Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots showing that knock-down of p62 in HeLa cells inhibits PorB K117Q-dependent degradation of TOM20 and TIM23. b Reduced p62 and HSP60 colocalization in HeLa cells expressing PorB K5 or K null compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; WT PorB-PorB K5: P < 10 −15 ; WT PorB-PorB K null : P < 10 −15 ). c Western blots of HeLa 4KO cells expressing WT or truncated p62 show that the p62 LIR domain and UBI domain are indispensable for PorB K117Q-induced degradation of TOM20 and TIM23. d Western blots showing co-immunoprecipitation of ubiquitin and p62 after immunoprecipitation of PorB WT and PorB K117Q. e Western blots showing co-immunoprecipitation of both K63-linked and K48-linked polyubiquitin, after immunoprecipitation of PorB from HeLa cells, which is enhanced with inhibitors for autolysosomal degradation (BafA1, 3-MA) for K63-linked ubiquitin or proteasomal degradation (MG132) for K48-linked ubiquitin. f Colocalization between HSP60 and K63-linked polyubiquitin, but not K48-linked polyubiquitin, is induced in HeLa cells expressing PorB. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test, K63 only Empty-PorB K117Q: P < 10 −15 . g Western blots after immunoprecipitation of HA-PorB from HeLa cells show co-immunoprecipitation of K63-linked polyubiquitin is dependent on PorB lysine 171 and co-immunoprecipitation of K48-linked polyubiquitin is dependent on PorB lysine 128. h Western blots showing PorB-induced degradation of TOM20 and TIM23 in HeLa cells is dependent on PorB lysines 117 and 171. i Western blots showing gonococcal OMVs expressing PorB K117Q/K171Q lost the ability to induce degradation of TOM20 and TIM23. Cells in b and f are from 3 independent experiments. Western blots in a , c , d , e , g , h , i are representative of 3 independent exper i ments. Source data are provided as a Source Data file.

Article Snippet: For experiments with chemical inhibitors, cells were pretreated with Dynasore (Selleck, #S8047, 80 μM, 1 h), Chlorpromazine (Selleck, #S5749, 15 μg/mL, 1 h), Wortmannin (MCE, #HY-10197, 200 nM, 1 h), Filipin III (Selleck, #HY-N6718, 10 μg/mL, 1 h), BafA1 (Selleck, #S1413, 50 nM, 4 h), 3-MA (10 mM, Selleck, #S2767, 2 h), MG132 (Selleck, #S2619, 5 μM, 6 h), CCCP (Selleck, #S6494, 10 μM, 2 h), Mdivi-1 (Selleck, #S7162, 20 μM, 2 h), or Mito-TEMPO (Selleck, #S9733, 10 μM, 2 h).

Techniques: Western Blot, Knockdown, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Asian journal of nanoscience and materials

Article Title: Effects of fluorodeoxyglucose magnetic nanoparticles on NCI-H727 and SH-SY5Y cancer cells

doi: 10.26655/AJNANOMAT.2021.1.5

Figure Lengend Snippet: Analysis of morphological changes to NCI-H727 spheroids after FDG-mNP treatment and Hydroxy Dynasore treatment A) Spheroids with an appropriate sphericity index (SI) were selected and treated with FDG-mNPs, with or without Hydroxy Dynasore 24 hours prior to FDG-mNP treatment, and an EVOS XL microscope was used to obtain images. B) Spheroids were reconstructed using the ReVISP software (https://sourceforge.net/projects/revisp/). C) Quantitative morphological information of the spheroids, including sphericity index (SI), roundness, and solidity. ImageJ software (https://imagej.net/) was used along with Macro S1 for the calculation of morphological parameters. Samples labeled as “control” received no treatment, samples labeled as “FDG-MNPs” were treated with FDG-mNPs only, and samples labeled as “Hydroxy Dynasore” were treated with Hydroxy Dynasore prior to FDG-mNP treatment

Article Snippet: Fasentin and Hydroxy Dynasore were obtained from TOCRIS Bioscience (Abingdon, UK).

Techniques: Microscopy, Software, Labeling, Control

Journal: Asian journal of nanoscience and materials

Article Title: Effects of fluorodeoxyglucose magnetic nanoparticles on NCI-H727 and SH-SY5Y cancer cells

doi: 10.26655/AJNANOMAT.2021.1.5

Figure Lengend Snippet: Fluorescence microscopy of NCI-H727 and corrected total cell fluorescence (CTCF). NCI-H727 cells were cultured for 24 hours to allow formation of spheroids and were treated with A) FDG-mNPs only or B) FDG-mNPs and Hydroxy Dynasore. The resulting spheroids were stained with propidium iodide solution (nucleus of dead cells appear red) in 100 mL of media for 10 minutes. Images were acquired using a Widefield microscope at 493 nm. C) CTCF was calculated using the formula Area×Intensity Density-Background using the ImageJ software

Article Snippet: Fasentin and Hydroxy Dynasore were obtained from TOCRIS Bioscience (Abingdon, UK).

Techniques: Fluorescence, Microscopy, Cell Culture, Staining, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors

doi: 10.1007/s00018-017-2733-4

Figure Lengend Snippet: Endocytic uptake of rIpaH9.8. a Flow cytometry-based analysis of HeLa cells incubated with 25 µg/mL Alexa Fluor®488-labeled rIpaH9.8 for up to 3 h at 37 and 4 °C, respectively. Data are means ± standard deviations from at least three independent experiments; ns non significant, **p < 0.01, ****p < 0.0001 comparison of cells incubated at 37 and 4 °C (two-way ANOVA followed by Bonferroni’s multiple comparisons test). AU arbitrary unit. b CLSM analysis of HeLa cells incubated with 25 µg/mL Cy3-labeled rIpaH9.8 for 1, 3 and 6 h, respectively. Merged images are shown in the left panels and single fluorescence channels are shown in the right panels. All images consist of one optical section of a z-series with a pinhole of 1 airy unit. Green, compartment-specific marker proteins for early endosomes (EE), late endosomes (LE) and lysosomes (Lys); red, Cy3-labeled rIpaH9.8; blue, nuclei; scale bars represent 10 µm. c Flow cytometry-based analysis of HeLa cells pre-treated with the indicated inhibitors for 1 h [200 µM cytochalasin D, 19 mM amiloride, 30 mM dynasore, 3.8 mM filipin, 50 mM methyl-β-cycodextrin (MβCD), 16.5 mM nocodazole] and subsequently incubated with 25 µg/mL Alexa Fluor®488-labeled rIpaH9.8 for 3 h. Data are means ± standard deviations from at least three independent experiments; ns, non significant, *p < 0.05, ****p < 0.0001 compared to rIpaH9.8-incubated cells without inhibitor (one-way ANOVA followed by Bonferroni’s multiple comparisons test). d Quantification of co-localization of rIpaH9.8-Cy3 with FITC-conjugated cholera toxin B-subunit (CTB-FITC) and Alexa Fluor®488-conjugated transferrin (Tf-Alexa Fluor®488), respectively, in HeLa cells co-incubated with either 20 µg/mL CTB-FITC or 20 µg/mL Tf-Alexa Fluor®488 and with 25 µg/mL Cy3-labeled rIpaH9.8 for 30 min. The percentages of co-localization of CTB-FITC/Tf-Alexa Fluor®488 with Cy3-labeled rIpaH9.8 were calculated using BioImageXD software. Data are means ± standard deviations after evaluating co-localization from six microscopic images; significance was calculated using the Student t test, ***p < 0.001 (left panel). Exemplary CLSM analysis of HeLa cells co-incubated with rIpaH9.8-Cy3 and CTB-FITC/Tf-Alexa Fluor®488. Merged images are shown in the left panels and single fluorescence channels are shown in the right panels. All images consist of one optical section of a z-series with a pinhole of 1 airy unit. Green, indicated fluorescently-labeled endocytic tracer; red, Cy3-labeled rIpaH9.8; blue, DNA; scale bars represent 10 µm (right panel). e Electron microscopy of cryosections of HeLa cells incubated with 50 µg/mL FLAG-tagged rIpaH9.8 for 3 h. Sections were stained with mouse-α-FLAG antibody, rabbit-α-mouse IgG antibody and Protein A Gold (PAG) (15 nm). PM plasma membrane, C cytosol, EE early endosome, N nucleus, Lys lysosome, M mitochondrion; scale bars represent 100 nm

Article Snippet: After pre-incubation with inhibitors (200 µM of cytochalasin D (Sigma-Aldrich), 19 mM of amiloride (Sigma-Aldrich), 30 mM of dynasore (Enzo Life Sciences, Farmingdale, NY, USA), 3.8 mM of filipin (Sigma-Aldrich), 50 mM of methyl-β-cycodextrin (Sigma-Aldrich) (MβCD), 16.5 mM of nocodazole (Sigma-Aldrich)) for 1 h, 25 µg/mL of Alexa Fluor ® 488-labeled rIpaH9.8 were added to the cells for further 3 h. Before measurement, cells were trypsinized and quenched with trypan blue as described above.

Techniques: Flow Cytometry, Incubation, Labeling, Comparison, Fluorescence, Marker, Software, Electron Microscopy, Staining, Membrane

Journal: PLoS ONE

Article Title: Uptake of nanowires by human lung adenocarcinoma cells

doi: 10.1371/journal.pone.0218122

Figure Lengend Snippet: a) Effects of the drugs used in this study to block specific pathways. b) Effects of the different drugs on nanowire uptake 8 h after the beginning of nanowire exposure corresponding to 4 h after the beginning of the drug application. The addition of Dynasore and Cytochalasin D leads to a decrease in the percentage of cells containing nanowires, suggesting that the internalization takes place via phagocytosis and macropinocytosis. (**: p<0.01, ***: p<0.001, one way ANOVA).

Article Snippet: Selected uptake mechanisms were blocked by adding cytochalasin D (Cayman Chemicals, USA; final concentration 0.005 μM), dynasore (Cayman Chemicals, USA; final concentration 100 μM), chlorpromazine (Cayman Chemicals, USA; final concentration 2.5 μM), or nystatin (Alfa Aesar, Germany; final concentration 25 μM) into the cell medium after 4 hours of exposure to nanowires.

Techniques: Blocking Assay

Journal: Scientific Reports

Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

doi: 10.1038/srep46069

Figure Lengend Snippet: Confluent HF monolayers were pretreated with dynasore, or pitstop 2 for 1 h, and then infected with Towne or BAD32GFP virus at an MOI of 3.0 in the medium containing the same drug for one hour, washed and thereafter incubated in the presence of the drug. Samples of infected-cells in the cell culture medium were harvested at 5 days post infection and stored at −80 °C before titration. Concentration of pitstop 2 ( A ) in the range of zero to 25 μM or of dynasore ( B ) in the range of zero to 250 μM led to dose dependent inhibition of final virus yields. Triplicate samples were used.

Article Snippet: Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

Techniques: Infection, Virus, Incubation, Cell Culture, Titration, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

doi: 10.1038/srep46069

Figure Lengend Snippet: Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for ( A ) 6 hours or ( B ) 5 days in the presence of the same concentration of the drug. DMSO-treated infected cells and uninfected untreated cells served as controls in this experiment. Cell viability was determined using trypan blue exclusion assay as described in materials and methods. Triplicate samples were used.

Article Snippet: Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

Techniques: Infection, Virus, Concentration Assay, Incubation, Trypan Blue Exclusion Assay

Journal: Scientific Reports

Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

doi: 10.1038/srep46069

Figure Lengend Snippet: Entry of HCMV laboratory strains AD169 (BAD32) ( A ) and Towne ( B ) in HF upon treatment with pitstop 2, or dynasore as determined by expression of viral immediate early 1 (IE1) protein in infected cells. Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with BAD32GFP or Towne virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 6 hours in the presence of the same concentration of drug before harvesting for immunoblots. Triplicate samples were used. DMSO-treated infected cells, untreated infected cells (UT) and untreated uninfected cells (UI) served as controls in this experiment. β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.

Article Snippet: Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

Techniques: Expressing, Infection, Virus, Concentration Assay, Incubation, Western Blot, Control

Journal: Scientific Reports

Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

doi: 10.1038/srep46069

Figure Lengend Snippet: Confluent HF monolayers were pretreated with dynasore (100 μM), pitstop 2 (25 μM), or mock (DMSO) for 1 h, then infected with BAD32GFP virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 72 hours in the presence of the same concentration of drug before harvesting for immunoblots using antibodies against early (pUL44) and late (gB) viral antigens. Duplicate samples were used for each treatment. DMSO-treated infected cells served as mock-control in this experiment and β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.

Article Snippet: Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

Techniques: Infection, Virus, Concentration Assay, Incubation, Western Blot, Control

Journal: Scientific Reports

Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

doi: 10.1038/srep46069

Figure Lengend Snippet: Transmission electron micrographs of HF cells that were either mock-treated ( A , B and C ) or treated with dynasore (100 μM) ( D , E and F ) or pitstop 2 (25 μM) ( G , H and I ) and then infected with HCMV (AD169) at an MOI of 3.0 and incubated in the presence of indicated drug for 4 days before processing for TEM. Representative whole cell ( A , D , G ), nuclear ( B , E , H ) or cytoplasmic ( C , F , I ) sections of the infected cell are shown in the micrographs. ( J ) Quantification of nuclear capsid types from the above treatments. A total of 400, 318 or 674 capsids were counted for mock, dynasore and pitstop 2 treatments, respectively. Scale: 2 μm ( A , C , D ), 1.0 μm ( F , G ), 0.5 μm ( B , E , H , I ). A- (white arrowheads), B- (black arrows), and C- capsids (black arrowheads).

Article Snippet: Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

Techniques: Transmission Assay, Infection, Incubation