures and the quantification of BrdU incorporation (E) and p16 INK4A (F) are shown. Scale bar, 100 μm. One-way ANOVA test was performed. All data show the means ± SEMs of 2–3 independent experiments. See also Figure S3 .

Article Snippet: p16 INK4A , Abcam , Cat# ab108349, RRID: AB_10858268.

Techniques: Inhibition, Incubation, Staining, Expressing, BrdU Incorporation Assay

Journal: Cell Reports

Article Title: Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

doi: 10.1016/j.celrep.2019.05.095

Figure Lengend Snippet: Increase in CD63 Staining and Multivesicular Body Formation during Senescence In Vivo (A and B) Immunoblot for endogenous expression of (A) ALIX and TSG101 and (B) CD63. p21 CIP and p16 INK4A upregulation are positive controls to confirm the induction of senescence. β-Actin represents the loading control. (C) Immunohistochemistry for SA-β-Gal (blue staining) and CD63 (brown signal) in a representative human sample of lung fibrosis. H&E staining is shown (violet). Pictures at top represent areas enriched in SA-β-Gal + cells, and pictures at bottom show areas with low SA-β-Gal + cells. (D) Quantification of positive pixels for CD63 per field, normalized by the H&E staining. The Mann-Whitney test was performed. (E) Representative transmission electron microscopy images of multivesicular bodies (MVBs) in wild-type ( WT ) and Kras G12D -derived PanIN ( Kras G12D ). Scale bar, 500 nm. (F) Quantification of MVB per cell in WT (n = 28 cells) and PanINs (n = 19 cells). See also Figure S4 .

Article Snippet: p16 INK4A , Abcam , Cat# ab108349, RRID: AB_10858268.

Techniques: Staining, In Vivo, Western Blot, Expressing, Immunohistochemistry, MANN-WHITNEY, Transmission Assay, Electron Microscopy, Derivative Assay

Journal: Cell Reports

Article Title: Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

doi: 10.1016/j.celrep.2019.05.095

Figure Lengend Snippet: IFITM3 within sEVs Is Partially Responsible for Inducing Paracrine Senescence (A and B) HFFF2s incubated with sEVs derived from iRAS cells show an increase in transcripts related to the interferon (IFN) pathway (A)—in particular, IFITM (in red) and IFIT mRNAs, which are downregulated when treated with SpE. (B) IFITM transcripts are specifically shown. Data in (A) and (B) have been normalized to the control and represent the mean of 3 independent experiments (RPKM-log 2 fold difference). (C and D) Immunoblotting analysis for IFITM3 in (C) cell lysates derived from iRAS HFFF2s transfected with Src or siIFITM3 and in (D) sEV (3 × 10 9 particles) from iC and iRAS cells. β-Actin and ALIX are used as loading controls. (E) sEVs from iC and iRAS were captured onto IFITM3-coated beads and the presence of CD81-PE determined by FACS. (F) Immunoblotting showing the absence of IFITM3 in sEVs (3 × 10 9 particles) derived from siIFITM3-treated cells. (G) IF staining for p16 INK4A and IL-8 in HFFF2s treated with the same number of sEV (1 × 10 7 particles) derived from iC or iRAS transfected with or without siIFITM3. (H) Immunoblotting for IFITM3 present in sEVs derived from HFFF2 expressing an ectopic IFITM3 construct. (I) IF staining for p16 INK4A and p-γH2AX in HFFF2s treated with sEVs derived from cells expressing IFITM3. (J and K) Immunoblotting (J) and quantification (K) for IFITM3 and CD63 protein expression levels in sEVs derived from the plasma of young (∼33 years old) and old (∼80 years old) donors. Gels were loaded based on equal protein levels. (A–I) Data representative of >3 experiments. See also Figure S7 .

Article Snippet: p16 INK4A , Abcam , Cat# ab108349, RRID: AB_10858268.

Techniques: Incubation, Derivative Assay, Western Blot, Transfection, Staining, Expressing, Construct

Journal: Cell Reports

Article Title: Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

doi: 10.1016/j.celrep.2019.05.095

Figure Lengend Snippet:

Article Snippet: p16 INK4A , Abcam , Cat# ab108349, RRID: AB_10858268.

Techniques: Recombinant, Software, Functional Assay, Microarray, Protein Concentration, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell Reports Medicine

Article Title: An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer

doi: 10.1016/j.xcrm.2022.100525

Figure Lengend Snippet:

Article Snippet: Human MDA-MB-468 , Characterized Cell Line Core (MDACC) , https://www.atcc.org/products/all/HTB-132.aspx ; RRID: CVCL_0419.

Techniques: Staining, Functional Assay, Recombinant, Saline, Modification, Blocking Assay, Plasmid Preparation, Electron Microscopy, Purification, dsDNA Assay, Hybridization, Diagnostic Assay, Protein Array, Sequencing, Next-Generation Sequencing, Gene Expression, Expressing, Software, Cytometry, Immunofluorescence, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: WDFY4 is up‐regulated in ox‐LDL‐treated HAEC and inhibits cell viability. HAECs were treated with 0 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL and 200 μg/mL ox‐LDL for 24 h. (A) The effects of different concentrations of ox‐LDL on the activity of HAEC were detected. (B) The expression level of WDFY4 protein in HAEC treated with different concentrations of ox‐LDL was detected. (C) The expression of WDFY4 mRNA in HAEC treated with different concentrations of ox‐LDL was detected. (D) The expression of WDFY4 was detected by immunofluorescence. Scale bar, 25 μm. n = 4. One‐way ANOVA was used for comparison between multiple groups. Data are presented as mean ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Activity Assay, Expressing, Immunofluorescence, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: Knockdown of WDFY4 inhibits ox‐LDL‐induced ferroptosis in HAECs. After HAECs were treated with 100 μg/mL ox‐LDL for 24 h, HAECs were transfected with sh‐WDFY4. (A) RT‐qPCR was used to detect the expression level of WDFY4 mRNA in HAECs. (B) Western blotting was used to detect the expression level of WDFY4 protein in HAECs. (C) CCK‐8 was used to detect the activity of HAECs. (D) The level of lipid ROS in HAECs was detected by DCFH‐DA fluorescent probe method. Scale bar, 100 μm. (E) MDA content was detected with MDA assay kit. (F) GSH level was detected with GSH assay kit. (G) ELISA was used to detect Fe 2+ content in HAECs. (H) Western blotting was used to detect the level of ferroptosis‐related proteins. (I) Representative transmission electron microscopy pictures of mitochondria ultrastructure in HAECs were showed. Scale bar, 1 μm. n = 4. One‐way ANOVA or Two‐way ANOVA was used for comparison between multiple groups. ** p < 0.01.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Activity Assay, Multiple Displacement Amplification, GSH Assay, Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: Interference with WDFY4 reduces ox‐LDL induced HAEC cell death and inflammatory response. After HAECs were treated with 100 μg/mL ox‐LDL for 24 h, HAECs were transfected with sh‐WDFY4. (A) The concentration of endothelial cell function marker NO in the supernatant of HAEC cells was detected by NO assay kit. (B) The activity of endothelial cell function marker eNOS in HAECs supernatant was detected by ELISA. (C) The level of endothelial cell function marker ET‐1 in the supernatant of HAECs was detected by ELISA. (D–F) The secretion level of inflammatory factors (TNF‐α, IL‐6 and IL‐1β) in the supernatant of HAECs was detected by ELISA. (G, H) PI staining was used to detect the mortality of HAECs treated with ox‐LDL. Scale bar, 100 μm. n = 4. One‐way ANOVA was used for comparison between multiple groups. * p < 0.05, ** p < 0.01.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Transfection, Concentration Assay, Cell Function Assay, Marker, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: WDFY4 interacts with LAPTM5 to promote LAPTM5 expression. After HAECs were treated with 100 μg/mL ox‐LDL for 24 h, HAECs were transfected with sh‐WDFY4. (A) WDFY4 interacting proteins were predicted. (B) Immunofluorescence staining of WDFY4 and LAPTM5 was showed. Scale bar, 25 μm. (C) Co‐IP assay was used to verify the interaction between WDFY4 and LAPTM5. (D) The effect of WDFY4 knockdown on the expression of LAPTM5 protein in HAEC was detected by Western blotting. n = 4. One‐way ANOVA was used for comparison between multiple groups. * p < 0.05, ** p < 0.01.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Expressing, Transfection, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Knockdown, Western Blot, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: WDFY4 interacts with LAPTM5 and promotes ferroptosis in HAEC by inhibiting CDC42/mTOR/4EBP1/SLC7A11 pathway. After HAECs were treated with 100 μg/mL ox‐LDL for 24 h. ox‐LDL treated HAECs were transfected with sh‐WDFY4 and pcDNA‐LAPTM5 or treated with 10 μM ML141. (A) Western blotting was used to detect the expression level of WDFY4 protein in HAECs. (B) CCK‐8 was used to detect the activity of HAECs. (C) Western blotting was used to detect the expression level of CDC42, mTOR and 4EBP1 protein in HAECs. (D) ELISA was used to detect Fe 2+ content in HAECs. (E) The level of lipid ROS in HAEC was detected by DCFH‐DA fluorescent probe method. Scale bar, 100 μm. (F) MDA content was detected with MDA assay kit. (G) GSH level was detected with GSH assay kit. (H) Western blotting was used to detect the level of ferroptosis‐related proteins. (I) Representative transmission electron microscopy pictures of mitochondria ultrastructure in HAECs were showed. Scale bar, 1 μm. n = 4. One‐way ANOVA or Two‐way ANOVA was used for comparison between multiple groups. ** p < 0.01, and ns means non‐significant.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Transfection, Western Blot, Expressing, CCK-8 Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, GSH Assay, Transmission Assay, Electron Microscopy, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: LAPTM5 and ML141 reversed the alleviation effect of sh‐WDFY4 on cell injury and inflammatory response in AS model cells. After HAECs were treated with 100 μg/mL ox‐LDL for 24 h. ox‐LDL treated HAECs were transfected with sh‐WDFY4 and pcDNA‐LAPTM5 or treated with 10 μM ML141. (A) The concentration of endothelial cell function marker NO in the supernatant of HAEC cells was detected by NO assay kit. (B) The activity of endothelial cell function marker eNOS in HAECs supernatant was detected by ELISA. (C) The level of endothelial cell function marker ET‐1 in the supernatant of HAECs was detected by ELISA. (D–F) The secretion level of inflammatory factors (TNF‐α, IL‐6 and IL‐1β) in the supernatant of HAECs was detected by ELISA. (G) PI staining was used to detect the mortality of HAECs treated with ox‐LDL. Scale bar, 100 μm. n = 4. One‐way ANOVA was used for comparison between multiple groups. ** p < 0.01.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Transfection, Concentration Assay, Cell Function Assay, Marker, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: Fluorescence co‐localization of WDFY4 and LAPTM5 In vivo. ApoE −/− mice were fed a high‐fat diet and injected with sh‐WDFY4, sh‐LAPTM5 or sh‐NC lentivirus every other day. Immunofluorescence staining of WDFY4 and LAPTM5 in the diseased area of the aortic root in mice were showed, and the fluorescence intensity was quantified. Scale bar, 25 μm. n = 8. One‐way ANOVA was used for comparison between multiple groups. ** p < 0.01, and ns means non‐significant.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Fluorescence, In Vivo, Injection, Immunofluorescence, Staining, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5 / CDC42 / mTOR / 4EBP1 / SLC7A11 Pathway

doi: 10.1111/jcmm.70729

Figure Lengend Snippet: Knockdown of WDFY4 and LAPTM5 alleviates AS associated with ferroptosis. ApoE −/− mice were fed a high‐fat diet and injected with sh‐WDFY4, sh‐LAPTM5 or sh‐NC lentivirus every other day. (A) Western blotting was used to detect the expression levels of WDFY4, LAPTM5 and ferroptosis‐related proteins in mouse aortic tissue. (B) ELISA was used to detect the Fe 2+ level in serum of mice. (C) The level of ROS in serum of mice was detected. (D) The expression levels of inflammatory factors TNF‐α and IL‐1β mRNA in serum of mice were detected by qPCR. (E) Oil red O staining was used to detect the degree of arterial plaque. Scale bar, 50 μm. (F) HE staining was used for pathological analysis of arterial tissue. Scale bar, 50 μm. (G) The serum TC, TG, LDL and HDL levels were detected by ELISA. n = 8. One‐way ANOVA was used for comparison between multiple groups. ** p < 0.01, and ns means non‐significant.

Article Snippet: Proteins were separated by SDS‐PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk powder (1 g skimmed milk powder: 20 mL 1 × TBST reagent) for 2 h. Subsequently, membranes were incubated overnight at 4°C with primary antibodies: WDFY4 (1: 500, orb512258, Biorbyt, UK), LAPTM5 (1: 1000, PA5‐23585, Thermo Fisher), GPX4 (1: 500, ab125066, Abcam, UK), SLC7A11 (1: 500, ab307601, Abcam), ACSL4 (1: 500, ab155282, Abcam), CDC42 (1: 500, ab187643, Abcam), mTOR (1:500, ab134903, Abcam), 4EBP1 (1: 500, ab32024, Abcam) and GAPDH (1: 1000, ab181602, Abcam).

Techniques: Knockdown, Injection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Comparison

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Tramadol impaired cell viability and induced transcriptional and pathway-level alterations in breast cancer cells. A Cell viability was measured using the MTT assay after treating MDA-MB-231, MCF-7, and MCF-10A cells with escalating doses of tramadol for 24 h. Results represent the mean ± SEM from three biologically independent replicates. Statistical comparisons were conducted using two-way ANOVA followed by Dunnett’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). B Volcano plots illustrating changes in gene expression profiles in MDA-MB-231 and MCF-7 cells after 24-hour exposure to 0.5 mg/mL tramadol. Differentially expressed genes were classified as upregulated (red) or downregulated (blue) based on a threshold of |log₂ fold change| > 1 and adjusted p -value < 0.05. C Hallmark pathway analysis was conducted using GSEA to compare tramadol-treated and untreated cells. Key enriched signatures included hypoxia signaling, ER stress/unfolded protein response, and oxidative stress pathways.

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: MTT Assay, Control, Gene Expression

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Tramadol promoted hypoxia signaling in a dose-dependent manner by stabilizing HIF-1α, enhancing its nuclear translocation, and upregulating HIF-1α target gene expression in breast cancer cells. A Hypoxia levels were elevated in MDA-MB-231 and MCF-7 cells following short-term tramadol exposure. Cells were incubated with 0, 0.5, or 1 mg/mL tramadol for 4 h, stained with a hypoxia-sensitive fluorescent dye (Hypoxia Red), and analyzed via flow cytometry. Quantification of the hypoxic population (M2 gate) is shown as mean ± SEM from three independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons (** p < 0.01, **** p < 0.0001 vs. untreated control). B Tramadol prolonged the stability of HIF-1α protein. MDA-MB-231 and MCF-7 cells were treated with 1 mg/mL tramadol or vehicle for 4 h prior to cycloheximide (CHX) addition. Protein degradation kinetics of HIF-1α were monitored at various time points (0–40 min) by western blot. β-actin served as the internal loading control. C Immunofluorescence staining revealed tramadol-induced nuclear localization of HIF-1α in breast cancer cells. MDA-MB-231 and MCF-7 cells were exposed to 1 mg/mL tramadol or vehicle control for 4 h, followed by staining with an anti-HIF-1α antibody (red) and nuclear counterstaining with DAPI (blue). Representative fluorescence images were acquired using a fluorescence microscope. Scale bar: 10 μm. D Subcellular fractionation followed by western blot analysis further confirmed the nuclear enrichment of HIF-1α following tramadol treatment (1 mg/mL, 4 h). Nuclear (Nuc) and cytoplasmic (Cyto) protein fractions were isolated from MDA-MB-231 and MCF-7 cells. GAPDH and histone H3 served as markers for cytoplasmic and nuclear compartments, respectively. E Tramadol induced the transcription of HIF-1α and its downstream targets BHLHE40, HMOX1, and VEGFA in a dose-dependent manner. Cells were treated with 0, 0.5, or 1 mg/mL tramadol for 4 h, with or without the HIF-1α inhibitor PX-478 (20 μM). Gene expression was measured by qPCR, normalized to ACTB, and shown as mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. untreated control).

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Translocation Assay, Targeted Gene Expression, Incubation, Staining, Flow Cytometry, Control, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Fractionation, Isolation, Gene Expression

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Effects of tramadol on the ER stress pathways and ROS levels in MDA-MB-231 and MCF-7 cells. A MDA-MB-231 and MCF-7 cells were treated with 0, 0.05, 0.1, 0.5, 1, or 1.5 mg/ml tramadol. β-Actin was used as a loading control. B MDA-MB-231 and MCF-7 cells were treated with 0 or 1 mg/ml tramadol. Western blot analysis was performed on the nuclear and cytosolic fractions, with Histone 3 and GAPDH serving as internal controls for the nuclear and cytosolic proteins, respectively. C MDA-MB-231 and MCF-7 cells were treated with 0 or 1 mg/ml tramadol. qPCR analysis was performed to assess the mRNA expression of downstream genes regulated by ATF4. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control). D MDA-MB-231 and MCF-7 cells were treated with 0, 0.5, 1, or 1.5 mg/ml tramadol. DCFH-DA staining was conducted to assess intracellular ROS levels, whereas MitoSox Red staining was used to evaluate mitochondrial ROS levels, both of which were analyzed via flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01 vs. control).

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, Western Blot, Expressing, Staining, Flow Cytometry

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: The mechanisms and types of cell death induced by tramadol in MDA-MB-231 and MCF-7 cells. A After treatment with various inhibitors, cells were treated with 1 mg/ml tramadol, and then, cell viability was assessed. Data are shown as mean ± SEM; *** p < 0.001 vs. tramadol alone (two-tailed unpaired Student’s t-test). B Phase-contrast images of breast cancer cells following treatment with 0, 0.5, or 1 mg/ml tramadol. Arrows indicate cytoplasmic vacuoles. C MDA-MB-231 and MCF-7 cells were treated with 1 mg/mL tramadol and then analyzed via electron microscopy. Representative regions (yellow squares) are enlarged and labeled as follows: M, mitochondria; N, nucleus; LD, lipid droplet; ER, endoplasmic reticulum; Arrow, autophagic vacuoles.

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Two Tailed Test, Electron Microscopy, Labeling

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Lipid droplet accumulation during tramadol-induced cell death in breast cancer cells. A MDA-MB-231 and MCF-7 cells treated with tramadol were stained with BODIPY to visualize lipid droplets via fluorescence microscopy; B the results were quantified via flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Effects of tramadol on autophagy in MDA-MB-231 and MCF-7 cells. A Fluorescence microscopy images of MDA-MB-231 and MCF-7 cells treated with 0, 0.5, or 1 mg/ml tramadol were captured after staining with AO. In these images, viable cells fluoresce green, whereas acidic compartments, such as those associated with autophagic or lysosomal activity, exhibit a red to orange hue. Squares indicate representative regions shown as enlarged areas in the fluorescent images. B MDA-MB-231 and MCF-7 cells were treated with 0, 0.5, 1, or 1.5 mg/mL tramadol and then stained with DALGreen to analyze autophagy; autophagy was quantified by flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, *** p < 0.001 vs. control). C MDA-MB-231 and MCF-7 cells were treated with 0, 0.05, 0.1, 0.5, 1, or 1.5 mg/ml tramadol. ACTN was used as a loading control.

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Fluorescence, Microscopy, Staining, Activity Assay, Flow Cytometry, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Effects of tramadol on mitochondrial function in MDA-MB-231 and MCF-7 cells. A Fluorescence microscopy images of MDA-MB-231 and MCF-7 cells treated with 0, 0.5, 1, or 1.5 mg/ml tramadol were captured after staining with MitoTracker. B MDA-MB-231 and MCF-7 cells were treated with 0, 0.5, 1, or 1.5 mg/mL tramadol and then stained with JC-1 to assess the mitochondrial membrane potential; mitochondrial membrane potential was quantified via flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (** p < 0.01, *** p < 0.001 vs. control). C MDA-MB-231 and MCF-7 cells were treated with 1 mg/mL tramadol, and the OCR was assessed using a Seahorse XF24 analyzer. Data are shown as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (two-tailed unpaired Student’s t-test).

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Fluorescence, Microscopy, Staining, Membrane, Flow Cytometry, Control, Two Tailed Test

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Tramadol influenced cell death pathways and paraptosis-associated signaling in breast cancer cells. MDA-MB-231 and MCF-7 cells were treated with 0, 0.05, 0.1, 0.5, 1, or 1.5 mg/ml tramadol. β-Actin was used as a loading control.

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Functional roles of HIF-1α, ATF4, and Alix in tramadol-induced cytotoxicity and morphological alterations in breast cancer cells. A-Β After HIF-1α, ATF4, and PDCD6IP were knocked out in MDA-MB-231 cells using the CRISPR-Cas9 system, the knockout efficiency at the mRNA level was assessed via qPCR. Data are shown as mean ± SEM; **** p < 0.0001 vs. control (two-tailed unpaired Student’s t-test). C MDA-MB-231 cells were treated with 0.375 or 0.75 mg/ml tramadol for 24 h. Cell viability was determined by CellTiter-Glo assay. Two-way ANOVA with Dunnett’s multiple comparisons was performed, and the results were compared with the vehicle group. * p < 0.05, ** p < 0.01. D MDA-MB-231 cells were treated with 0, 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 mg/ml tramadol for 24 h. Cell viability was determined by CellTiter-Glo assay. Two-way ANOVA with Dunnett’s multiple comparisons was performed, and the results were compared with the vehicle group. **** p < 0.0001. E MDA-MB-231 cells were exposed to tramadol (0, 0.5, or 1 mg/mL) for 4 h, and morphological alterations were examined using phase-contrast microscopy. Arrows indicate cytoplasmic vacuoles.

Article Snippet: MDA-MB-231 (HTB-26TM) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Functional Assay, CRISPR, Knock-Out, Control, Two Tailed Test, Glo Assay, Microscopy