Journal: International Journal of Molecular Sciences

Article Title: Transport and Toxicity of Methylmercury-Cysteine in Cultured BeWo Cells

doi: 10.3390/ijms23010394

Figure Lengend Snippet: Indicators of autophagy in BeWo cells. BeWo cells were exposed to various concentrations of MeHg-Cys for 30 min at 37 °C, and the formation of autophagosomes was measured ( A ). For analyses of mRNA expression and protein levels, cells were exposed to MeHg-Cys for 16 h at 37 °C. RNA and protein were isolated from the control and exposed cells, and quantitative PCR (qPCR) was used to analyze levels of ATG13 ( B ), PINK ( C ), and BNIP3 ( D ). Western blotting ( E ) measured protein levels of ATG13 (72 kDa), PINK (63 kDa), and BNIP3 (22 kDa). β-actin (42 kDa) was used as the control. Results are presented as mean ± SE. Data represent 3 experiments performed in triplicate. *, significantly different ( p < 0.05) from unexposed cells.

Article Snippet: Quantitative PCR analyses of superoxide dismutase 1 (SOD1), caspase 8, tumor necrosis factor alpha (TNFα), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), hypoxia-inducible factor 1 (HIF-1), autophagy-related protein 13 (ATG13), PTEN-induced putative kinase 1 (PINK1), and BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3L) expression were performed using an ABI Prism 7300 sequence detection system and commercially available gene expression assays (SOD1: Hs00533490_m1; Caspase 8: Hs01018151_m1; TNFα: Hs00174128_m1; NF-κB: Hs00765730_m1; HIF-1: Hs00153153_m1; RIPK3: Hs00179132_m1; ATG13: Hs00207186_m1, PINK1: Hs00260868_m1, BNIP3L: Hs00188949_m1, Life Technologies).

Techniques: Expressing, Isolation, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific reports

Article Title: Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes.

doi: 10.1038/s41598-020-76227-7

Figure Lengend Snippet: Figure 2. CVB3-induced LC3 lipidation occurs independent of FIP200 and ATG13. (A) FIP200-KO HEK293A cells were established through CRISPR-Cas9 gene editing. Knockout efficiency was validated by western blotting (left panel). WT-HEK293A or FIP200-KO cells were cultured in either normal medium, HBSS starvation medium, or starvation medium supplemented with 200 nM bafilomycin (BAF) for 2 h (middle panel). WT or FIP200-KO cells were sham- or CVB3-infected for 16 h (right panel). Western blotting was performed for analysis of LC3 lipidation. (B,C) Schematic of siRNA-based gene silencing and CVB3 infection schedule (left), HEK293A cells were transiently transfected with scrambled siRNA control (siCON) or siRNAs against FIP200 (B) or ATG13 (C) for 48 h. Cells were then subjected to sham or CVB3 infection for an additional 16 h. Western blotting was conducted to determine knockdown efficiency and LC3 lipidation. Densitometry was measured as above and the results are presented either underneath the blots or in the right panel (mean ± SD, n = 3, analyzed by one-way ANOVA with Tukey’s post-test). (D) HEK293A cells were treated with a selective ULK1/2 kinase inhibitor (MRT68921, 5 µM) under starvation for 2 h in the presence or absence of 200 nM BAF (left panel). HEK293A cells were sham- or CVB3-infected for 16 h with or without 5 µM MRT68921 (right panel). Western blotting was performed to examine LC3 levels and the quantified results are shown underneath the blots.

Article Snippet: The scrambled siRNA (sc-37007) and the siRNAs targeting FIP200 (sc-38211), ATG13 (sc-97013), BECN1 (sc-29797), PIK3C3 (sc-62802), WIPI2 (sc-72212), ATG9 (sc72586), and PIKfyve (sc-39142) were purchased from Santa Cruz Biotechnology.

Techniques: CRISPR, Knock-Out, Western Blot, Cell Culture, Infection, Transfection, Control, Knockdown

Journal: Scientific reports

Article Title: Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes.

doi: 10.1038/s41598-020-76227-7

Figure Lengend Snippet: Figure 5. CVB3 targets several autophagy proteins in HEK293A cells. (A–C) HEK293A cells were sham- or CVB3-infected for 8 h, 16 h, or 24 h as indicated. Cell lysates were harvested and probed for ULK1, ATG13, FIP200, and ULK2, components of the ULK1/2 complex (A), BECN1, UVRAG, PIK3C3, and ATG14, components of the PI3K complex (B), and WIPI2 and ATG9A, components of the WIPI complex (C). Protein levels were quantified and presented underneath each western blot as above.

Article Snippet: The scrambled siRNA (sc-37007) and the siRNAs targeting FIP200 (sc-38211), ATG13 (sc-97013), BECN1 (sc-29797), PIK3C3 (sc-62802), WIPI2 (sc-72212), ATG9 (sc72586), and PIKfyve (sc-39142) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Western Blot

Journal: Open Biology

Article Title: Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast

doi: 10.1098/rsob.220366

Figure Lengend Snippet: The NTDs of GSDMD and MLKL cause a cell cycle arrest through the inhibition of TORC1. ( a ) Cell cycle profiles obtained by measuring DNA content (FL2-A) of cells stained with PI and subsequently analysed by flow cytometry ( n = 10 000) (left panels), and graph showing the percentage of cells in phase G0/G1 for each population (right panel) of BY4741 strain bearing plasmids as in b , f , after 5 h of induction in SG medium. ( b ) Immunoblot showing Sch9 phosphorylation (upper panel) and quantification of P-Sch9 relative to total Sch9 (lower panel) in yeast lysates of BY4741 strain bearing the plasmids pJU676 (Sch9-5xHA) and pAG413-GSDMD-EGFP, pAG413-GSDMD(NT)-EGFP, pAG413-MLKL-EGFP, pAG413-MLKL(PM)-EGFP or pAG413-MLKL(1–182)-EGFP after 7 h of induction in SG medium. pAG413-EGFP empty vector was used as a control. ( c ) Immunoblot showing Atg13 phosphorylation (upper panel) and quantification of P-Atg13 relative to total Atg13 (lower panel) in yeast lysates of BY4741 strain bearing the plasmids HC078 (3xHA-Atg13) and the same plasmids as in ( b ). ( d ) Immunoblot showing Maf1 phosphorylation (upper panel) and quantification of P-Maf1 relative to total Maf1 (lower panel) in yeast lysates of BY4741 strain bearing plasmids pAH099 (Maf1-3xHA) and the same plasmids as in ( b ). In ( b–d ), cells treated with 100 nM rapamycin for 5 h were used as a positive control of TORC1 inhibition. Membranes were hybridized with anti-HA antibody. Anti-G6PDH antibody was used for loading control. A representative blot from three different experiments with different transformants is shown. In ( a–d ), results correspond to the mean of three biological replicates performed on different transformants. Error bars represent the standard deviation. Asterisks (*, **, ***) indicate p -values of < 0.05, < 0.01 and < 0.001, respectively, by Tukey's HSD test.

Article Snippet: HC078 (pRS315-3xHA-Atg13) plasmid, used also as a readout of TORC1 activity, was obtained from Addgene (Addgene_ 59544).

Techniques: Inhibition, Staining, Flow Cytometry, Western Blot, Phospho-proteomics, Plasmid Preparation, Control, Positive Control, Standard Deviation

Journal: PLoS ONE

Article Title: The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

doi: 10.1371/journal.pone.0059292

Figure Lengend Snippet: (A,B) HT29 cells were pretreated for 1 h with the indicated concentrations of TPCA-1 and Bay 11-7082 and were subsequently challenged with TNF (50 ng/ml) for 3 and 10 min (A) or with Flag-TWEAK (200 ng/ml) for 8 hours (B). Total cell lysates were finally analyzed by Western blotting with respect to TNF-induced phosphorylation and degradation of IκBα (A) and TWEAK-induced p100 processing (B). (C) HT29 cells (40,000/chamber) were grown on glass slides and were pretreated with Bay 11-7082 (30 µM) or TPCA-1 (20 µM) for 30 min. Cells were then challenged with 100 ng/ml TNF for 1 h. After immunofluorescence staining for p65, the ratio of nuclear to cytoplasmic fluorescence intensity (FI) was determined. Data shown corresponds to 95–111 analyzed cells per experimental condition derived from a total of four independent experiments. (D) HT29 cells (20,000/well, 96 well-plate, triplicate values) were pretreated with the IKK inhibitors Bay 11-7082 (30 µM) or TPCA-1 (20 µM) for 30 min and were then stimulated with 100 ng/ml TNF for 6 h. The IL8 content of supernatants was subsequently determined by ELISA. To minimize the background signal related to constitutive IL8 production, medium was changed prior to inhibitor treatment. (E) The effects of TPCA-1 and Bay 11-7082 on TNF-induced phosphorylation and degradation of IκBα were analyzed in KMS-12-BM myeloma cells as described under “A”; n.s. = non specific. (F) Equivalent analysis on TNF-induced IL8 production as described in “D” using the RPMI8226 MM cell line. For statistical analysis of data shown in C, D and F one-way ANOVA with a Tukey post-test was performed. Asterisks indicate p-values≤0.01.

Article Snippet: Suppliers of antibodies: NIK (4994) and phospho-IκBα (2859): Cell Signaling Technology (Frankfurt am Main, Germany); p100 (05-361): Merck Millipore (Darmstadt, Germany); tubulin (MS-581-P): Dunn Labortechnik (Asbach, Germany); IκBα (sc847) and p65 (sc109): Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Staining, Fluorescence, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

doi: 10.1371/journal.pone.0059292

Figure Lengend Snippet: (A) HT29 and KMS-12-BM cells were pretreated for 1 h with the indicated concentrations of MLN4924 and challenged with TNF (50 ng/ml) for 3 and 10 min. Phosphorylation and degradation of IκBα were subsequently analyzed by Western blotting of total cell lysates. (B) HT29 cells (20,000/well, 96 well-plate, triplicate values) and RPMI8226 cells (50,000/well, 96 well-plate, triplicate values) were pretreated for 30 min with different concentrations of MLN4924. Cells were then stimulated with 100 ng/ml TNF for 6 h and the IL8 content of supernatants was determined by ELISA. For statistical analysis a one-way ANOVA with a Tukey post-test was performed. Groups showing significant inhibition (p-values≤0.01) of TNF-induced IL8 production are indicated by asterisks. (C) HT29 cells were pretreated for 1 h with the indicated concentrations of MLN4924 and then stimulated with Flag-TWEAK (200 ng/ml) for 18 h. The levels of p52, p100 and NIK were analyzed by Western blotting of total cell lysates. (D) MM cell lines were treated for 18 h with 10 µM MLN4924 and assayed for viability using the MTT assay. Significant (unpaired, two-tailed t-test, p-values≤0.01) induction of cell death is highlighted by asterisks. (E) Primary MM samples (n = 11) in co-culture with BMSCs were treated with 10 µM MLN4924 for 3 days and cell death was assessed by annexin V-FITC/PI staining and FACS analysis.

Article Snippet: Suppliers of antibodies: NIK (4994) and phospho-IκBα (2859): Cell Signaling Technology (Frankfurt am Main, Germany); p100 (05-361): Merck Millipore (Darmstadt, Germany); tubulin (MS-581-P): Dunn Labortechnik (Asbach, Germany); IκBα (sc847) and p65 (sc109): Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, MTT Assay, Two Tailed Test, Co-Culture Assay, Staining

Journal: Cell reports

Article Title: Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

doi: 10.1016/j.celrep.2024.115016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Monoclonal phospho-Atg13 Ser355 , Cell Signaling Technology , Cat# 46329; RRID:AB_3064843.

Techniques: Ubiquitin Proteomics, Recombinant, Modification, Membrane, Electron Microscopy, Protease Inhibitor, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Plasmid Preparation, Software, Imaging

Journal: Communications Biology

Article Title: Lysosomal trafficking mediated by Arl8b and BORC promotes invasion of cancer cells that survive radiation

doi: 10.1038/s42003-020-01339-9

Figure Lengend Snippet: a , b Matrigel chemoinvasion assay of MDA-MB-231 cells ( a ) and Hs578T cells ( b ) with or without treatment of lysosomal inhibitors bafilomycin A1 (Baf A1) or chloroquine (CQ) and 4 Gy IR. Representative images of the results were obtained from the Matrigel invasion assays. c Lysosomal exocytosis was quantified by the measurement of dextran-488 in culture medium. d Cell-surface LAMP1 was measured by flow cytometry. e Cell-surface LAMP1 fluorescence intensity was measured using an image scanner. The fluorescence intensity was normalized to the surviving cell viability in each group. f Immunofluorescence images of LAMP1 in MDA-MB-231 cells treated with or without IR treatment. Green, LAMP1; red, α-tubulin; yellow, γ-tubulin (centrosome); blue, DAPI (nucleus). Bar, 10 μm. g Total LAMP1 in whole-cell lysates of MDA-MB-231 cells after IR treatment was detected by immunoblotting. h The lysosomal distribution from the centrosome to the cell membrane was quantified. The cells were divided into regions based on the relative LAMP1 intensity as follows: region 0 (0–20%), region 1 (20–40%), region 2 (40–60%), region 3 (60–80%), and region 4 (80–100%). i LAMP1 fluorescence intensities in each region of MDA-MB-231 cells. The intensity of region 0 was excluded. Points and connecting lines, means; bars, SEMs. j LAMP1 fluorescence intensity in the cell periphery (regions 3 and 4). More than 20 cells per group were assessed in three independent experiments, and the results are shown as a scatter plot. Bars, SEMs. k Matrigel chemoinvasion assay of MCF-7 cells with or without IR treatment. l Immunofluorescence images of LAMP1 in MCF-7 cells. Green, LAMP1; red, α-tubulin; blue, DAPI. Bar, 10 μm. m LAMP1 fluorescence intensities in each region of MCF-7 cells. n LAMP1 fluorescence intensity in the cell periphery (regions 3 and 4) of MCF-7 cells. Bars, SEMs. All column graphs show the means with SEMs of three ( d , e , k ) or four ( a – c ) independent experiments. The results of each group in column graphs were normalized against that of the untreated group. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Cells were trypsinized, resuspended in flow cytometry buffer (2% FBS in PBS) and incubated with anti-LAMP1 antibody (Cell Signaling Technology, Danvers, USA; recognizes the luminal/extracellular domain [29-382] of LAMP1) in flow cytometry buffer at 4 °C for 30 min. After washing two times with flow cytometry buffer, cells were stained with Alexa Fluor-conjugated secondary antibodies at 4 °C for 30 min. As an isotype control, cells were stained with secondary antibodies alone.

Techniques: Flow Cytometry, Fluorescence, Immunofluorescence, Western Blot, Membrane

Journal: Communications Biology

Article Title: Lysosomal trafficking mediated by Arl8b and BORC promotes invasion of cancer cells that survive radiation

doi: 10.1038/s42003-020-01339-9

Figure Lengend Snippet: a Arl8b in the lysosomal fraction of MDA-MB-231 cells. Markers of the nucleus (Histone H3), lysosomes (Rab9) and the cytoskeleton (β-actin) were stained with antibodies. The amount of Arl8b in the lysosomal fraction was normalized against LAMP1. b Total Arl8b in whole-cell lysates of MDA-MB-231 cells after IR treatment was detected by immunoblotting. c , d Arl8b-mVenus overexpression was induced by doxycycline ( c ) and assessed by confocal fluorescence microscopy ( d ). Dox, doxycycline. Green, Arl8b-mVenus; red, LysoTracker. Bar, 10 μm. e Lysosomal distribution in Arl8b-mVenus-overexpressing MDA-MB-231 cells. Pseudocolor micrographs with lysosomes shown in red (LAMP1), microtubules shown in blue (α-tubulin), and nuclei shown in white (DAPI). Bar, 10 μm. f LAMP1 fluorescence intensities in each region. The intensity of region 0 was excluded. Points and connecting lines, means; bars, SEMs. g LAMP1 fluorescence intensity in the cell periphery (regions 3 and 4). Bars, SEMs. h Matrigel chemoinvasion assay of Arl8b-mVenus-overexpressing MDA-MB-231 cells. Representative images of the results were obtained from the Matrigel invasion assays. i Immunofluorescence images showing essential proteases in Arl8b-positive lysosomes. Blue, proteases; green, Arl8b-mVenus; red, LysoTracker. Bar, 10 μm. All column graphs show the means with SEMs of three independent experiments. The results of each group in column graphs were normalized against that of the untreated group. * P < 0.05; *** P < 0.001; ns, not significant.

Article Snippet: Cells were trypsinized, resuspended in flow cytometry buffer (2% FBS in PBS) and incubated with anti-LAMP1 antibody (Cell Signaling Technology, Danvers, USA; recognizes the luminal/extracellular domain [29-382] of LAMP1) in flow cytometry buffer at 4 °C for 30 min. After washing two times with flow cytometry buffer, cells were stained with Alexa Fluor-conjugated secondary antibodies at 4 °C for 30 min. As an isotype control, cells were stained with secondary antibodies alone.

Techniques: Staining, Western Blot, Over Expression, Fluorescence, Microscopy, Immunofluorescence

Journal: Communications Biology

Article Title: Lysosomal trafficking mediated by Arl8b and BORC promotes invasion of cancer cells that survive radiation

doi: 10.1038/s42003-020-01339-9

Figure Lengend Snippet: a Arl8b was knocked down with shRNAs (shArl8b human #1 and #2) in MDA-MB-231 cells. b LAMP1 in control or Arl8b-knockdown MDA-MB-231 cells. Cells were or were not treated with 4 Gy IR. Green, LAMP1; red, α-tubulin; blue, DAPI. Bar, 10 μm. c Quantification of the lysosomal distribution in MDA-MB-231 cells. The intensity of region 0 was excluded. Points and connecting lines, means; bars, SEMs. d Scatter plot of the lysosomal distribution in the cell periphery (regions 3 and 4). Bars, SEMs. e The fluorescence intensities of dextran-488 in the culture medium of control or Arl8b-knockdown MDA-MB-231 cells, with or without IR treatment, were determined with a plate reader. f Surface LAMP1 on control and Arl8b-knockdown MDA-MB-231 cells was detected with flow cytometry. g Surface LAMP1 on control and Arl8b-knockdown MDA-MB-231 cells was detected with an image scanning system. h – k Matrigel chemoinvasion assay of control or Arl8b-knockdown MDA-MB-231 ( h ), Hs578T ( i ), MCF-7 ( j ) and 4T1 ( k ) cells (shArl8b mouse #1 and #2). l Matrix degradation activity was determined using a mixture of 3D lrECM and DQ-Collagen IV. Control or Arl8b-knockdown Hs578T cells were cultured in 3D lrECM containing DQ-Collagen IV for 48 h after IR. Blue: blue fluorescent protein (BFP) expressed by cells as a marker of cell margins in live-cell imaging. Green: area of DQ-collagen IV cleaved by proteases. m Ratios of extracellular DQ-collagen IV degradation area to the total cell area. Bars, SEMs. All column graphs show the means with SEMs of three ( g – k ) or four ( e , f ) independent experiments. The results of each group in column graphs were normalized against that of the control group. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Cells were trypsinized, resuspended in flow cytometry buffer (2% FBS in PBS) and incubated with anti-LAMP1 antibody (Cell Signaling Technology, Danvers, USA; recognizes the luminal/extracellular domain [29-382] of LAMP1) in flow cytometry buffer at 4 °C for 30 min. After washing two times with flow cytometry buffer, cells were stained with Alexa Fluor-conjugated secondary antibodies at 4 °C for 30 min. As an isotype control, cells were stained with secondary antibodies alone.

Techniques: Control, Knockdown, Fluorescence, Flow Cytometry, Activity Assay, Cell Culture, Marker, Live Cell Imaging

Journal: Communications Biology

Article Title: Lysosomal trafficking mediated by Arl8b and BORC promotes invasion of cancer cells that survive radiation

doi: 10.1038/s42003-020-01339-9

Figure Lengend Snippet: a Kaplan–Meier survival curves were generated based on the expression levels of ARL8B and BORC-subunit genes using TCGA data from 1075 human breast cancer patients. b Lymph node metastasis (number of positive metastatic lymph nodes/total lymph node count) was found to correlate with the combined expression of ARL8B and BORC-subunit genes using the same TCGA data set and stratification as in ( a ). Box-and-whisker plots. ** P < 0.01. c Heat map of fold-changes in the expression of ARL8B , PLEKHM2 and BORC-subunit genes in MDA-MB-231 cells in 3D lrECM produced by 2 Gy × 4 IR treatment. d GST-Arl8b-WT pulldown assays of HEK293T cells transfected with siRNA against BLOS2 or Myrlysin that were then cotransfected with Xpress-SKIP and GST-Arl8b-WT. The Xpress-SKIP signal intensity was normalized by that of GST-Arl8b-WT. Columns, means ( n = 4); bars, SEMs. * P < 0.05. e BLOS2 or Myrlysin was knocked down by siRNA in Arl8b-HA-expressing MDA-MB-231 cells. Green, Arl8b-HA; red, LAMP1; blue, DAPI. Bar, 10 μm. f Lysosomal (LAMP1) distribution in MDA-MB-231 cells in which BLOS2 or Myrlysin was knocked down with siRNA. Green, LAMP1; red, α-tubulin; blue, DAPI. Bar, 20 μm. g Lysosomal distribution in each region. The intensity of region 0 was excluded. Points and connecting lines, means; bars, SEMs. h LAMP1 distribution in the cell periphery (regions 3 and 4). Bars, SEMs. i Matrigel chemoinvasion assay of MDA-MB-231 cells treated with siRNA against BLOS2 or Myrlysin. Columns, means ( n = 3); bars, SEMs. * P < 0.05.

Article Snippet: Cells were trypsinized, resuspended in flow cytometry buffer (2% FBS in PBS) and incubated with anti-LAMP1 antibody (Cell Signaling Technology, Danvers, USA; recognizes the luminal/extracellular domain [29-382] of LAMP1) in flow cytometry buffer at 4 °C for 30 min. After washing two times with flow cytometry buffer, cells were stained with Alexa Fluor-conjugated secondary antibodies at 4 °C for 30 min. As an isotype control, cells were stained with secondary antibodies alone.

Techniques: Generated, Expressing, Whisker Assay, Produced, Transfection