Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inducible IL-33 expression by mast cells is regulated by a calcium-dependent pathway 1

doi: 10.4049/jimmunol.1201224

Figure Lengend Snippet: BMMCs were transfected with scrambled (top), Sphk1 (middle) or Sphk2 (bottom) shRNA plasmids that also express GFP for 7 days and activated with IgE/DNP. Cells transfected with shRNA plasmids were gated on GFP+ cells. IL-33 protein expression of shRNA transfected cells (GFP+ gated) was analyzed by intracellular staining 24 hours after stimulation. Data is representative of 4 independent experiments.

Article Snippet: APC-anti-rat IgG F(ab′) was from eBioscience. pGFP-V-RS plasmids with or without scrambled, Sphk1 or Sphk2 shRNA were purchased from Origene AMAXA mouse dendritic cell transfection buffer was obtained from Lonza.

Techniques: Transfection, shRNA, Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Domain organization and mitochondrial localization of ChChd3. A, amino acid sequence and exon organization of ChChd3. Different exons are shown in alternate black and gray colors. The myristoylation motif at the N terminus is underlined . The consensus site for the PKA phosphorylation is shown in the box, and the previously identified PKA phosphorylation site is underlined . The C X 9 C-C X 9 C motif cysteines in the chch domain are highlighted with asterisk. B, schematic diagram of the ChChd3 protein. DUF, domain of unknown function, CHCH, coiled-coil helix-coiled-coil helix domain. C and D, ChChd3 in mitochondria is primarily localized to the IM facing toward the IMS. Mouse liver mitochondrial subfractions were separated by SDS-PAGE and analyzed by immunoblotting by using antibodies against ChChd3 and known mitochondrial marker proteins. ChChd3 is enriched in the IM fraction similar to that of the IM marker protein, NDUFS3. M, matrix ( C ). SDS-PAGE and immunoblot analysis of trypsin-treated samples of mitochondria, swollen mitochondria, and submitochondrial particles ( D ).

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Sequencing, SDS Page, Western Blot, Marker

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Loss of ChChd3 in HeLa cells results in abnormal mitochondrial morphology. A, mitochondria in ChChd3 knockdown cells are clumpy, fragmented, and clustered around the nucleus. Representative confocal microscopic images of HeLa-control, control-shRNA, and ChChd3-shRNA HeLa cells expressing matrix-targeted RFP ( mito-RFP ). Two independent clones of ChChd3-shRNA ( ChChd3-shRNA clone 1 and ChChd3-shRNA clone 2 ) were analyzed to avoid the selection artifacts. Scale bar, 10 μm. B, quantification of mitochondrial abnormalities in ChChd3 knockdown cells. 300 cells each from the control and ChChd3 knockdown cells were analyzed under confocal microscope for mitochondrial fragmentation and clumping. Mean ± S.D. from three independent experiments is shown. C, HeLa control, control-shRNA, and ChChd3-shRNA cell lysates were assessed for the changes in the protein levels of the key regulators of mitochondrial fusion and fission. ChChd3-shRNA1 and shRNA2 represent two different clones derived from a single shRNA sequence.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: shRNA, Expressing, Clone Assay, Selection, Microscopy, Derivative Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: ChChd3 is required for mitochondrial fusion in HeLa cells. Inhibition of fission by K38A-Drp1 does not restore tubular mitochondrial network in the absence of ChChd3. Representative confocal micrographs of control-shRNA or ChChd3-shRNA HeLa cells expressing mito-RFP ( red ) and GFP or Drp1 WT GFP or dominant negative mutant of Drp1, Drp1 K38A GFP ( green ) are shown. Scale bars, 5 μm.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Inhibition, shRNA, Expressing, Dominant Negative Mutation

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: ChChd3 depletion impairs cellular bioenergetics. A and B, ChChd3 knockdown cells show a drastic decrease in the OCR and ECAR. OCR and ECAR were measured simultaneously by using the Seahorse extracellular flux analyzer in real time. Rates shown are the averages of four wells measured for 3 min after every 5 min. Also shown are the OCR and ECAR values after addition of 1 μ m oligomycin, 200 n m carbonyl cyanide p -trifluoromethoxyphenylhydrazone ( FCCP ), and 100 n m rotenone + myxothiozol ( Rot/Myx ). C, cellular ATP levels in control-shRNA and ChChd3-shRNA HeLa cells were measured by luminescence-based CellTiter-Glo® cell viability assay. ATP levels were normalized to the protein levels. ChChd3-shRNA1 and shRNA2 represent two different clones derived from a single shRNA sequence, Error bars represent standard deviation from triplicate samples. p < 0.01.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: shRNA, Viability Assay, Clone Assay, Derivative Assay, Sequencing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Loss of ChChd3 results in crista remodeling and perinuclear clumping and fragmentation of mitochondria. A, EM analysis of control-shRNA and ChChd3-shRNA HeLa cells showed that mitochondria in ChChd3-shRNA cells cluster around the nucleus. Altered cristae and an increase in autophagy/mitophagy were also prevalent. Panel a, control-shRNA-expressing HeLa cells have mitochondria distributed throughout the cytoplasm. Panel b, in contrast, ChChd3-shRNA cells show clustering of mitochondria around the nucleus. Panel c, mitochondria of control cells have predominantly lamellar crista, whereas the mitochondria in the ChChd3 knockdown cells have lower crista density and more tubular crista ( panel d ). Panel e, common in these cells is mitochondria devoid or nearly devoid of cristae. Panel f, an autophagosome in a ChChd3 knockdown cell engulfing mitochondria. Panel g, multivesicular bodies are seen high in number in ChChd3 knockdown cells. B–I, quantification of mitochondrial abnormalities in ChChd3 knockdown cells from EM analysis of control and ChChd3 knockdown cells. ChChd3 knockdown cells have significantly reduced crista surface area ( B ), higher percentage of mitochondria without crista ( C ), and altered crista ( D ) with no change in number of cristae per mitochondria ( E ). B, bar graph showing the ratio of crista membrane surface area to the OM surface area in control and ChChd3 knockdown cells. OM surface area and the crista membrane surface area from 25 mitochondria each from control and ChChd3 knockdown cells were measured. p < 0.001. C, bar graph showing the percentage of mitochondria completely devoid of cristae measured from 10 different cells as the total number of mitochondria completely devoid of cristae in a cell divided by the total number of mitochondria in the cell. A total of 414 mitochondria in control and 822 mitochondria in ChChd3 knockdown cells were counted. p = 0.0015. D , bar graph showing the percentage of mitochondria with small and rounded cristae. Measured as above in C from 100 mitochondria from control and ChChd3 knockdown cells, 10 were randomly chosen from each cell from the same 10 cells used in C. p = 0.0015. The average number of cristae per mitochondria were counted from the same 10 cells and 100 mitochondria used in C–E. F–I, mitochondria in ChChd3 knockdown cells are fragmented and clustered around the nucleus. F, statistical analysis of the closest distance between the mitochondrial outer membrane and nuclear membrane. 448 mitochondria from five control cells and 339 mitochondria from five ChChd3 knockdown cells were used for measurements. p < 0.01. G, bar graph of the average number of mitochondria per μm 2 in control and ChChd3 knockdown HeLa cells. The number of mitochondria per cell was measured as the number/cytoplasm cross-sectional area for 10 cells each of control and ChChd3 knockdown. There were a total of 414 mitochondria in the control cells and 822 mitochondria in the knockdown cells. p = 0.019. H, bar graph of quantification of mitochondrial volume over total cytoplasmic volume measured from 10 cells each of control and ChChd3 knockdown. I, bar graph of average length of mitochondria measured from the same 10 cells used in H . 165 mitochondria from each control and ChChd3 knockdown were measured. p < 0.001. Means ± S.E. used throughout.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: shRNA, Expressing

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Electron tomography reveals that crista junctions in ChChd3 knockdown mitochondria are smaller than those in control mitochondria. a, 2.4-nm-thick slice through the tomographic volume of a control mitochondrion. Control mitochondria are generally longer than their ChChd3 knockdown counterpart. Two crista junctions are shown ( arrowheads ) with the one at bottom having a very large opening. Scale bar , 200 nm and applies to all panels. b, 2.4-nm thick slice through the tomographic volume of five ChChd3 knockdown mitochondria. These mitochondria are generally smaller and often found close to each other suggesting fission has occurred. The amount of cristae present can differ remarkably (compare L , similar cristae complement to control mitochondria, with M , tiny mitochondrion devoid of cristae), and the matrix density can also differ substantially (compare M with R ). Their crista junction openings are characteristically smaller ( arrowheads ). c and d, side views of a segmented and surface-rendered inner membrane from a control mitochondrial volume with numbered crista junctions. Six crista junctions are shown on one side and nine on the other side. e and f, side views of a segmented and surface-rendered inner membrane from a ChChd3 knockdown mitochondrial volume showing smaller crista junctions ( numbered ) than the control. Six crista junctions are shown on one side and four on the other side. g, crista junction openings in ChChd3 knockdown mitochondria are about half the size of control openings. The mean crista junction width at its largest opening in tomographic reconstructions is compared. The number of measurements is shown above each bar. Error bar, S.E. (**, p < 0.01). h, density of crista junctions is no different in control and ChChd3 knockdown mitochondria. The total number of crista junctions per mitochondrial volume was counted and divided by the mitochondrial surface area derived from the tomographic volume to determine the crista junction density. Error bar, S.E.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Tomography, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: ChChd3 interacts with Mitofilin, Sam50, HSP70, and OPA1. A–C, FLAG-tagged ChChd3 ( A ), mitofilin ( B ), and Sam50 ( C ) were transiently expressed in HEK293 cells and immunoprecipitated ( IP ) with FLAG resin. Eluted samples were analyzed on SDS-PAGE followed by immunoblot (IB) with the indicated antibodies. D, ChChd3 binds to mitofilin through the chch domain and to Sam50 through N-terminal myristoylation or myristoylated confirmation. E–H, ChChd3 and mitofilin preferentially interact with the shorter isoform of OPA1 in HEK293 cells ( E–G ) and in mitochondria ( H ). FLAG-tagged full-length WT ChChd3 ( E ) or FLAG-tagged mitofilin ( F ) or ChChd3 mutant proteins ( G ) were expressed in HEK293 cells and immunoprecipitated with FLAG resin. Sam50 levels are not shown in the input as the antibodies available for Sam50 failed to detect endogenous protein in total cell lysates of HEK293 cells. H, ChChd3 protein, immunoprecipitated from mouse liver mitochondria by using ChChd3 antibody, binds efficiently with mitofilin and the soluble IMS isoform of OPA1. 5% of the total input is shown throughout. I, proposed model for localization of ChChd3 in mitochondria. ChChd3 is synthesized in the cytoplasm and kept in a reduced and soluble form with the help of HSP70, before it is imported and folded in the mitochondria. In mitochondria, ChChd3 may exist at two discrete foci: 1) at CJ and (2) at contact sites ( CS ). At the CJs, ChChd3 would form a complex with mitofilin and OPA1, and at the contact sites it would associate with mitofilin and Sam50 thereby influencing the regulation of crista biogenesis and mitochondrial protein import, respectively. MF, mitofilin; IBM, inner boundary membrane.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Mutagenesis, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Depletion of ChChd3 leads to major loss of mitofilin and Sam50 and reduction in specific mitochondrial proteins. A, equal amounts of protein from control and ChChd3 knockdown HeLa cell lysates were analyzed on the immunoblot against the antibodies indicated. Protein load was normalized against tubulin. B, ChChd3 knockdown cells show reduced levels of mitochondrial inner membrane proteins AIF, prohibitin, Cox-IV, and Cox-II and the outer membrane protein VDAC. Cytoplasmic and mitochondrial fractions from control and ChChd3 knockdown cells were separated as described under “Experimental Procedures,” and equal amounts of protein (15 μg) from each of these fractions were analyzed on SDS-PAGE followed by Western blotting. HSP90 and MFN1 were used as loading controls for cytoplasmic and mitochondrial fractions, respectively. ChChd3-shRNA1 and shRNA2 represent two different clones derived from a single shRNA sequence.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Western Blot, SDS Page, Clone Assay, Derivative Assay, shRNA, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function *

doi: 10.1074/jbc.M110.171975

Figure Lengend Snippet: Loss of ChChd3 results in reduced cellular proliferation. A, rate of cellular proliferation in control and ChChd3 knockdown cells measured by plating an equal number of cells and counting them periodically for 4 days. Error bars represent standard deviation from triplicate samples. B, reduced growth rate is not due to the increased apoptosis or sensitivity toward apoptosis in ChChd3 knockdown cells. Control and ChChd3 knockdown cells were treated with staurosporine to induce apoptosis, and the cells were lysed after the indicated time periods and analyzed for the changes in poly(ADP-ribose) polymerase ( PARP ) cleavage. C, ChChd3 knockdown cells show reduced p70 S6 kinase ( S6k ) and phospho-p70 S6 kinase protein levels. Equal amounts of protein from the control and ChChd3 knockdown cells were analyzed for p70 S6 kinase and phospho-p70 S6 kinase. D, ChChd3 knockdown cells show elevated autophagy. Equal amounts of protein from the total cell lysates of control and ChChd3 knockdown cells were separated on a SDS-PAGE and immunoblotted against LC3 antibody. Tubulin is used as the loading control.

Article Snippet: HeLa cells maintained in DMEM with 10% FBS and 1% GlutaMAX grown to 40% confluency in 35 mm plates were transfected with ChChd3-shRNA or scrambled-control using Turbofectin (OriGene) according to the manufacturer's protocol, and stable cell lines were selected in 0.5 μg/ml puromycin (Sigma).

Techniques: Standard Deviation, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ

doi: 10.3390/ijms161226093

Figure Lengend Snippet: Knockdown of PPARγ by its specific siRNA alleviated the inhibitory effects of evodiamine on PDGF-BB-stimulated VSMC migration. ( A ) Knockdown efficiency of siRNA tested by Western blot; ( B , C ) Quantification of VSMC migration. VSMCs were transfected with scrambled siRNA (negative control, or NC) or siRNA against PPARγ (siPPARγ). 24 h after transfection, cells were pretreated with 0.5 μM evodiamine for 6 h and then stimulated with 10 ng/mL PDGF-BB for 24 h (wound healing assay, B ) or 6 h (transwell assay, C ). In Panel C, blue color indicates cell nuclei stained with DAPI. Evo, evodiamine. Data are represented as mean values ± SD of three independently prepared samples each with five measurements. ** p < 0.01 compared with the control group; ## p < 0.01 compared with the PDGF-BB-stimulated group; $$ p < 0.01 compared with the group treated with both evodiamine and PDGF-BB.

Article Snippet: The rat PPARγ-specific siRNA (5’-GCUCCAAGAAUACCAAAGUTT-3’) and non-specific siRNA (Scrambled siRNA, 5’-UUCUCCGAACGUGUCACGUTT-3’) were designed and synthesized by GenePharma (Shanghai, China).

Techniques: Knockdown, Migration, Western Blot, Transfection, Negative Control, Wound Healing Assay, Transwell Assay, Staining, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients.

doi: 10.1158/1078-0432.CCR-19-2912

Figure Lengend Snippet: A - D: Transcript levels of hyaluronidase gene family members in bladder tissues. Normal bladder: NBL (n=31); low-grade BC: LG (n=7); high-grade non-muscle invasive BC: HG NMIBC (n=6); HG MIBC (n=39). (A): HYAL-2, HYAL-3, PH20; (B): HYAL-4 (C): HYAL-4 Wt; (D): V1. E: V1 levels of HG MIBC specimens further stratified by clinical outcome (metastasis and CSS). Data: Mean ± SEM. P-values two-tailed; Mann-Whitney U test. F and G: Kaplan-Meier plot showing risk-stratification of the cohort by V1 mRNA levels for metastasis and CSS.

Article Snippet: HYAL-4 shRNA transfectants: HYAL-4 shRNA constructs were purchased from Origene.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients.

doi: 10.1158/1078-0432.CCR-19-2912

Figure Lengend Snippet: A: Immunoblot analysis of bladder tissue extracts using anti-HYAL-4 antibody. B: Chase levels (U/mg protein) in bladder tissue extracts measured by Chase assay. C: HYAL-4 staining in representative NBL and BC tissues. Magnification: 400X. See Figure S2 for enlarged photos and quantification. D: Immunoblot anlaysis of cell lysates and CM of EV, Wt, and V1, Ctr, and HYAL-4 shRNA transfectants for indicated proteins; loading control: Actin. HYAL-4 peptide block: the peptide was added during incubation with the anti-HYAL-4 antibody. Note for A and D: In each blot, all sample lanes were run on the same gel with the same exposure time; a gap denotes those samples that were not contiguous within the gel. E: Chase activity (U) was measured in the CM of bladder cell transfectants (left panel) and in the CM of control (Ctr) and HYAL-4 shRNA transfectants (right panel). The activity was normalized to cell number. Data: Mean ± SEM; quadruplicate. F: Normalized Chase activity in the CM of HT1376 transfectants and in a high-grade bladder tumor tissue extract (HG-TBL) at indicated pH. Data: Mean ± SEM; quadruplicate. G: Chase activity in the CM of HT1376 V1 transfectants and a HG-TBL extract measured against different chondriotin sulfate substrates. Data: Mean ± SEM; quadruplicate.

Article Snippet: HYAL-4 shRNA transfectants: HYAL-4 shRNA constructs were purchased from Origene.

Techniques: Western Blot, Staining, shRNA, Blocking Assay, Incubation, Activity Assay

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Chronic TGF-β treatment results in reversal of EMT and Dab2 cleavage by CTSB. (a) Morphological analysis of NMuMG cells treated with TGF-β for the times indicated. Images were captured using a digital camera mounted on an inverted microscope. Scale bars, 100 µm. (b) Whole-cell lysates from cells treated with TGF-β for the times indicated were subjected to immunoblot analysis. Hsp90 expression served as a loading control. (c) In vitro protease inhibitor screen for p96 Dab2 cleavage. In vitro synthesized and [35S]-methionine-labelled p96 Dab2 was incubated for 1 h at 37 °C with extracts from cells treated with TGF-β for the times indicated. Each inhibitor (20 µM) was added to cells 6 h before the preparation of whole-cell lysates and autoradiographic analysis. (d) Amino acid sequence of the wild-type Dab2 and mutant LVL Dab2 cleavage sites. (e) In vitro cleavage analysis of [35S]-methionine-labelled wild-type Dab2 and LVL Dab2 mutant. (f) Real-time PCR analysis of CTSB mRNA and immunoblot analysis of CTSB protein levels in NMuMG cells treated with TGF-β for the times indicated; data are shown as mean ± s.d., n = 5 independent experiments. (g) CTSB activity assay in vitro using extracts from NMuMG cells treated with TGF-β for the times indicated; data are shown as mean ± s.d., n = 3 independent experiments. (h) Immunofluorescence analysis of Dab2 and CTSB in NMuMG cells treated with TGF-β for the times indicated. Scale bars, 10 µm. All experiments were repeated at least three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: Inverted Microscopy, Western Blot, Expressing, In Vitro, Protease Inhibitor, Synthesized, Incubation, Sequencing, Mutagenesis, Real-time Polymerase Chain Reaction, Activity Assay, Immunofluorescence

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 enhances DOXO-mediated cell death and attenuates tumour cell metastasis in vivo. (a) Parental (control) BT-20 and MDA-MB-468 breast cancer cells (2 × 105 cells) were injected into the right fourth mammary fat pad, and their Dab2 and Beclin-1 mutant derivatives (2 × 105 cells) were injected into the left fourth mammary fat pad. Tumours were excised and imaged. Scale bars, 5 mm. (b,c) Box-and-whisker plots depicting the weight of the respective tumours. Centre values are shown as horizontal lines in the boxes. The vertical lines extending from the top and bottom of the box indicate the maximum and minimum values, respectively. The top horizontal line indicates the score at the 95th percentile, and the lowest horizontal line represents the score at the 5th percentile. n = 10 mice pooled from three independent experiments. One-way ANOVA; P values are indicated. (d) Representative bioluminescence images of metastatic tumours and non-metastatic tumours in mice after the cells in a with stable luciferase expression were implanted into mice mammary gland fat pads. Post implantation (7–8 weeks), mice were subjected to bioluminescence imaging using the IVIS200 image system. Liver and lung metastasis are indicated by green arrows. Scale bars, 5 mm. (e) Kaplan–Meier survival plots showing survival rates for mice implanted with the aforementioned breast cancer cells in mice mammary gland fat pads. (f) Immunohistochemical analysis was performed on 15 invasive and 15 non-invasive human breast cancer tissues using anti-CTSB and anti-Dab2 antibodies. Slides were scanned with a ScanScope CS using bright-field imaging at ×20 magnification. Specimen areas were selected and individual images were saved in a 24-bit RGB TIFF file format with a resolution of 1 µm per pixel using the ImageScope software. Automated analysis of the TIFF image files was performed using the programming language IDL 6.3. Error bars are mean ± s.e.m. n = 15 breast cancer tissues pooled from three independent experiments. Student two-tailed paired test; P <0.05 indicates a significant difference. Scale bars, 20 µm. The experiments were repeated three times and similar results were observed.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: In Vivo, Injection, Mutagenesis, Whisker Assay, Luciferase, Expressing, Imaging, Immunohistochemical staining, Software, Two Tailed Test

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: CTSB-mediated Dab2 degradation promotes TGF-β-induced autophagy. (a) NMuMG cells and their derivatives were treated with TGF-β for the times indicated before the preparation of whole-cell lysates and immunoblot analyses using anti-Dab2 and anti-CTSB antibodies. The resultant images are the product of time average data. The lines on the western blots demarcate individual blots that were run in parallel. (b) The same as in a, but with anti-p62, anti-LC3B and anti-Beclin-1 antibodies. (c) The same as in a, but with anti-Bim, anti-cleaved caspase-3 and anti-Hsp90 antibodies. Hsp90 expression served as a loading control. (d) Morphological analysis of modified cell lines after 3 days and 7 days of TGF-β treatment and 2 days after the removal of TGF-β from cells treated for 7 days with TGF-β. Scale bars, 100 µm. The experiments were repeated three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: Western Blot, Expressing, Modification

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 inhibits chronic TGF-β-induced autophagy by blocking Vps34–Beclin-1 interaction and promotes apoptosis by attenuating ERK–Bim interaction. (a) Beclin-1 immunoprecipitation using whole-cell lysates from cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Dab2, Vps34, Bim, ERK and p-ERK. (b) Immunofluorescence analysis of Dab2 and Beclin-1 in NMuMG cells treated with TGF-β for the times indicated. Photos were taken by confocal microscope. Scale bars, 10 µm. (c) Co-immunoprecipitation analysis using anti-ERK1/2 antibody from whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of p-ERK1/2 and Bim. (d) Immunofluorescence analysis was performed to detect the co-localization between ERK and Bim before and after NMuMG cells were treated for 7 days. Scale bars, 10 µm. (e) Top: p-ERK1/2 immunoprecipitation using whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Bim. As a control, p-Bim ubiquitylation and degradation is inhibited by PYR-41, a cell-permeable inhibitor of the ubiquitin-activating enzyme E1 (20 µM, 4 h before cell lysates were collected). Bottom: immunoblotting analysis was employed to detect the Bim and p-Bim expression from whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated in the presence of 20 µM PYR-41. (f) Immunofluorescence analysis was performed to detect the co-localization between p-ERK and Bim before and after NMuMG cells were treated for 7 days in the presence of PYR-41. Scale bars, 10 µm. The experiments were repeated three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: Blocking Assay, Immunoprecipitation, Expressing, Immunofluorescence, Microscopy, Western Blot

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 blocks ERK–Bim interaction and attenuates ERK-mediated Bim phosphorylation in NMuMG cells. (a) Immunoblotting analysis of apoptosis markers Bim and cleaved caspase-3 from whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated. ERK inhibitor II was applied to cell media 3 h before collecting cell lysates of 5-day-and 7-day-treated cells (left). Flow cytometric analyses of apoptosis using Annexin V staining were evaluated in NMuMG cells treated with TGF-β for 7 days in the presence or absence of ERK inhibitor II (right). (b) Flow cytometric analyses of apoptosis using Annexin V staining were evaluated in NMuMG cells with lentivirus-mediated Bim expression. (c) Co-immunoprecipitation analysis using anti-Beclin-1 antibody of whole-cell lysates from CTSB/OE and CTSB/OE+LVL Dab2 NMuMG cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were analysed to detect the expression of Vps34, Dab2, ERK, p-ERK, Bim and Beclin-1. Immunoblots (lower panels) depict expression levels of Vps34, Dab2, ERK, p-ERK, Bim and Beclin-1 in whole-cell lysates. (d) Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in CTSB/OE and CTSB/OE + LVL Dab2 cells treated ± TGF-β for 7 days. Photos were taken by confocal microscope. Scale bars, 10 µm. The experiments were repeated three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: Western Blot, Staining, Expressing, Immunoprecipitation, Immunofluorescence, Microscopy

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 promotes CK2-mediated Beclin-1 phosphorylation resulting in Beclin-1–Vps34 dissociation. (a) Schematic representation of the three CK2 consensus phosphorylation sites in Beclin-1 (Ser64, Ser337 and Ser341). (b) Beclin-1 immunoprecipitation analysis using whole-cell lysates from cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Dab2, CK2, Beclin-1, Atg14 and Vps34. Straight immunoblot analysis depicts the expression level of CK2 and Atg14 in whole-cell lysates. (c) Immunoblot analysis of whole-cell lysates from CTSB/OE + LVL Dab2 cells treated with TGF-β for the times indicated. The CK2 inhibitor apigenin (20 µM) was added 6 h before the preparation of whole-cell lysates. (d) In vitro kinase assay using recombinant CK2 and in vitro-synthesized WT Beclin-1 and Beclin-1 mutants S64A, S337A and S341A as substrates. Reactions were subjected to SDS–PAGE and autoradiography. Apigenin was used as a specific inhibitor of CK2. (e) Co-immunoprecipitation analysis using either anti-Flag or anti-Vps34 antibody of whole-cell lysates from NMuMG CTSB/OE cells transfected with Flag-tagged wild-type Beclin-1 and three phosphomimetic Beclin-1 mutants. Immunoprecipitated complexes were immunoblotted with anti-Vps34, anti-Atg14 and anti-Flag antibodies to determine the interaction between Vps34, Atg14 and Beclin-1. (f) Flag immunoprecipitation using whole-cell lysates from 7-day-TGF-β-treated CTSB/OE+LVL Dab2 cells transfected with Flag-tagged wild-type Beclin-1 and its mutants. Immunoprecipitated complexes were immunoblotted to determine the interaction between Dab2, CK2, Vps34, Atg14 and Flag-tagged-Beclin-1. (g) Immunoblot analysis of whole-cell lysates prepared from TGF-β-treated NMuMG LVL Dab2 cells silenced for endogenous Beclin-1 and re-expressing either Flag-tagged wild-type or the indicated Beclin-1 phosphomutants. Blots were probed with the autophagic markers (LC3B and p62) and apoptosis markers (Bim and cleaved caspase-3). Hsp90 was used as a loading control. (h) Immunoblot analysis of whole-cell lysates from NMuMG LVL Dab2 and Beclin-1 KD cells stably re-expressing Flag-tagged exogenous WT or the indicated Beclin-1 mutants. All experiments were repeated at least three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: Immunoprecipitation, Expressing, Western Blot, In Vitro, Kinase Assay, Recombinant, Synthesized, SDS Page, Autoradiography, Transfection, Stable Transfection

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 promotes chemotherapy-drug-induced cell death by attenuating drug-induced autophagy. (a) Immunoblot analysis of whole-cell lysates prepared from BT-20 and MDA-MB-468 cells, and their modified derivatives, depicting the expression levels of Dab2, CTSB, endogenous Beclin-1 and Flag-tagged muBeclin-1. (b) Clonogenic assays depicting the dose-dependent effects of DOXO on cell growth in NMuMG, BT-20 and MDA-MB-468 cells, and their modified derivatives. (c) Flow cytometric analysis of autophagic flux in NMuMG, BT-20 and MDA-MB-468 cells, and their modified derivatives, incubated in the absence (left peak) or presence (right peak) of DOXO (2.0 µg ml−1) for 24 h. FITC-conjugated Cyto-ID was employed to measure autophagic vacuoles with representative histograms. (d) Flow cytometric analysis of apoptosis using Annexin V staining evaluated in NMuMG, BT-20 and MDA-MB-468 cells, and their modified derivatives, incubated in the absence (top panels) or presence (bottom panels) of DOXO (2.0 µg ml−1). The flow cytometry profile represents Annexin V–FITC staining on the x axis and PI on the y axis. The pink numbers represent the percentages of non-apoptotic cells (lower left quadrant), early apoptotic cells (lower right quadrant), later apoptotic cells (upper right quadrant) and dead cells (upper left quadrant). All experiments were repeated at least three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: Cathepsin B shRNA and Dab2 shRNA constructs were purchased from OriGene Technologies.

Techniques: Western Blot, Modification, Expressing, Incubation, Staining, Flow Cytometry